RNA interference of pax2 inhibits growth of transplanted human endometrial cancer cells in nude mice

The development of human endometrial carcinoma(HEC) is a complex pathologic process involves several oncogenes and tumor suppressor genes.The full-length paired-box gene 2(pax2),a recently discovered oncogene,promotes cell proliferation and growth and inhibits apoptosis of HEC cells.Here,we examined the effect of pax2 small interfering RNA(siRNA) on the growth of transplanted HEC cells in nude mice.The expression of Pax2 in 21 cases of normal endometrium and 38 cases of HEC was examined by immohistochemistry(IHC).HEC models were developed by subcutaneously transferring HEC cells into nude mice,followed by treatment with empty lentivirus vector,lentivirus vector-based pax2 siRNA,and phosphate buffered saline,respectively.Four weeks later,tumor size was measured,tumor inhibition rate was calculated,and histological analyses were conducted after staining with hematoxylin and eosin.The expression of Pax2 and Bcl-2 was detected by Western blot;proliferating cell nuclear antigen(PCNA) was detected by IHC.Significant differences were observed in the positive rate of Pax2 between normal endometrium and HEC(14.2% vs.60.5%,P<0.01).The expression index of Pax2 in well differentiated tumors was 1.88±1.68,much lower than that in tumors of moderate(3.07±1.96,P<0.05) or poor differentiation(5.45±2.76,P<0.01).Tumor necrosis increased,nuclear basophilia stain decreased,tumor growth was inhibited,and PCNA,Pax2,and Bcl-2 expression was reduced in HEC models treated with pax2 siRNA.These results indicate that Pax2 expression is related to HEC tumor biology with the increased expression of Pax2 correlated to malignancy.pax2 siRNA down-regulates Pax2 expression and inhibits tumorigenesis of HEC in nude mice,possibly due to cell apoptosis and the inhibition of tumor proliferation induced by down-regulation of Bcl-2.

Evaluation of Tumor Formation of Three Bladder Cancer Cell Lines in Nude Mice

This study examined the differences in tumor formation of three bladder tumor cell lines （BIU-87, T24 and EJ） after subcutaneously transplanted into nude mice, in order to find the best technique for establishing in vivo bladder tumor model. BIU-87, T24 and EJ cells at logarithmic phase were re-suspended in serum-free medium. The cells suspensions of the identical concentration were subcutaneously transplanted into nude mice and then the success rate and tumor growth were compared among the three cell groups. The results of tumor formation were pathologically evaluated. Lung, liver and kidney tissues were also pathologically examined for distant metastasis. The proliferation of the three cells were determined by immunohistochemically detecting the PCNA expression in the tumors. The results showed that the success rates of EJ and T24 cells were significantly higher than that of BIU-87 cells and no distant metastasis was noted among the three groups. The proliferation levels of EJ and T24 cells was significantly higher than that of BIU-87. But at the later stage of tumor formation, as compared with T24 cells, EJ grew more vigorously, soon resulting in the central necrosis of tumor, which affected the measurement of the actual size of the tumors. Moreover, PCNA staining exhibited that the proliferation of EJ and T24 was significantly higher than that of BIU-87 cells. It is concluded that as compared with BIU-87 cells, EJ and T24 cells had higher success rates, with not significant differences in death rate and distant metastasis found among them. There existed no significant difference in tumor formation between EJ and T24 cells and T24 cells do not rupture easily, which makes it a better cell line for the establishment of in vivo bladder tumor model.

Effects of epidermal growth factor on the growth of human gastric cancer cell and the implanted tumor of nude mice

AIM: Epidermal growth factor (EGF) plays an important role in the regulation of gastrointestinal tissue growth and development, and it can stimulate epithelial proliferation, cell differentiation and growth. It has been established that the EGF can promote gastric cytoprotection and ulcer healing. But the potential ability of EGF to regulate the gastric cancer growth is unknown. This study is to investigate the influence of EGF on human gastric cancer cell and the implanted tumor growth of nude mice. METHODS: The cell growth rates of human gastric adenocarcinoma cell lines MKN-28, MKN-45, SGC-7901 and normal human gastric epithelial cells 3T3 were assessed when incubated with recombinant human EGF (rhEGF, 0.05, 0.1, 0.5, 1.0, 10, 50, 100 mg.L(-1)) using MTT method. The cells of MKN-28, MKN-45, SGC-7901 (gastric cancer tissue 1.5mm(3)) were implanted in the BALB/cA nude mice for 10 days.The EGF was given intraperitoneally (15, 30, 60 microg.kg(-1)) for 3 weeks. The body weights of the tumor-bearing animals and their tumor mass were measured afterwards to assess the mitogenic effect of rhEGF in the nude mice. RESULTS: Within the concentration range of 0.05-100mg.L(-1), rhEGF could increase the cell growth of normal 3T3 cells (cell growth rate 100% vs 102.8%, P【0.05), but partially restrain the gastric cancer cell growth. The latter effect was related to cell differentiation. In 15-60 microg/kg rhEGF groups, the mean implanted tumor mass of MKN-28 cell were 1.75 g, 1.91 g, 2.08 g/NS group 1.97 g (P】0.05), the mean tumor mass of SGC-7901 cell were 1.53 g, 1.07 g, 1.20 g/NS group 1.07 g (P】0.05), and for MKN-45 cell, the tumor mass were respectively 1.92 g, 1.29 g, 1.77 /NS group 1.82 g (P】0.05). So rhEGF had no obvious effect on implanted MKN-28, SGC-7901 and MKN-45 tumor growth. CONCLUSION: EGF has no stimulating effect on the human gastric cancer cell growth neither in vitro nor in vivo.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

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.

Kanglaite combined Gemcitabine inhibits growth of nude mouse subcutaneous transplantation tumor of human PC-3 pancreatic cancer cell

Objective:To study the mechanisms of pancreatic cancer treatment with Kanglaite combined Gemcitabine by investigating the relationship between the apoptosis and the expression of bcl-2, Bax and VEGF in pancreatic cancer cells.Methods:Nude mouse subcutaneous transplantation tumor model of Human PC-3 pancreatic cancer was established; the expressions of bcl-2, Bax and VEGF of transplantation tumor cell were determined; the earlier apoptosis rate of pancreatic cancer cell and the gross tumor volume were determined. Results:Kanglaite combined Gemcitabine remarkably decreased the protein expression of bcl-2,raised the expression of Bax,increased the apoptosis rate of the pancreatic cancer and contract the gross tumor volume. Kanglaite greatly decreased the protein expression of VEGF of the tumor cell. Conclusion:Therapeutic efficacy of Kanglaite combined Gemcitabine is far better than separate use of the two medicines in the pancreatic cancer transplantation tumor treatment.

Antitumor activities of human autologous cytokineinduced killer(CIK)cells against hepatocellular carcinoma cells in vitro and in vivo

AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation rate, phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1), IL-2 and monoclonal antibody (mAb) against CD3. The phenotype and characterization of CIK cells were identified by flow cytometric analysis. The cytotoxicity of CIK cells was determined by (51)Cr release assay. RESULTS: The CIK cells were shown to be a heterogeneous population with different cellular phenotypes. The percentage of CD3+/CD56+ positive cells, the dominant effector cells, in total CIK cells from healthy donors and HCC patients, significantly increased from 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study. After 28 day in vitro incubation, the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number, respectively. CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivo animal experiment, CIK cells had stronger effects on the inhibition 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 inhibitory rate, 84.7% vs 37.1%, P【0.01). CONCLUSION: Autologous CIK cells are of highly efficient cytotoxic effector cells against primary hepatocellular carcinoma cells and might serve as an alternative adoptive therapeutic strategy for HCC patients.

OB glue paste technique for establishing nude mouse human gastric cancer orthotopic transplantation models

AIM： To establish nude mouse human gastric cancer orthotopic transplantation models using OB glue paste technique. METHODS： Using OB glue paste technique, orthtopic transplantation models were established by implanting SGC-7901 and NKN-45 human gastric cancer cell strains into the gastric wall of nude mice. Biological features, growth of the implanted tumors, the success rate of transplantation and the rate of auto-metastasis of the two models were observed. RESULTS： The success rates of orthotopic transplanration of the two models were 94.20% and 96%. The rates of hepatic metastasis, pulmonary metastasis, peritoneal metastasis, lymphocytic metastasis and splenic metastasis were 42.13% and 94.20%, 48.43% and 57.97%, 30.83% and 36.96%, 67.30% and 84.06%, and 59.75% and 10.53%, respectively. The occurrence of ascites was 47.80% and 36.96%. CONCLUSION： OB glue paste technique is easy to follow. The biological behaviors of the nude mouse human gastric cancer orthotopic transplantation models established with this technique are similar to the natural processes of growth and metastasis of human gastric cancer, and, therefore, can be used as an ideal model for experimental research of proliferative metastasis of tumors.

Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.

苦参碱联合顺铂对人肝癌细胞株HepG2裸鼠移植瘤模型瘤体生长及caspase-3和Survivin表达的影响

目的分析苦参碱联合顺铂对人肝癌细胞株Hep G2裸鼠移植瘤模型瘤体生长及半胱氨酸天冬氨酸蛋白酶-3(caspase-3)和存活素(Survivin)表达的影响。方法于BALB/c裸鼠腋部皮下注入人肝癌细胞株Hep G2以此构建移植瘤模型。在成瘤后,将28只裸鼠随机分为对照组(等量生理盐水)、顺铂组(顺铂2 mg/kg)、苦参碱组(苦参碱100 mg/kg)、联用组(苦参碱100 mg/kg+顺铂2 mg/kg)各7只,各组均通过腹腔注射给药。观察各组瘤体生长状况,对各组抑瘤率进行计算。通过免疫组化法对各组瘤组织中caspase-3、Survivin表达水平进行检测,计算和比较各组阳性细胞率。结果联用组裸鼠抑瘤率为80. 00%,较顺铂组(64. 00%)、苦参碱组(36. 00%)明显升高(P <0. 05)。联用组裸鼠caspase-3阳性细胞率为(77. 89±7. 35)%,较对照组(19. 89±4. 14)%、顺铂组(64. 86±9. 34)%、苦参碱组(36. 97±9. 35)%均显著上升(P <0. 05)。联用组裸鼠Survivin阳性细胞率为(20. 04±4. 37)%,较对照组(84. 85±14. 75)%、顺铂组(39. 05±7. 69)%、苦参碱组(64. 06±9. 14)%均显著降低(P <0. 05)。结论单用顺铂或苦参碱对人肝癌细胞株Hep G2裸鼠移植瘤均具有一定的抑瘤作用,但二者联用的抑瘤作用更为明显。其作用机制可能与苦参碱联合顺铂处理可对caspase-3、Survivin表达水平造成影响,进而促使肿瘤细胞凋亡存在密切关系。

E1A基因对裸鼠移植瘤细胞凋亡及Survivin、Caspase-3基因表达的影响

目的探讨E1A基因对裸鼠移植瘤肿瘤细胞凋亡的影响及其相关作用机制。方法采用免疫组织化学染色检测E1A基因对裸鼠移植瘤肿瘤细胞凋亡及Survivin基因和Caspase-3基因表达的影响。结果免疫组织化学染色显示:E1A基因组肿瘤细胞凋亡数量较多,Caspase-3基因呈高表达,Survivin基因表达较弱;而Hela和Hela-vect组肿瘤细胞凋亡数量较少,Survivin基因呈高表达,Caspase-3基因表达较弱。结论E1A基因能够明显促进裸鼠移植瘤肿瘤细胞的凋亡,该作用可能与E1A基因激活Caspase-3基因和抑制Survivin基因的表达有关。

二氧化碳气腹环境对裸鼠卵巢癌移植瘤中Beclin1表达的研究

目的:探讨二氧化碳(CO_(2))气腹在相同压力不同时间作用下裸鼠卵巢癌移植瘤组织中自噬基因Beclin1表达的影响,推断腹腔镜手术对卵巢癌的影响,为腹腔镜手术在卵巢癌治疗中的应用提供动物实验依据。方法:采用实时荧光定量PCR(Real Time-PCR)、免疫组织化学法及蛋白质免疫印迹(western blot)检测四组(单纯麻醉即对照组、开腹组、CO_(2)气腹0.5 h组、CO_(2)气腹1 h组)裸鼠卵巢癌移植瘤组织中Beclin1基因mRNA和蛋白的表达情况。结果:(1)RT-PCR结果:在对照组、开腹组、CO_(2)气腹0.5 h组、CO_(2)气腹1 h组四组裸鼠卵巢癌移植瘤组织中,Beclin1 m RNA呈现逐渐降低趋势;CO_(2)气腹0.5 h组及CO_(2)气腹1 h组与对照组或开腹组比较均有统计学差异(P<0.05);而CO_(2)气腹0.5 h组与CO_(2)气腹1 h组之间比较无统计学差异(P>0.05)。(2)免疫组化结果表明:在40例卵巢癌组织中,Beclin1表达阳性的有29例,其表达率及表达强度在对照组、开腹组、CO_(2)气腹0.5 h组、CO_(2)气腹1 h组中无明显差异。(3)Western blot结果:与RT-PCR结果一致,在对照组、开腹组、CO_(2)气腹0.5 h组、CO_(2)气腹1 h组四组裸鼠卵巢癌移植瘤组织中,Beclin1蛋白呈现逐渐降低趋势;CO_(2)气腹0.5 h组及CO_(2)气腹1 h组与对照组或开腹组比较均有统计学差异(P<0.05);而CO_(2)气腹0.5 h组与CO_(2)气腹1 h组之间比较无统计学差异(P>0.05)。结论:CO_(2)气腹可能通过降低卵巢癌移植瘤中Beclin1的表达促进卵巢癌的增殖转移。CO_(2)气腹的作用时间对自噬相关基因Beclin1表达的影响不大。

二氢杨梅素对裸鼠腹腔移植人胃癌细胞中Caveolin-1表达的影响

目的观察二氢杨梅素(DMY)对裸鼠腹腔移植人胃癌细胞生长的抑制作用及对腹腔瘤组织中Caveolin-1表达的影响。方法复制人胃癌腹腔移植的裸鼠模型。将复模成功的24只裸鼠随机分为4组,即移植瘤模型组,DMY低、中及高剂量处理组。分别观察荷瘤裸鼠的腹腔移植瘤的生长和成瘤情况,测量肿瘤的重量、长短径并计算肿瘤的体积。采用免疫组织化学法和Western blot检测人胃癌腹腔移植瘤腹腔瘤体中Caveolin-1和Ki67的表达。结果与移植瘤模型组比较,100和200 mg/(kg·d)DMY中、高剂量组腹腔移植瘤成瘤个数,腹腔瘤体的重量及腹腔瘤体的体积均降低,呈现剂量依赖性(P<0.05),裸鼠的食欲、活动情况和精神状态等一般情况改善。与移植瘤模型组比较,100和200 mg/(kg·d)DMY中、高剂量组腹腔移植瘤体表达Caveolin-1的阳性细胞百分率和Caveolin-1的蛋白表达水平均增加,呈现剂量依赖性(P<0.05);而Ki67的阳性细胞百分率和Caveolin-1的蛋白表达水平均降低,也呈现剂量依赖性(P<0.05)。结论二氢杨梅素以剂量依赖的方式抑制人胃癌腹腔移植瘤的生长,其作用机制可能与上调Caveolin-1的表达有关。

橄榄苦苷调控Wnt/β-catenin信号通路对裸鼠子宫颈癌移植瘤生长的作用

目的探讨橄榄苦苷对裸鼠子宫颈癌移植瘤生长的作用及对Wnt/β-catenin信号通路的调控。方法将成功建立的24只子宫颈癌裸鼠随机分为实验组、阳性对照组和模型组,每组8只,实验组腹腔注射10 mg·mL^-1橄榄苦苷溶液100μL,阳性对照组腹腔注射顺铂2 mg·kg^-1,模型组腹腔注射90%NaCL+10%二甲基亚砜溶液(DMSO)混合液100μL,每日1次,连续给药30 d。观察各组裸鼠移植瘤生长情况,计算肿瘤抑瘤率;TdT介导的dUTP缺口末端标记(TUNEL)法检测裸鼠移植瘤组织细胞凋亡情况,蛋白免疫印迹(Western blot)法检测裸鼠移植瘤组织中Wnt/β-catenin通路蛋白表达情况。结果实验组和阳性对照组皮下移植瘤生长速度较模型组减缓;药物干预后第15d,实验组和阳性对照组裸鼠移植瘤体积均小于模型组(P均<0.01)。药物干预结束后,实验组和阳性对照组裸鼠移植瘤质量均低于模型组(P均<0.01),其中实验组和阳性对照组的抑瘤率分别为(52.47±3.16)%,(49.81±5.26)%,且两组差异无统计学意义(P>0.05)。TUNEL细胞凋亡检测,实验组和阳性对照组裸鼠移植瘤组织细胞凋亡指数分别为(8.26±0.36)%和(7.26±0.54)%,均高于模型组(3.69±0.52)%(P均<0.01)。Western blot检测结果显示,与模型组比较,实验组和阳性对照组Wnt1蛋白相对表达量均升高,c-Myc、β-catenin、Axin2蛋白相对表达量均降低(P均<0.01)。结论橄榄苦苷可通过调控Wnt/β-catenin信号通路抑制裸鼠子宫颈癌移植瘤生长,促进移植瘤组织细胞凋亡。

Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines

AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. Egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.

新城疫病毒作用下卵巢癌裸鼠移植瘤Survivin、Caspase-3的表达

目的研究新城疫病毒(Newcastle disease virus,NDV)对卵巢癌裸鼠移植瘤Survivin、Caspase-3表达的影响。方法①细胞培养:人卵巢癌细胞SKOV-3细胞的培养;②建立裸鼠移植瘤模型;③实验分组:分为实验组与对照组,实验组为肿瘤内注射NDV病毒抗卵巢癌;④应用免疫组化SP法观察Survivin、Caspase-3的表达。结果 Survivin表达实验组与对照组均为阳性,实验组明显弱于对照组,差异有显著性(P<0.05);Caspase-3表达实验组均为阳性,对照组1例表达,实验组明显强于对照组,差异有显著性(P<0.05)。结论 NDV可诱导肿瘤细胞凋亡,抑制其生长;其机制可能通过激活Caspase-3凋亡,抑制Survivin抗调亡作用。

超声造影评估裸鼠食管癌移植瘤血管生成情况的实验研究

目的探讨超声造影(CEUS)对裸鼠食管癌移植瘤血管生成情况的评估价值。方法选取18只裸鼠,于其背部皮下注射人食管癌细胞株建立食管癌移植瘤模型,分别于移植后4、6、8周均随机选取6只裸鼠,先应用二维超声观察移植瘤回声、形态和边界,再行CEUS检查并获取时间-强度曲线(TIC)。然后处死所有裸鼠,使用HE染色观察移植瘤细胞结构和组织形态,免疫组化观察移植瘤组织血管内皮生长因子(VEGF)蛋白表达和微血管密度(MVD),比较移植后不同时间移植瘤CEUS表现、TIC定量参数及病理学检查结果的差异。Pearson相关分析法分析TIC定量参数与VEGF蛋白表达、MVD的相关性。结果所有裸鼠食管癌移植瘤模型均成功建立,未出现死亡。移植后4周,移植瘤二维超声表现为类椭圆形低回声肿块,边界清晰,内部回声不均匀,CEUS表现为移植瘤内部呈均匀增强;移植后6周,移植瘤二维超声表现为肿块边界欠清晰,内部回声不均匀,CEUS表现为移植瘤内部呈不均匀高增强;移植后8周,移植瘤二维超声表现为肿块边界不清晰,内部回声不均匀,CEUS表现为移植瘤内部出现灌注缺损。移植后4、6、8周移植瘤峰值强度(PI)、曲线下流入面积(AWI)及曲线下流出面积(AWO)比较,差异均有统计学意义(均P<0.05);与移植后4周比较,移植后6、8周移植瘤PI、AWI和AWO均升高(均P<0.05);与移植后6周比较,移植后8周移植瘤PI和AWO均升高(均P<0.05)。HE染色显示,移植后4周移植瘤见角化珠且细胞间见细胞间桥;移植后6周移植瘤角化珠和细胞间桥均稍减少,毛细血管增多;移植后8周,移植瘤角化珠和细胞间桥均明显减少,毛细血管增多,可见病理性核裂变。免疫组化显示,移植后4、6、8周MVD和VEGF蛋白表达比较,差异均有统计学意义(均P<0.05);与移植后4周比较,移植后6、8周移植瘤MVD均升高,移植后8周移植瘤VEGF蛋白表达升高,差异均有统计学意义(均P<0.05)。相关性分析显示,移植瘤PI、AWI、AWO与MVD、VEGF蛋白表达均呈正相关(均P<0.001)。结论CEUS能够有效观察裸鼠食管癌移植瘤内部血流灌注情况,TIC定量参数PI、AWI、AWO随着移植瘤的生长而逐渐升高,其与移植瘤MVD、VEGF蛋白表达均呈正相关,可以较好地评估移植瘤血管生成情况。

Human breast carcinoma xenografts in nude mice

OBJECTIVE: To investigate spontaneous metastasis, micrometastasis and genetic stability in human breast carcinoma xenografts in nude mice. METHODS: Intact tissue from surgical specimens from breast carcinoma patients was xenografted into nude mice and transplanted from generation to generation. Cells from the xenografts were cultured in vitro and retransplanted into nude mice. Microsatellite DNA in the genome of human breast carcinomas, xenotransplanted tumors and metastases in nude mice were analyzed at three microsatellite loci. RESULTS: The tumorigenicity of orthotopic xenotransplantation was 88.6% (31/35), with a metastatic rate of 41.9% (13/31). Cells from xenotransplants were successfully cultured in vitro. The taking rate of retransplantation into nude mice and the spontaneous lung metastasis rate were both 100% (10/10). Microsatellite DNA sequences in the genome of xenotransplanted tumors and metastases in nude mice were identical with that of the original human breast carcinoma at three microsatellite loci. CONCLUSIONS: Tumorigenicity and metastatic potential can be improved in human breast carcinoma xenografts using intact fresh tumor tissue and orthotopic grafts. Xenotransplanted tumors and tumors after serial passage maintained the genetic stability. The detection of microsatellite DNA may identify micrometastases in a nude mouse model.

菌群移植对氟尿嘧啶抗鼠源结肠肿瘤的协同作用

目的:观察菌群移植对氟尿嘧啶抑制结肠癌MC38荷瘤小鼠移植瘤生长的协同作用效果。方法:选取雌性C57BL/6N小鼠21只作为研究对象,分为空白组、对照组、实验组。培养MC38小鼠结肠癌细胞接种于小鼠右前肢背部皮下构建结肠癌荷瘤模型。空白组无任何处理,取其粪便制作干预菌液,实验组和对照组自分组后每两天1次给予0.1 mL菌液/生理盐水灌胃至实验结束。成瘤后第6天,按照65 mg/kg剂量连续5天腹腔注射氟尿嘧啶,3天后处死实验动物,计算肿瘤体积和抑瘤率。采用HE染色观察结直肠组织情况。采用酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法检测白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子Alpha(tumor necrosis factor alpha,TNF-α)血清水平,通过16S测序进行肠道菌群检测和分析。结果:与空白组相比,实验组和对照组模型小鼠体重均明显降低(P<0.05),但对照组和实验组体重变化未见明显差异(P>0.05)。对照组荷瘤平均体积大于实验组荷瘤平均体积(P<0.05)。与对照组相比,实验组结直肠组织病理镜下可见更多的隐窝细胞,绒毛更完整。实验组IL-6血清水平高于对照组和空白组(P<0.05);实验组和对照组TNF-α血清水平与空白组相比升高明显(P<0.05),但实验组和对照组差异无统计学意义(P>0.05)。16S测序结果提示自体菌群移植干预后,实验组肠道菌群更接近空白组,对照组菌群丰富度最低,但三组未见明显区别(P>0.05)。进一步分析三组菌群差异,发现在属(Genus)水平上,与空白组相比,实验组的Mar-Odoribacter和Tan-Parabacteroide富集增多,对照组Ent-Escherichia-Shigella富集增多。结论:菌群移植后上调Mar-Odoribacter和Tan-Parabacteroide菌,下调Ent-Escherichia-Shigella菌,可保护肠道黏膜,改善机体代谢,激活机体免疫系统,促进了IL-6和TNF-α细胞因子表达,提高抗肿瘤免疫能力,对氟尿嘧啶抑制MC38荷瘤小鼠移植瘤增殖起到了协同作用。

灵菌红素对耐阿霉素人乳腺癌细胞MCF-7/ADR的作用研究

目的探讨灵菌红素对耐阿霉素人乳腺癌细胞MCF-7/ADR的作用。方法采用平板发酵粘质沙雷氏菌WA12-1-18生产灵菌红素,CCK-8测定灵菌红素的体外活性及MCF-7/ADR的耐药指数。构建裸鼠皮下移植瘤模型,灵菌红素高、低剂量组分别腹腔注射5.0、2.5 mg/kg灵菌红素,生理盐水对照位腹腔注射等体积生理盐水,每4天1次,连续用药24 d,观察移植瘤的体积、质量及裸鼠体质量,HE染色观察移植瘤组织及裸鼠主要脏器的病理情况,免疫组织化学检测移植瘤Ki-67的表达。结果获得的灵菌红素质量分数为95.18%,对MCF-7和MCF-7/ADR的IC50分别为0.484μg/mL和0.264μg/mL。MCF-7/ADR耐药指数13,符合细胞耐药株的要求。灵菌红素处理组裸鼠移植瘤增长速度较生理盐水组慢,肿瘤细胞排列稀疏,核小且着色较浅,Ki-67染色后棕色明显减少。灵菌红素2.5 mg/kg组和灵菌红素5 mg/kg组,在荷MCF-7/ADR裸鼠中抑瘤率分别为37.23%和53.72%。另外,各组裸鼠体质量差异无统计学意义,主要脏器无明显病理变化。结论灵菌红素可抑制裸鼠体内耐阿霉素人乳腺癌细胞MCF-7/ADR的生长,对心、肝、脾、肺和肾等主要脏器无明显毒副作用,为灵菌红素在耐阿霉素乳腺癌的临床应用提供了实验依据。

基质金属蛋白酶12在胃癌组织、细胞中的表达及对裸鼠移植瘤生长的影响实验研究

目的:观察基质金属蛋白酶12(MMP-12)在胃癌组织和胃癌细胞株SGC-7901中的表达及对裸鼠移植瘤生长的影响。方法:收集胃癌患者42例,手术后留取肿瘤组织和距肿瘤边缘>3 cm的正常胃黏膜组织,应用免疫组化EnVision法检测MMP-12的表达。合成sh-MMP12质粒,应用慢病毒载体转染胃癌细胞株SGC-7901,设为sh-MMP12组,同时设立未行任何干预的细胞为空白对照组(NC组),应用Western blot法检测MMP-12和增殖细胞核抗原(PCNA)蛋白的表达,应用实时荧光定量PCR(RT-qPCR)检测MMP-12和PCNA mRNA的表达。选择4~5周龄的BALB/C雌性裸鼠5只,分别将sh-MMP12组和NC组细胞悬液注射于同一只裸鼠的左右两侧腋下(左侧为sh-MMP12组,右侧为NC组),间隔7 d测量并记录肿瘤体积,28 d后处死小鼠对瘤体称重。结果:免疫组化结果显示,胃癌组织MMP-12阳性率高于正常胃黏膜组织,胃癌组织MMP-12和PCNA表达呈正相关(均P<0.05)。Western blot结果显示,sh-MMP12组MMP-12和PCNA蛋白表达量于NC组(均P<0.05)。RT-qPCR结果显示,sh-MMP12组MMP-12和PCNA mRNA表达量低于NC组(均P<0.05)。裸鼠移植瘤实验结果显示,sh-MMP 12组肿瘤体积在7、14 d时与NC组比较无统计学差异(均P>0.05),而在21、28 d时与NC组比较明显缩小(均P<0.05);28 d后,sh-MMP12组腋下肿瘤重量低于NC组(P<0.05)。结论:胃癌组织和细胞株SGC-7901中MMP-12表达升高,抑制MMP-12可减缓裸鼠移植瘤生长。