Prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay

AIM: To investigate the prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay.METHODS: Clonogenic assay, flow cytometry, and CB micronuclei assay were used to survey the cell survival rate, radiation-induced apoptosis and micronucleus frequency of hepatocarcinoma cell lines SMMC-7721,HL-7702, and HepG2 after being irradiated by X-ray at the dosage ranging 0-8 Gy.RESULTS: After irradiation, there was a dose-effect relationship between micronucleus frequency and radiation dosage among the three cell lines (P＜0.05). A positive relationship was observed between apoptosis and radiation dosage among the three cell lines. The HepG2 cells had a significant correlation (P＜0.05) but apoptosis incidence had a negative relationship with micronucleus frequency. There was a positive relationship between apoptosis and radiation dosage and the correlation between SMMC-7721 and HL-7702 cell lines had a significant difference (P＜0.01). After irradiation,a negative relationship between cell survival rate and radiation dosages was found among the three cell lines(P＜0.01). There was a positive relationship between cell survival rate and micronucleus frequency (P＜0.01). No correlation was observed between apoptosis and cell survival rate.CONCLUSION: The radiosensitivity of hepatocarcinoma cells can be reflected by apoptosis and micronuclei.Detection of apoptosis and micronuclei could enhance the accuracy for predicting radiosensitivity.

Effects of dendritic cells from cord blood CD34^+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCTD) mice.METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocarcinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect.RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%,47.92% vs 19.44%, P＜0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P＞0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80%vs 100%, P＜0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11 d vs 7 d, P＜0.01).CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

Cell cycle and radiosensitivity of progeny of irradiated primary cultured human hepatocarcinoma cells

AIM: To evaluate the change of growth characteristics and radiosensitivity of irradiated primary cultured human hepatocarcinoma cells.METHODS: All tumor tissue samples were obtained from 39 hepatocarcinoma patients with a mean age of 49.6 years (range 22-76 years). We divided the samples into irradiated group and non-irradiated group and measured their plating efficiency (PE), population doubling time (PDT), radiosensitivity index SF2 and cell cycle.RESULTS: The PDT of primary culture of hepatocarcinoma cells was 91.0±6.6 h, PE was 12.0±1.4%, SF2 was 0.41±0.05%.The PDT of their irradiated progeny was 124.8±5.8 h, PE was5.0±0.7%, SF2 was 0.65±0.09%. The primary cultured human hepatocarcinoma cells showed significant S redutiion and G2arrest in a dose-dependent manner. The progeny of irradiated primary cultured hepatocarcinoma cells grew more slowly and its radiosensitivity increased.CONCLUSION: The progeny of irradiated primary cultured human hepatocarcinoma cells grows more slowly and its radiosensitivity increases.

Protein profile of human hepatocarcinoma cell line SMMC-7721:Identification and functional analysis

AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS: Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol- cytochrome C reductase complex core proteinⅠ, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip

AIM: In order to obtain lymphogenous metastasisassociated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential.METHODS: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip() MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics.RESULTS: Out of the 14 500 known genes investigated,110 (0.8%) were up regulated at least 23 fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 23 fold. According to the Gene Ontology and TreeView analysis, the 110genes were further classified into two groups: differential biological process profile and molecular function profile.CONCLUSION: Using high-throughput gene chip method,a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription,chaperone activity, motor activity, protein kinase activity,receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation.Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments.ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.

Inhibitory Effect of Melatonin on the Growth of H22 Hepatocarcinoma Cells by Inducing Apoptosis

Whether melatonin not only inhibits the growth of H22 hepatocarcinoma cells but also induces apoptosis in vitro was assessed. The anti proliferative effects of melatonin on tumor cells was observed by MTT assay and tumor cells growth curve assay. And the apoptosis of the cells was studied by acridine orange fluorescence assay and flow cytometry. The cell cycle of the tumor cells was also observed by flow cytometry. It was found that melatonin could significantly inhibit the growth of H22 hepatocarcinoma cells. Incubated with melatonin, chromatin condensation of the tumor cells was observed by fluorescence microscopy. Compared with control, the percentage of apoptotic cells was increased, and the proportion of G 0/S increased but that of G 2/M decreased. It was suggested that melatonin could directly inhibit the growth of H22 hepatocarcinoma cells by inducing apoptosis and extending the length of cell cycle of the tumor cells.

Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L_(6))

In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells,a new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(Hca-P/L_(6))was established and the effect of curcumin on biological behavior of Hca-P/L_(6) was observed.Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential(Hca-P)was subcutaneously inoculated into the medioventral line of a mouse 615 and thefirst generation of metastatic tumor cells of inguinal lymph node(Hca-P/L_(1))was obtained.Then,Hca-P/L_(1) was screened by the route of mouse foot pad subcutaneously!lymph node!scale-up culture in vitro!mouse foot pad subcuta-neously forfive times consecutively.The sensitivity of two murine ascites hepatocarcinoma cell lines(Hca-P and Hca-P/L_(6))and two anchorage-dependent human hepato-carcinoma cell lines(SMC7721 and HepG_(2))to curcumin were studied by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay after these cells had been pretreated by curcumin at the concentration of 15–240μmol/L for 48 h.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15μmol/L in vitro,the effect of curcumin against cell proliferation of Hca-P and Hca-P/L6 was observed by inverted micro-scope,cell growth curve and cell population doubling time;the effects of curcumin on cell cycles of Hca-P/L6 and Hca-P were studied byflow cytometry(FCM).The results showed Hca-P/L_(6) spreading to the lymph nodes at multiple sites in mice was screened from Hca-P.The lymph node metastatic rate was 100%.Curcumin had significant growth inhibiting effect on both murine ascites and human hepatocarcinoma cell lines in a dose-dependent manner(P<0.05).At concentrations of 30–120μmol/L,curcu-min had more inhibition on murine ascites hepatocarci-noma cell lines than on human anchorage-dependent hepatocarcinoma cell lines.At concentrations of 60–240μmol/L,curcumin had more inhibition on Hca-P/L_(6) with the 50%inhibitory concentration(IC50)of 51.48μmol/L than on Hca-P with IC50 of 90.87μmol/L.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days,the proliferations of Hca-P/L_(6) and Hca-P were inhibited(P<0.05)in a time-dependent manner(P<0.01)and the population doubling time of Hca-P/L6 and Hca-P was prolonged(P<0.01),and curcumin had more inhibition on Hca-P/L6 than on Hca-P(P<0.05).After pretreatment by 15μmol/L curcumin for 48 h,the morphous of Hca-P/L_(6) was influenced more seriously than that of Hca-P and the cell cycle was redistributed with Hca-P/L6 being blocked in the S phase and Hca-P in the S and G_(2)/M phases.Hca-P/L_(6) was validated to be more sensitive to curcumin than Hca-P.Hca-P/L_(6) is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.

EXPRESSION OF ONCOGENES DURING INDUCED DIFFERENTIATION OF HUMAN HEPATOCARCINOMA CELL LINE

There was no detectable expression of c-fos,but a little c-myc,high c-fms and mederate high IGF-ⅡmRNA in the untreated human hepatocarcinoma cell SMMC- 7721.After treatment with 10 μmol/L retinoic acid or 0.5 mmol/L dibutyryl cyclic-3',5'adenosine monophosphate(db-cAMP),the c-fos was transiently expressed within 20- 60mins.If the treatment of RA or db-cAMP prolonged to 1-5 days, the transcriptions of c- myc were increased,reaching the highest level on the 2nd and 4th day.Simultaneously the transcriptions of c- fms and IGF- Ⅱwere gradually decreased.On the 5th day of the treatment,c-fms and IGF-ⅡmRNA were decreased to 32% and 14%respectively of the control (untreated cell) value by RA,and 35% and 22%respectively by db-cAMP.The biological significance of the above mentioned results was discussed.

Therapeutic efficacy and bone marrow protection of the mdr1 gene and over-dose chemotherapy with doxorubicin for rabbits with VX2 hepatocarcinoma

BACKGROUND : Malignant tumors are common diseases threatening to the health and life of human being. Clinically, the multidrug resistance of tumor cells and bone marrow depression caused by chemotherapeutic agents are the main obstacles to the treatment of tumors, and both are related to the mdr1 gene. The over expression of the mdr1 gene in tumor cells contributes to the multidrug resistance of malignant tumor cells. With little expression of the mdr1 gene, bone marrow cells particularly susceptible to multidrug resistance-sensitive agents, which cause serious toxicity in bone marrow. This study was undertaken to assess therapeutic efficacy of transplantation of bone marrow mononuclear cells transferred with the mdr1 gene and over-dose chemotherapy with doxorubicin for VX2 hepatocarcinoma of rabbits. METHODS: The mdr1 gene was transferred into the bone marrow mononuclear cells of rabbits, which was co- cultured with retroviral vector-containing supernatant, and the cells were autotransplanted into a rabbit model with VX2 hepatocarcinoma. After chemotherapy with doxorubicin, the protective effects of the mdr1 gene and therapeutic efficacy of over-dose chemotherapy were observed. RESULTS: The mdr1 gene was transferred successfully into the bone marrow mononuclear cells, with a transduction efficiency of 35%. After autotransplantation, the mdr1 gene was expressed functionally in bone marrow with a positive rate of 8%, indicating that the gene played animportant role in bone marrow protection. The rabbits with VX2 hepatocarcinoma, which had received the mdr1 gene-transduced cells, survived after chemotherapy with a 3-fold dose of adriamycin, and their white blood cell counts were (4.26±1.03)×104/L. Since hepatocarcinoma cells were eradicated, the survival time (97.00±46.75 d) of the rabbits was extended (P<0.05) and the healing rate of the tumor was increased (P<0.05). CONCLUSIONS: The transferring of the mdr1 gene into bone marrow mononuclear cells could confer chemoprotection to bone marrow, and over-dose chemotherapy could be prescribed for the treatment of malignant tumors.

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.

EFFECT OF ACUPUNCTURE ON IL 2-IFN-NKC IMMUNOREGULATORY NETWORK IN MICE WITH TRANSPLANTED HEPATOCARCINOMA

Objective: To study the effect of acupuncture on interleukin 2 (IL 2)-interferon(IFN)-natural killer cells (NKC) immunoregulatory network of mice with transplanted hepatocarcinoma(HAC) in order to provide new evidence for acupuncture treatment of hypofunction of immune system. Methods: The 28 HAC-vaccinated BALB/C mice are randomly divided into control group (n = 14) and acupunctrue group (n = 14). In the latter group bilateral"Dazhui" (BL 11 )and "Zusanili" (ST 36) which are located according to the same positions indicated in Comparative Anatomy of Macroanimals, are needled once every day, twelve sessions altogether. Twenty-four hours after the needling treatment the mice are killed and the spleen is taken out to be made into cell suspension for assaying concentrations of IL2 (MTT method) and NKC (colorimetric method) respectively. Obital Serum IFN is determined by using (CPE microplate staining), and the tumor mass is taken out and balanced with an analytical balance (1/10000) to calculate the tumor inhibition rate according to the formula. Results: The tumor weight of the mice of aupuncture group is obviously decreased (inhibition rate 43.06%) while the activity of the IL2 and NKC, and the IFN titer are increased greatly, which have significant differences compared with those of control group (P ＞0.01). Conclusion:The results of the study show that acupuncture can strengthen the positive immunoregulatory function of the IL2 -IFN-NKC network in immune hypofunction mice bearing HAC.

Effect of dendritic cell modified by gp96-peptide complex on antitumor effect in H22 cell

Objective:To investigate the antitumor effect of dendritic cell （DC） modified by gp96-peptide complexes both in vitro and in vivo.Methods:Gp96-peptide complexes were acquired from H22 liver cancer cells in mice. DC were cultured from bone marrow cells and modified by gp96-peptide complexes. Spleen lymphocytes of mice were activated by modified DC and the cytotoxicity were detected by (（）51Cr) release method. Modified DC, gp96-peptide complexes and inactivated H22 cells were injected into mice bearing H22 liver cancer cells to observe the levels of IL-10, IFN-γ in serum and the alteration of proportions of CD8+-IFNγ+ and CD8+- IL-10+ cells, CD4+-IFNγ+ and CD4+- IL-10+ cells. Results:DC modified by gp96-peptide complexes can activate spleen lymphocyte and the latter can specifically kill H22 cells but not Ehrilich ascites carcinoma cells. Modified DC can improve the host’s antitumor immune response and the proportions of Th1 cells, inhibiting tumor growth. Conclusion: Gp96-peptide complexes can activate DC effectively, making DC a good vaccine.

OBSCN突变对于肝癌细胞增殖和迁移能力的影响

目的构建细胞骨架蛋白OBSCN基因过表达和敲除的稳转细胞系,初步探究OBSCN突变对肝癌细胞增殖和迁移能力的影响。方法qPCR技术确定HepG2肝癌细胞有无OBSCN本底表达,慢病毒感染HepG2细胞构建过表达细胞系;Crispr cas9技术构建敲除细胞系;并利用Western blot技术检测敲除及过表达细胞Obscurin蛋白表达量,CCK-8法和Transwell小室法探究OBSCN突变细胞株的增殖和迁移能力。结果qPCR验证HepG2细胞无OBSCN本底表达,慢病毒感染得到HepG2 H21157过表达稳转细胞系和HepG2 GL119对照空载体稳转细胞系;菌落PCR、质粒PCR以及基因测序结果验证敲除重组质粒reOBSCN构建成功,转染得到OBSCN敲除的HepG2 OBSCN KO稳转细胞系和对照空载体HepG2 ecas稳转细胞系;Western blot技术验证OBSCN敲除及过表达细胞系构建成功;CCK-8法结果显示OBSCN过表达加快了HepG2细胞增殖,差异有统计学意义(P<0.0001),OBSCN基因的敲除可以减慢HepG2细胞增殖,差异有统计学意义(P<0.0001);Transwell小室法得到HepG2 H21157、HepG2 GL119的迁移细胞数分别为150.30±14.95、136.50±15.02,HepG2 OBSCN KO、HepG2 ecas的迁移细胞数分别为112.70±20.30、147.8±11.55,差异有统计学意义(P<0.01)。结论OBSCN基因的过表达会加快HepG2肝癌细胞的增殖和迁移,OBSCN基因的敲除会减慢HepG2肝癌细胞的增殖和迁移。

胆木抗肝癌活性的网络药理学及初步细胞筛选研究

目的:基于网络药理学探究胆木抗肝癌的作用机制,并通过细胞筛选初步验证胆木抗肝癌活性。方法:利用网络药理学筛选胆木与肝癌的共同靶点,构建蛋白互作网络以及进行富集分析及作用机制预测,并将胆木的主要活性成分与核心靶点进行分子对接。利用CCK8、细胞凋亡及PCR等体外细胞实验进行初步验证。结果:经筛选获得胆木活性成分14种,相关的靶点587个,与肝癌靶点映射后共同靶点有288个,主要有TP53、SRC、STAT3等核心靶点。其中异长春花苷内酰胺、短小蛇根草苷、喜果苷等化合物可能是胆木抗肝癌的潜在活性成分,可能通过介导癌症通路、PI3K/Akt和EGFR酪氨酸激酶抑制剂抵抗等信号通路,参与蛋白质磷酸化、凋亡过程的负调控等过程,发挥抗肝癌作用;分子对接结果显示胆木活性成分与肝癌核心靶点产生稳定的结合;体外细胞实验结果表明,胆木中主要成分异长春花苷内酰胺对肝癌细胞具有细胞毒性,抑制肝癌细胞增殖(P<0.001),内在机制是下调肝癌HepG2细胞SRC、STAT3、MAPK3的基因表达(P<0.05),并诱导肝癌细胞凋亡(P<0.001)。结论:本研究初步探讨了胆木活性成分抗肝癌的潜在机制及初步药效作用,为胆木抗肝癌作用机制研究提供理论依据。

丹参酮对人肝癌细胞某些表型的逆转作用

目的观察丹参酮促人肝癌细胞株在体外向正常方向分化的效果。方法体外培养的人肝癌细胞（ＳＭＭＣ┐７７２１）经０．５μｇ／ｍｌ丹参酮处理４天后，作光电镜观察，ＢｒｄＵ掺入试验，ＰＣＮＡ免疫组化及ＦＣＭ检测。结果在镜下，细胞形态趋向良性分化，细胞生长明显被抑制；ＢｒｄＵ标记率和ＰＣＮＡ阳性率均明显低于对照组；流式细胞仪检测显示丹参酮处理组的细胞被阻止于Ｇ０／Ｇ１期，而Ｓ期细胞数量明显减少，ｃ┐ｍｙｃ癌基因蛋白表达降低，ｃ┐ｆｏｓ癌基因蛋白表达明显增加。结论丹参酮可诱导人肝癌细胞某些表型的逆转，可能是一种有前途的分化诱导剂。

苦参碱对TIM2转基因小鼠肝癌细胞的作用研究

目的:观察苦参碱对TIM2转基因修饰小鼠H22肝癌细胞瘤苗的体内作用。方法:构建小鼠TIM2基因真核表达载体并用以转染H22细胞,经体外稳定筛选后获得TIM2转基因H22肝癌细胞全细胞瘤苗(H22-TIM2),用以建立小鼠肝癌移植瘤模型,观察该瘤苗在小鼠体内的成瘤性和免疫原性;加用药物苦参碱(matrine,M)治疗后,观察苦参碱对其体内抗癌活性的影响。结果:筛选得到的TIM2转基因H22肝癌细胞瘤苗有TIM2 mR-NA及EGFP的稳定表达,此瘤苗接种后,在小鼠体内的成瘤率为41%,远低于H22对照组和H22-EGFP空载体组(H22-EGFP)(后两者成瘤率在92%以上),对小鼠肿瘤的抑制率为69.2%,明显高于苦参碱治疗组(67.5%)和其他各组。同时苦参碱可进一步增强H22-TIM2瘤苗的肿瘤抑制率。H22-TIM2组小鼠的脾指数,CD4+T细胞亚群和CD4/CD8较其他实验组明显增高。结论:TIM2基因修饰H22细胞瘤苗可显著降低H22肝癌细胞在小鼠体内的致瘤性,在体内具有一定的免疫原性,苦参碱可明显改善其体内抗癌活性,为进一步研究TIM2基因在肿瘤免疫中的作用及苦参碱的抗癌机制奠定了基础。

中国鲎鲎素诱导人肝癌SMMC-7721细胞分化的观察

背景与目的:海洋生物活性物质抗肿瘤活性研究是海洋生物活性物质开发与抗癌药物研究的一个重要领域。诱导肿瘤细胞分化则是肿瘤药物治疗的新策略。本文拟研究海洋生物活性物质鲎素对人肝癌SMMC-7721细胞分化的影响,以为鲎素抗肿瘤作用及其机理的深入研究提供实验依据。方法:从中国鲎血细胞酸抽提液提取鲎素,应用光镜和透射电镜观察3.0μg/ml鲎素处理前后人肝癌SMMC-7721细胞形态和超微结构的变化,应用细胞化学或免疫细胞化学方法观察鲎素处理前后细胞碱性磷酸酶活性与甲胎蛋白和增殖细胞核抗原表达的变化。结果:经3.0μg/ml鲎素处理的SMMC-7721细胞形态和超微结构发生恢复性变化,碱性磷酸酶活性减弱,甲胎蛋白和增殖细胞核抗原表达降低。结论:鲎素能有效改变肝癌细胞恶性形态和超微结构特征,改变肝癌细胞相关酶活性和抗原表达,对肝癌细胞具有一定的诱导分化作用。

甲基莲心碱逆转肝癌HepG2/thermotolerance细胞对阿霉素耐受性的作用

背景与目的:如何成功地逆转耐热癌细胞的多药耐药性(multidrug resistance,MDR)是当前肿瘤热疗的研究热点。本研究探讨耐热肝癌细胞能否对阿霉素(adriamycin,ADR)产生耐受性,以及甲基莲心碱(neferine,Nef)能否逆转耐热肝癌细胞的阿霉素耐药性。方法:MTT法检测肿瘤细胞存活率,PI染色流式细胞仪检测细胞凋亡率,间接免疫荧光流式细胞术检测bcl-2表达。结果:在43℃环境中培养24h后,耐热肝癌细胞HepG2/thermotolerance的细胞存活率和细胞凋亡率分别为(89.6±5.4)%和(13.6±5.4)%,而非耐热肝癌细胞HepG2的细胞存活率和细胞凋亡率分别为(23.9±3.6)%和(68.9±7.3)%。正常培养环境情况下(37℃),ADR对HepG2/thermotolerance细胞的IC50为(113.7±12.7)μmol/L,而对HepG2细胞的IC50为(10.5±2.3)μmol/L,耐药倍数达10.8倍。1、10、100μmol/LADR分别作用24h后,HepG2/thermotolerance细胞的凋亡率分别为(9.3±2.6)%、(17.8±7.3)%和(32.9±8.6)%,而HepG2细胞的凋亡率分别为(14.3±3.9)%、(38.9±6.8)%和(62.7±5.9)%。在37℃培养环境下,10、40μmol/LNef对HepG2细胞和HepG2/thermotolerance细胞无增殖抑制作用和凋亡诱导作用,但可使ADR对HepG2/thermotolerance细胞的IC50分别下降至(63.7±5.6)μmol/L和(16.8±2.8)μmol/L,逆转倍数分别为1.78和6.79,并可使10μmol/LADR对HepG2/thermotolerance细胞凋亡的诱导作用升高至(26.8±5.9)%和(34.9±8.7)%;HepG2/thermotolerance细胞较HepG2细胞高表达Bcl-2蛋白,而Nef能下调HepG2/thermotolerance细胞的Bcl-2表达。结论:HepG2/thermotolerance细胞对ADR可产生耐受性,Nef可逆转HepG2/thermotolerance细胞对ADR的耐受性,其机制可能与其下调HepG2/thermotolerance细胞Bcl-2蛋白表达有关。

细胞周期相关基因微阵列在中药抑制肝癌细胞增殖作用机理研究中的应用

目的 :设计DNA微阵列并利用其研究中药抗肿瘤的分子机制。方法 :制备了包含有与细胞周期和DNA损伤调控检测相关的 2 4个基因的cDNA微阵列 ,选择了本实验室已证明对肝癌细胞有抑制效果的中药 ,通过流式细胞仪检测其对细胞周期的影响 ,提取药物作用后的RNA ,反转录后标记探针与微阵列杂交 ,检测中药作用细胞后对细胞周期及损伤检测相关基因转录的影响。结果 :4种中药作用SMMC 772 1后 ,细胞周期基因和损伤检测点基因均有不同程度改变 ,表现为部分上调 ,部分下调 ,这些和流式细胞术检测的结果是相符合的。结论