AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F(ab’)<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimag...AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F(ab’)<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging(RⅡ).METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F(ab’)<sub>2</sub>.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis(SDS-PAGE)and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate( EDTA )and L-cysteine(L-cys)solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma(HHCC)xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat <sup>99m</sup>Tc-labeled HAb18 F(ab’)<sub>2</sub> in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of <sup>99m</sup>Tc-labeled HAb18 F(ab’)<sub>2</sub>.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01(%ID/g),respectively.CONCLUSION <sup>99m</sup>Tc-HAb18 F(ab’)<sub>2</sub> conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.展开更多
The improved tumoricidal effect of the radioatibody mixture ("cocktail") has been reported recently for the treatment of colon tumor. In the present study, we demonstrated the enhanced radioimmunotherapeutic...The improved tumoricidal effect of the radioatibody mixture ("cocktail") has been reported recently for the treatment of colon tumor. In the present study, we demonstrated the enhanced radioimmunotherapeutic efficacy of a monoclonal atibody (MAb) cocktail against human hepatocellular carcinoma. Therapeutic efficacy was determined by measuring the change in tumor size over a period, determining the percentage of growth inhibition of each treatment at various times after radioantibody therapy. boioimmunotherapy of SMMC-7721 human hepatoma xenografts in athymic nude mice with combination of 131I labeled Hepama-1 and 131Llabeled 9403 mouse MAbs was more effective than using either Hepeam-1 or 9403 Mab alone The MAb cocktail could target a greater number of hepstoma cells and increase the magnitude of hepatoma cen uptde of radioamibodies. The in vjtro results explain the enhanced effect of the MAb cocktail in in vjvo model system.展开更多
I-labeled anti-CEA monoclonal antibody injected intrasplenically has better effects on inhibiting liver metastasis from human colonic adenocarcinoma than that through tail vein in nu/nu mice model.
MGb<sub>2</sub> is a monoclonal antibody againstgastric cancer.It can be well localized in thetumour tissue and has been successfully used inthe radioimmunoimaging in the patients withgastric cancer.The co...MGb<sub>2</sub> is a monoclonal antibody againstgastric cancer.It can be well localized in thetumour tissue and has been successfully used inthe radioimmunoimaging in the patients withgastric cancer.The conjugate of MGb<sub>2</sub> withchemotherapeutic drug mitomycin C(MMC)al-so shows highly selective cytotoxic effect uponhuman gastric cancer cells in vitro.展开更多
In the present study,an anti-gastric cancer monoclonal antibody MGb<sub>2</sub>-daunomycin(DM)conjugate was prepared by cis-aconitic anhydride method.The distribution of the conjugatein nude mice bearing...In the present study,an anti-gastric cancer monoclonal antibody MGb<sub>2</sub>-daunomycin(DM)conjugate was prepared by cis-aconitic anhydride method.The distribution of the conjugatein nude mice bearing human gastric carcinoma SGC-7901 was investigated.Four to five moleculesof DM were introduced into each molecule of antibody while the antigen-binding capacity ofthe antibody was well-retained.This conjugate could be satisfactorily localized in the tumortissue.Ninety-six hours after intraperitoneal injection of <sup>125</sup>I-MGb<sub>2</sub>-DM conjugate,the ratio ofradioactivity per milligram of tumor to that per milligram of blood reached 203,the result beingquite similar to that of unmodified MGb<sub>2</sub>.展开更多
The monoclonal antibody (MAb) RWS4 specific to membrane-associated antigen of human gastric adenocarcinoma was purified by protein A-Sepharose 4B affinity chromatography and labeled with 131I by chloramine-T method. 1...The monoclonal antibody (MAb) RWS4 specific to membrane-associated antigen of human gastric adenocarcinoma was purified by protein A-Sepharose 4B affinity chromatography and labeled with 131I by chloramine-T method. 131-RWS,, was injected (65 μCi/10μg/0.2 ml, intraperitoneally) into the stomach cancer-bearing nude mice (solid tumor about 1 cm in diameter), and its biodistribution was studied by SPECT and gamma-counter over a peroid of 7 days. A clear image of transplanted tumor was observed on the 4th day, and the image became more clear on the 6th day. After SPECT scanning, the animals were killed on the 3rd to 7th day separately and radioactivity was detected in various organs. The ratios of T/NT were calculated. The results were shown as follows: tumor/blood, was 3.41±0.29 on the 6th day and the tumor/other organs (liver, spleen, stomach, lung, heart, kidney and brain etc.) were>3. The specificity of the 131I-RWS4 was 7.74±0.65.展开更多
Objective To study the role of cell adhesion molecule P-selectin monoclonal antibody (MAb) in tumor metastasis of an orthotopic metasta tic model. Methods SUD mice were implanted orthotopically SGC-7901 human gastric ...Objective To study the role of cell adhesion molecule P-selectin monoclonal antibody (MAb) in tumor metastasis of an orthotopic metasta tic model. Methods SUD mice were implanted orthotopically SGC-7901 human gastric cancer tissue. 3d later, animals received i. v. injections of PBS or P-selectin MAb (100μg/injection) twice weekly for 3 weeks. 42d after operation, all animals were sacrificed. Tissues from all organs were obtained for histopathological evaluation. Results 10 of the animals ( n = 11) treat-ed with PBS were found to develop metastatic tumors in the regional lymph nodes, liver, and lung. In contrast, 2 of the animals ( n = 9) treated with P-selectin MAb developed metastatic tumors in the organs examined. The expression of P-selectin mRNA in gastric cancer tissue of SCID mice with tumor metastasis was higher than that without such metastasis. Conclusion P-selectin expression is associated with tumor metastasis, and the metastasis may be inhibited by the MAb.展开更多
AIM: To investigate the inhibitory efficacy of ^(125)I-labeled anti-basic fibroblast growth factor(b FGF) monoclonal antibody(m Ab) in hepatocellular carcinoma(HCC).METHODS: b FGF m Ab was prepared by using the 1G9B9 ...AIM: To investigate the inhibitory efficacy of ^(125)I-labeled anti-basic fibroblast growth factor(b FGF) monoclonal antibody(m Ab) in hepatocellular carcinoma(HCC).METHODS: b FGF m Ab was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with ^(125)I through the chloramine-T method, b FGF m Ab was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of ^(125)I-b FGF m Ab. The murine H22 HCC xenograft model was established and randomized to interventions with control(phosphate-buffered saline), ^(125)I-b FGF m Ab,^(125)I plus b FGF m Ab, b FGF m Ab, or ^(125)I. The ratios of tumor inhibition were then calculated. Expression of b FGF, fibroblast growth factor receptor(FGFR), plateletderived growth factor, and vascular endothelial growth factor(VEGF) m RNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction. RESULTS: The purified b FGF m Ab solution was 8.145 mg/m L with a titer of 1:2560000 and was stored at-20 ℃. After coupling, ^(125)I-b FGF m Ab was used at a 1: 1280000 dilution, stored at 4 ℃, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, ^(125)I, b FGF m Ab, ^(125)I plus b FGF m Ab, and ^(125)I-b FGF m Ab groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the ^(125)I, b FGF m Ab, ^(125)I plus b FGF m Ab, and ^(125)I-b FGF m Ab groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the ^(125)I-b FGF m Ab group than in the other groups(P < 0.05). Expression of b FGF and FGFR m RNA in the ^(125)I-b FGF m Ab group was significantly decreased in comparison with other groups(P < 0.05). Groups under interventions revealed increased expression of VEGF m RNA(except for ^(125)I group) compared with the control group.CONCLUSION: ^(125)I-b FGF m Ab inhibits growth of HCC xenografts. The coupling effect of ^(125)I-b FGF m Ab is more effective than the concomitant use of ^(125)I and b FGF m Ab.展开更多
Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient like m...Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient like metastatic model of human HCC in nude mice (LCI-D20)and a Iow metastatic model of human HCC in nude mice LCI-D35 ) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding.Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-Iincreased gradually following tumor progression in LCID20 model, and correlated with tumor size and AFP level,Phasic expression of tissue intercellular adhesion molecule-I in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including antiangiogenesis, antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.展开更多
AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in ...AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.展开更多
AIM: To investigate whether the regulation of aquaporin 3(AQP3) and AQP9 induced by Auphen and dibutyryl c AMP(dbc AMP) inhibits hepatic tumorigenesis. METHODS: Expression of AQP3 and AQP9 was detected by Western blot...AIM: To investigate whether the regulation of aquaporin 3(AQP3) and AQP9 induced by Auphen and dibutyryl c AMP(dbc AMP) inhibits hepatic tumorigenesis. METHODS: Expression of AQP3 and AQP9 was detected by Western blot, immunohistochemistry(IHC), and RT-PCR in HCC samples and paired non-cancerous liver tissue samples from 30 hepatocellular carcinoma(HCC) patients. A xenograft tumor model was used in vivo. Nine nude mice were divided into control, Auphen-treated, and dbc AMP-treated groups(n = 3 for each group). AQP3 and AQP9 protein expression after induction of xenograft tumors was detected by IHC and m RNA by RT-PCR analysis. The terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling assay and histological evaluation were used to detect apoptosis of tumor cells, and the concentration of serum α-fetoprotein(AFP) was measured using RTPCR and an ELISA kit.RESULTS: The volumes and weights of tumors decreased significantly in the Auphen- and dbc AMP-treated mice compared with the control mice(P < 0.01). The levels of AQP3 were significantly lower in the Auphen treatment group, and levels of AQP9 were significantly higher in thedbc AMP treatment mice than in the control mice(P < 0.01). The reduction of AQP3 by Auphen and increase of AQP9 by dbc AMP in nude mice suppressed tumor growth of HCC, which resulted in reduced AFP levels in serum and tissues, and apoptosis of tumor cells in the Auphen- and dbc AMP-treated mice, when compared with control mice(P < 0.01). Compared with para-carcinoma tissues, AQP3 expression increased in tumor tissues whereas the expression of AQP9 decreased. By correlating clinicopathological and expression levels, we demonstrated that the expression of AQP3 and AQP9 was correlated with clinical progression of HCC and disease outcomes. CONCLUSION: AQP3 increases in HCC while AQP9 decreases. Regulation of AQP3 and AQP9 expression by Auphen and dbc AMP inhibits the development and growth of HCC.展开更多
AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hep...AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphologicalchanges were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR. CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.展开更多
Recently, serum Golgi protein 73 (GP73) levels have been found to be elevated in patients with hepatocellu- lar carcinoma (HCC), and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patien...Recently, serum Golgi protein 73 (GP73) levels have been found to be elevated in patients with hepatocellu- lar carcinoma (HCC), and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say (ELISA). Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody (mAb) designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls (P = 0.0036). Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls (P = 0.0172).展开更多
AIM:To investigate the role of osteopontin(OPN) and its splice variants in the proliferation of hepatocellular carcinoma(HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and ...AIM:To investigate the role of osteopontin(OPN) and its splice variants in the proliferation of hepatocellular carcinoma(HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and nontumour tissue was studied using polymerase chain reaction.OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines.OPN expression was studied in these cells using Western blotting,immunofluoresnce and enzyme linked immunosorbent assay.A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture.Huh-7 cells stably expressing either OPN-A,-B or-C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth.Expression of OPN mRNA and protein in these tumours was examined using reverse transcriptionpolymerase chain reaction and immunohistochemistry.RESULTS:OPN is expressed in HCC in 3 forms,the full length OPN-A and 2 splice variants OPN-B and-C.OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue.Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium.Transfer of OPN conditioned media to na ve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner.Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect.OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model,with Huh-7 cells expressing OPN-A showing the greatest proliferative effect.CONCLUSION:This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44.Modulation of this interaction could represent a novel strategy for the control of HCC.展开更多
AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carc...AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.展开更多
AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptiveimmunotherapy for the patients with primary hepatocellularcarcinoma (HCC), we evaluated the proliferation rate...AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptiveimmunotherapy for the patients with primary hepatocellularcarcinoma (HCC), we evaluated the proliferation rate,phenotype and the antitumor activity of human CIK cellsfrom healthy donors and HCC patients in vitro and in vivo.METHODS: Peripheral blood mononuclear cells (PBMC) fronhealthy donors and patients with primary HCC were incubatedin vitro and induced into ClK cells in the presence of variouscytokines such as interferon-gamma (IFN-γ), interleukin-1(IL-1), IL-2, and monoclonal antibody (mAb) against CD3.The phenotype and characterization of CIK cells wereidentified by flow cytometric analysis. The cytotoxicity of CIKcells was determined by 51 Cr release assay.RESULTS: The CIK cells were shown to be a heterogeneouspopulation with different cellular phenotypes. Thepercentage of CD3+/CD56+ positive cells, the dominanteffector cells, in total CIK cells from healthy donors andHCC patients, significantly increased from 0.1-0.13 % at day0 to 19.0-20.5 % at day 21 incubation, which suggested thatthe CD3+ CD56+ positive cells proliferated faster than othercell populations of CIK cells in the protocol used in thisstudy. After 28 day in vitro incubation, the ClK cells frompatients with HCC and healthy donors increased by morethan 300-fold and 500-fold in proliferation cell number,respectively. CIK cells originated from HCC patientspossessed a higher in vitro antitumor cytotoxic activity onautologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivoanimal experiment, CIK cells had stronger effects on theinhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate,84.7 % vs 52.8 %, P < 0.05) or PBMC (mean inhibitoryrate, 84.7% vs37.1%, P<0.01).CONCLUSION: Autologous CIK cells are of highly efficientcytotoxic effector cells against primary hepatocellularcarcinoma cells and might serve as an alternative adoptivetherapeutic strategy for HCC patients.展开更多
AIM: To study the expression level and localization of insulin-like growth factor -I receptor (IGF-IR) in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody (αIR3) on the ...AIM: To study the expression level and localization of insulin-like growth factor -I receptor (IGF-IR) in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody (αIR3) on the growth of HepG2 cells. METHODS: The expression of IGF-IR in HepG2 cells and Chang liver cells was detected by immunohistochemistry. The influences of aIR3 on proliferation and apoptosis were examined by the 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and electron microscopy, respectively. Flow cytometry (FCM) was applied for the analysis of cell cycle and apoptosis was observed under electron microscope. RESULTS: IGF-IR was located in the membranes of both HepG2 and Chang liver cell lines, and the expression level of IGF-IR was higher in HepG2 cells than in Chang liver cells. Treated with 0.1μg/mLαIR3 for 48 h in vitro, the cell growth index (GI) of HepG2 cells was significantly higher than that of control (103.41% vs 100%, P<0.01). However, the aIR3 for 24 h at final concentration of 4.0μg/mL made the GI of HepG2 cells lower than that of control (93.37% vs 100%, P < 0.01). Compared with control, treated with aIR3 for 48 h at final concentrations ranging from 1.0μg/mL to 4.0μg/mL markedly reduced the GIs of HepG2 cells (97.63%, 97.16%, 95.13%, 92.53% vs 100%, P < 0.05 or P < 0.01), treated withαIR3 for 72 h at final concentrations ranging from 0.2μg/mL to 4.0μg/mL decreased the GIs of HepG2 cells obviously (95%, 91.63%, 90.77%, 89.84%, 88.51% vs 100%, P<0.01), and treated with aIR3 for 96 h at final concentrations ranging from 0.5μg/mL to 4.0μg/mL made GIs of HepG2 cells lower significantly (88.86%, 83.97%, 79.81%, 77.24%, 70.51% vs 100%, P<0.05 or P<0.01). Moreover, treated withαIR3 from 24 h to 96 h at final concentrations ranging from 0.2μg/mL to 4.0μg/mL reduced the GI of HepG2 cells from 97.63% to 70.51% in a dose- and time-dependent manner. Also,αIR3 treatment for 72 h at final concentration from 0.5μg/mL to 2.0μg/mL increased the proportion of G0/G1 phase cells(61.73%, 67.1%, 83.7%,76.87% vs 44.47%, P<0.01) and significantly decreased that of S phase cells(28.63%, 25.13%, 15.63%, 23.13% vs 53.17%, P<0.01), in contrast to the proportion of G2/M phase cells. The apoptotic rates of HepG2 cells were increased more than that of control (7.83%, 16.13%, 21.1%, 37.73% vs 4.13%, P< 0.01). CONCLUSION: The malignant cell phenotype of human hepatocarcinoma cell is related to overexpression of IGF-IR. The blockage of IGF-IR with aaaaaIR3 may contribute to the inhibition of proliferation and induction of apoptosis in HepG2 cells.展开更多
OBJECTIVES: To detect blood AFPmRNA in the nude mice bearing with human hepatocellular carcinoma (HCC) by using nested reverse transcriptase polymerase chain reaction (nested RT-PCR), and assess its significance in HC...OBJECTIVES: To detect blood AFPmRNA in the nude mice bearing with human hepatocellular carcinoma (HCC) by using nested reverse transcriptase polymerase chain reaction (nested RT-PCR), and assess its significance in HCC distant metastasis. METHOD: We detected 20 blood samples from the nude mice bearing with human HCC by nested RT-PCR to find out AFPmRNA. RESULT: AFPmRNA was detected in 6 blood samples from the nude mice hearing with human HCC (30. 0%), in which 4 mice developed distant metastasis. CONCLUSION: AFPmRNA may be used as an efficient and sensitive marker to detect blood spread of HCC cells. It can predict the occurrence of HCC distant metastasis.展开更多
基金National Natural Science Foundation of China,No.39700175
文摘AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F(ab’)<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging(RⅡ).METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F(ab’)<sub>2</sub>.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis(SDS-PAGE)and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate( EDTA )and L-cysteine(L-cys)solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma(HHCC)xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat <sup>99m</sup>Tc-labeled HAb18 F(ab’)<sub>2</sub> in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of <sup>99m</sup>Tc-labeled HAb18 F(ab’)<sub>2</sub>.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01(%ID/g),respectively.CONCLUSION <sup>99m</sup>Tc-HAb18 F(ab’)<sub>2</sub> conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.
文摘The improved tumoricidal effect of the radioatibody mixture ("cocktail") has been reported recently for the treatment of colon tumor. In the present study, we demonstrated the enhanced radioimmunotherapeutic efficacy of a monoclonal atibody (MAb) cocktail against human hepatocellular carcinoma. Therapeutic efficacy was determined by measuring the change in tumor size over a period, determining the percentage of growth inhibition of each treatment at various times after radioantibody therapy. boioimmunotherapy of SMMC-7721 human hepatoma xenografts in athymic nude mice with combination of 131I labeled Hepama-1 and 131Llabeled 9403 mouse MAbs was more effective than using either Hepeam-1 or 9403 Mab alone The MAb cocktail could target a greater number of hepstoma cells and increase the magnitude of hepatoma cen uptde of radioamibodies. The in vjtro results explain the enhanced effect of the MAb cocktail in in vjvo model system.
文摘I-labeled anti-CEA monoclonal antibody injected intrasplenically has better effects on inhibiting liver metastasis from human colonic adenocarcinoma than that through tail vein in nu/nu mice model.
文摘MGb<sub>2</sub> is a monoclonal antibody againstgastric cancer.It can be well localized in thetumour tissue and has been successfully used inthe radioimmunoimaging in the patients withgastric cancer.The conjugate of MGb<sub>2</sub> withchemotherapeutic drug mitomycin C(MMC)al-so shows highly selective cytotoxic effect uponhuman gastric cancer cells in vitro.
文摘In the present study,an anti-gastric cancer monoclonal antibody MGb<sub>2</sub>-daunomycin(DM)conjugate was prepared by cis-aconitic anhydride method.The distribution of the conjugatein nude mice bearing human gastric carcinoma SGC-7901 was investigated.Four to five moleculesof DM were introduced into each molecule of antibody while the antigen-binding capacity ofthe antibody was well-retained.This conjugate could be satisfactorily localized in the tumortissue.Ninety-six hours after intraperitoneal injection of <sup>125</sup>I-MGb<sub>2</sub>-DM conjugate,the ratio ofradioactivity per milligram of tumor to that per milligram of blood reached 203,the result beingquite similar to that of unmodified MGb<sub>2</sub>.
文摘The monoclonal antibody (MAb) RWS4 specific to membrane-associated antigen of human gastric adenocarcinoma was purified by protein A-Sepharose 4B affinity chromatography and labeled with 131I by chloramine-T method. 131-RWS,, was injected (65 μCi/10μg/0.2 ml, intraperitoneally) into the stomach cancer-bearing nude mice (solid tumor about 1 cm in diameter), and its biodistribution was studied by SPECT and gamma-counter over a peroid of 7 days. A clear image of transplanted tumor was observed on the 4th day, and the image became more clear on the 6th day. After SPECT scanning, the animals were killed on the 3rd to 7th day separately and radioactivity was detected in various organs. The ratios of T/NT were calculated. The results were shown as follows: tumor/blood, was 3.41±0.29 on the 6th day and the tumor/other organs (liver, spleen, stomach, lung, heart, kidney and brain etc.) were>3. The specificity of the 131I-RWS4 was 7.74±0.65.
基金the Youth Science Foundation of Shanghai Public Ad-ministration (131984Y15)
文摘Objective To study the role of cell adhesion molecule P-selectin monoclonal antibody (MAb) in tumor metastasis of an orthotopic metasta tic model. Methods SUD mice were implanted orthotopically SGC-7901 human gastric cancer tissue. 3d later, animals received i. v. injections of PBS or P-selectin MAb (100μg/injection) twice weekly for 3 weeks. 42d after operation, all animals were sacrificed. Tissues from all organs were obtained for histopathological evaluation. Results 10 of the animals ( n = 11) treat-ed with PBS were found to develop metastatic tumors in the regional lymph nodes, liver, and lung. In contrast, 2 of the animals ( n = 9) treated with P-selectin MAb developed metastatic tumors in the organs examined. The expression of P-selectin mRNA in gastric cancer tissue of SCID mice with tumor metastasis was higher than that without such metastasis. Conclusion P-selectin expression is associated with tumor metastasis, and the metastasis may be inhibited by the MAb.
基金Supported by the National Natural Science Foundation of China,No.81273814Guangdong Province Key Scientific Research Grant,No.2013A022100031
文摘AIM: To investigate the inhibitory efficacy of ^(125)I-labeled anti-basic fibroblast growth factor(b FGF) monoclonal antibody(m Ab) in hepatocellular carcinoma(HCC).METHODS: b FGF m Ab was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with ^(125)I through the chloramine-T method, b FGF m Ab was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of ^(125)I-b FGF m Ab. The murine H22 HCC xenograft model was established and randomized to interventions with control(phosphate-buffered saline), ^(125)I-b FGF m Ab,^(125)I plus b FGF m Ab, b FGF m Ab, or ^(125)I. The ratios of tumor inhibition were then calculated. Expression of b FGF, fibroblast growth factor receptor(FGFR), plateletderived growth factor, and vascular endothelial growth factor(VEGF) m RNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction. RESULTS: The purified b FGF m Ab solution was 8.145 mg/m L with a titer of 1:2560000 and was stored at-20 ℃. After coupling, ^(125)I-b FGF m Ab was used at a 1: 1280000 dilution, stored at 4 ℃, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, ^(125)I, b FGF m Ab, ^(125)I plus b FGF m Ab, and ^(125)I-b FGF m Ab groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the ^(125)I, b FGF m Ab, ^(125)I plus b FGF m Ab, and ^(125)I-b FGF m Ab groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the ^(125)I-b FGF m Ab group than in the other groups(P < 0.05). Expression of b FGF and FGFR m RNA in the ^(125)I-b FGF m Ab group was significantly decreased in comparison with other groups(P < 0.05). Groups under interventions revealed increased expression of VEGF m RNA(except for ^(125)I group) compared with the control group.CONCLUSION: ^(125)I-b FGF m Ab inhibits growth of HCC xenografts. The coupling effect of ^(125)I-b FGF m Ab is more effective than the concomitant use of ^(125)I and b FGF m Ab.
基金Partly supporled by the State Key Basic Research Program Grant of China(G1998051211)Leading Speciality Grant of Shanghai Health Bureau.
文摘Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient like metastatic model of human HCC in nude mice (LCI-D20)and a Iow metastatic model of human HCC in nude mice LCI-D35 ) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding.Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-Iincreased gradually following tumor progression in LCID20 model, and correlated with tumor size and AFP level,Phasic expression of tissue intercellular adhesion molecule-I in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including antiangiogenesis, antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.
基金Supported by the National 863 Project of China,No.102-09-01-02National Natural Science Foundation of China,No.39770827
文摘AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.
基金Supported by Science and Technology Commission of Shanghai,No.13ZR1406700 and No.13DZ1930908
文摘AIM: To investigate whether the regulation of aquaporin 3(AQP3) and AQP9 induced by Auphen and dibutyryl c AMP(dbc AMP) inhibits hepatic tumorigenesis. METHODS: Expression of AQP3 and AQP9 was detected by Western blot, immunohistochemistry(IHC), and RT-PCR in HCC samples and paired non-cancerous liver tissue samples from 30 hepatocellular carcinoma(HCC) patients. A xenograft tumor model was used in vivo. Nine nude mice were divided into control, Auphen-treated, and dbc AMP-treated groups(n = 3 for each group). AQP3 and AQP9 protein expression after induction of xenograft tumors was detected by IHC and m RNA by RT-PCR analysis. The terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling assay and histological evaluation were used to detect apoptosis of tumor cells, and the concentration of serum α-fetoprotein(AFP) was measured using RTPCR and an ELISA kit.RESULTS: The volumes and weights of tumors decreased significantly in the Auphen- and dbc AMP-treated mice compared with the control mice(P < 0.01). The levels of AQP3 were significantly lower in the Auphen treatment group, and levels of AQP9 were significantly higher in thedbc AMP treatment mice than in the control mice(P < 0.01). The reduction of AQP3 by Auphen and increase of AQP9 by dbc AMP in nude mice suppressed tumor growth of HCC, which resulted in reduced AFP levels in serum and tissues, and apoptosis of tumor cells in the Auphen- and dbc AMP-treated mice, when compared with control mice(P < 0.01). Compared with para-carcinoma tissues, AQP3 expression increased in tumor tissues whereas the expression of AQP9 decreased. By correlating clinicopathological and expression levels, we demonstrated that the expression of AQP3 and AQP9 was correlated with clinical progression of HCC and disease outcomes. CONCLUSION: AQP3 increases in HCC while AQP9 decreases. Regulation of AQP3 and AQP9 expression by Auphen and dbc AMP inhibits the development and growth of HCC.
文摘AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphologicalchanges were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR. CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.
基金supported by the Science and Technology Foundation of Nanjing Medical University (No. 07NMUZ005).
文摘Recently, serum Golgi protein 73 (GP73) levels have been found to be elevated in patients with hepatocellu- lar carcinoma (HCC), and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say (ELISA). Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody (mAb) designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls (P = 0.0036). Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls (P = 0.0172).
文摘AIM:To investigate the role of osteopontin(OPN) and its splice variants in the proliferation of hepatocellular carcinoma(HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and nontumour tissue was studied using polymerase chain reaction.OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines.OPN expression was studied in these cells using Western blotting,immunofluoresnce and enzyme linked immunosorbent assay.A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture.Huh-7 cells stably expressing either OPN-A,-B or-C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth.Expression of OPN mRNA and protein in these tumours was examined using reverse transcriptionpolymerase chain reaction and immunohistochemistry.RESULTS:OPN is expressed in HCC in 3 forms,the full length OPN-A and 2 splice variants OPN-B and-C.OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue.Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium.Transfer of OPN conditioned media to na ve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner.Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect.OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model,with Huh-7 cells expressing OPN-A showing the greatest proliferative effect.CONCLUSION:This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44.Modulation of this interaction could represent a novel strategy for the control of HCC.
基金Supported by National Natural Science Foundation of China, No.81201963Inner Mongolia Natural Science Foundation of China,No.2010MS1123
文摘AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.
基金Science and Technology Development Foundation of Beijing Institute of Infectious Diseases,No.01 Z094
文摘AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptiveimmunotherapy for the patients with primary hepatocellularcarcinoma (HCC), we evaluated the proliferation rate,phenotype and the antitumor activity of human CIK cellsfrom healthy donors and HCC patients in vitro and in vivo.METHODS: Peripheral blood mononuclear cells (PBMC) fronhealthy donors and patients with primary HCC were incubatedin vitro and induced into ClK cells in the presence of variouscytokines such as interferon-gamma (IFN-γ), interleukin-1(IL-1), IL-2, and monoclonal antibody (mAb) against CD3.The phenotype and characterization of CIK cells wereidentified by flow cytometric analysis. The cytotoxicity of CIKcells was determined by 51 Cr release assay.RESULTS: The CIK cells were shown to be a heterogeneouspopulation with different cellular phenotypes. Thepercentage of CD3+/CD56+ positive cells, the dominanteffector cells, in total CIK cells from healthy donors andHCC patients, significantly increased from 0.1-0.13 % at day0 to 19.0-20.5 % at day 21 incubation, which suggested thatthe CD3+ CD56+ positive cells proliferated faster than othercell populations of CIK cells in the protocol used in thisstudy. After 28 day in vitro incubation, the ClK cells frompatients with HCC and healthy donors increased by morethan 300-fold and 500-fold in proliferation cell number,respectively. CIK cells originated from HCC patientspossessed a higher in vitro antitumor cytotoxic activity onautologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivoanimal experiment, CIK cells had stronger effects on theinhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate,84.7 % vs 52.8 %, P < 0.05) or PBMC (mean inhibitoryrate, 84.7% vs37.1%, P<0.01).CONCLUSION: Autologous CIK cells are of highly efficientcytotoxic effector cells against primary hepatocellularcarcinoma cells and might serve as an alternative adoptivetherapeutic strategy for HCC patients.
基金Supported by the Gansu Province's Natural Science Fund, No.ZS021-A25-079-Y
文摘AIM: To study the expression level and localization of insulin-like growth factor -I receptor (IGF-IR) in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody (αIR3) on the growth of HepG2 cells. METHODS: The expression of IGF-IR in HepG2 cells and Chang liver cells was detected by immunohistochemistry. The influences of aIR3 on proliferation and apoptosis were examined by the 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and electron microscopy, respectively. Flow cytometry (FCM) was applied for the analysis of cell cycle and apoptosis was observed under electron microscope. RESULTS: IGF-IR was located in the membranes of both HepG2 and Chang liver cell lines, and the expression level of IGF-IR was higher in HepG2 cells than in Chang liver cells. Treated with 0.1μg/mLαIR3 for 48 h in vitro, the cell growth index (GI) of HepG2 cells was significantly higher than that of control (103.41% vs 100%, P<0.01). However, the aIR3 for 24 h at final concentration of 4.0μg/mL made the GI of HepG2 cells lower than that of control (93.37% vs 100%, P < 0.01). Compared with control, treated with aIR3 for 48 h at final concentrations ranging from 1.0μg/mL to 4.0μg/mL markedly reduced the GIs of HepG2 cells (97.63%, 97.16%, 95.13%, 92.53% vs 100%, P < 0.05 or P < 0.01), treated withαIR3 for 72 h at final concentrations ranging from 0.2μg/mL to 4.0μg/mL decreased the GIs of HepG2 cells obviously (95%, 91.63%, 90.77%, 89.84%, 88.51% vs 100%, P<0.01), and treated with aIR3 for 96 h at final concentrations ranging from 0.5μg/mL to 4.0μg/mL made GIs of HepG2 cells lower significantly (88.86%, 83.97%, 79.81%, 77.24%, 70.51% vs 100%, P<0.05 or P<0.01). Moreover, treated withαIR3 from 24 h to 96 h at final concentrations ranging from 0.2μg/mL to 4.0μg/mL reduced the GI of HepG2 cells from 97.63% to 70.51% in a dose- and time-dependent manner. Also,αIR3 treatment for 72 h at final concentration from 0.5μg/mL to 2.0μg/mL increased the proportion of G0/G1 phase cells(61.73%, 67.1%, 83.7%,76.87% vs 44.47%, P<0.01) and significantly decreased that of S phase cells(28.63%, 25.13%, 15.63%, 23.13% vs 53.17%, P<0.01), in contrast to the proportion of G2/M phase cells. The apoptotic rates of HepG2 cells were increased more than that of control (7.83%, 16.13%, 21.1%, 37.73% vs 4.13%, P< 0.01). CONCLUSION: The malignant cell phenotype of human hepatocarcinoma cell is related to overexpression of IGF-IR. The blockage of IGF-IR with aaaaaIR3 may contribute to the inhibition of proliferation and induction of apoptosis in HepG2 cells.
文摘OBJECTIVES: To detect blood AFPmRNA in the nude mice bearing with human hepatocellular carcinoma (HCC) by using nested reverse transcriptase polymerase chain reaction (nested RT-PCR), and assess its significance in HCC distant metastasis. METHOD: We detected 20 blood samples from the nude mice bearing with human HCC by nested RT-PCR to find out AFPmRNA. RESULT: AFPmRNA was detected in 6 blood samples from the nude mice hearing with human HCC (30. 0%), in which 4 mice developed distant metastasis. CONCLUSION: AFPmRNA may be used as an efficient and sensitive marker to detect blood spread of HCC cells. It can predict the occurrence of HCC distant metastasis.