【作者】 向征；

【导师】 张才全；

【作者基本信息】 重庆医科大学 ， 外科学， 2007， 博士

【摘要】 结肠癌是的常见恶性肿瘤之一,术后化疗是结肠癌治疗的一个重要步骤,对患者生存期的延长和生活质量的改善有重要的作用。其中奥沙利铂（oxaliplatin, L-OHP）是目前临床治疗结肠癌的最重要的药物之一,具有较广泛的抗瘤谱。但是,肿瘤细胞对L-OHP产生耐受严重地影响了它的疗效,是临床上急待解决的问题之一。为了了解恶性肿瘤对L-OHP产生耐药的特征规律和耐药机理,本研究采用逐步递增L-OHP浓度,间歇作用体外诱导法培养建立了人结肠癌奥沙利铂耐药细胞模型—HT29/L-OHP。然后采取RNA干扰（RNA interferece,RNAi）技术,成功构建了TSG101（tumour susceptibility gene101,TSG101）小干扰RNA（TSG101-siRNA）的真核表达载体,顺利地逆转了结肠癌耐药细胞株HT29/L-OHP对L-OHP的耐药性。第一部分人结肠癌奥沙利铂耐药细胞模型的建立及耐药机理的初步探讨目的奥沙利铂（Oxaliplatin,L-OHP）是目前临床使用的最重要的抗癌药物之一,但肿瘤细胞对L-OHP产生耐受严重地影响了它的疗效。本研究建立人结肠癌奥沙利铂耐药细胞系HT29/L-OHP,了解恶性肿瘤细胞对L-OHP产生耐药的特征规律和耐药机理。方法本研究用逐步递增L-OHP浓度,间歇作用体外诱导法培养建立了人结肠癌L-OHP耐药细胞模型—HT29/L-OHP。MTT法测定生长曲线、交叉耐药性和耐药指数;并用光镜、电镜、流式细胞仪（FCM）等方法观察其一般生物学特性的改变;采用免疫组化检测GST-π、bcl-2等的表达水平了解其耐药的可能机理;通过RT-PCR和Western blotting检测tsg101mRNA和TSG101基因表达蛋白的水平。结果历时3个月建成了L-OHP耐药细胞模型—HT29/L-OHP,其耐药性能稳定,耐药指数为10.6430;并且与5-氟尿嘧啶、长春新碱、阿霉素等多种抗癌药有不同程度的交叉耐药性。HT29/L-OHP的细胞形态及染色体数目发生了改变;体外细胞群体倍增时间较亲代细胞延长29.7107小时;细胞周期分析发现其S期与G₀/G₁期细胞减少、G₂/M期细胞增多;tsg101mRNA和TSG101蛋白表达均较HT29亲代细胞增高;HT29/L-OHP细胞GST-π、MRP、NF-kB、INOS、HSP70、MTP53、Bcl-2和E-cd的表达升高,Bax和AE1/AE3等则无明显变化。结论HT29/L-OHP细胞是模拟临床用药特点建立的耐药性较稳定的多药耐药细胞,是研究L-OHP耐药机理及筛选逆转剂的理想细胞模型。HT29/L-OHP细胞具有耐药细胞的基本生物学特性。奥沙利铂耐药与GST-π、MRP、NF-kB、INOS、HSP70、MTP53、Bcl-2和TSG101蛋白表达升高有关。第二部分TSG101-siRNA表达载体的构建及对结肠癌耐药细胞株的逆转作用目的构建TSG101（tumour susceptibility gene101,TSG101）小干扰RNA（TSG101-siRNA）的真核表达载体,观察RNA干扰沉默TSG101基因后化疗药物奥沙利铂（L-OHP）对结肠癌耐药细胞株HT29/L-OHP增殖的影响。方法根据小干扰RNA的设计原则由计算机软件辅助设计TSG101的靶序列,应用DNA重组技术将目的序列克隆入小干扰RNA的表达载体;RT-PCR检测TSG101-siRNA对HT29/L-OHP细胞tsg101mRNA表达的影响;Western blotting检测TSG101-siRNA对TSG101基因表达蛋白的抑制效率;采用MTT法测定TSG101-siRNA联合化疗药物奥沙利铂对HT29/L-OHP的增殖抑制作用;FCM分析TSG101-siRNA转染HT29/L-OHP细胞前后的细胞周期分布情况。结果测序显示干扰载体构建成功,TSG101-siRNA能抑制tsg101mRNA表达,使HT29/L-OHP细胞TSG101蛋白的表达下降,明显增强奥沙利铂对HT29/L-OHP的增殖抑制;TSG101-siRNA转染HT29/L-OHP细胞后G₀/G₁期和S期细胞数有增加,而G₂/M期细胞数有减少。结论TSG101-siRNA的真核表达载体能有效地抑制HT29/L-OHP细胞的tsg101mRNA、TSG101蛋白的表达,提高化疗药物奥沙利铂对HT29/L-OHP的抑制率,能逆转结肠癌耐药细胞株HT29/L-OHP对L-OHP的耐药性。更多还原

【Abstract】 Human colon carcinoma is one of most common maligmant tumors. Chemotherapy is an important step in treatment of colon carcinoma, and have important effect on prolong of exist and improving life’s quality. Oxaliplatin （L-OHP） is one of the most important anticancer agents now used to colon carcinoma. Despite its broad clinical application, however, the resistance of cancer cells to oxaliplatin often culminates in chemotherapeutic failure and becomes one of the urgent questions to resolve in clinical chemotherapy.In order to explore its resistant mechanisms, a resistant cell line—HT29/L-OHP was established by stepwise increasing dose of L-OHP and intermittent administration. The eukaryotic expression plasmids of TSG101-siRNA was constructed successfully by RNA interference technology. The eukaryotic expression vector of TSG101-siRNA combined with oxaliplatin inhibits the proliferation of HT29/L-OHP and increases the sensitivity to chemotherapy. PART ONE ESTABLISHMENT OF OXALIPLATIN-INDUCED RESISTANT HUMAN COLON CANCER CELL MODEL AND PRELIMINARY STUDY ON THE RESISTANT MECHANISMSObjective Oxaliplatin（L-OHP） is one of the most important anticancer agents now used. Despite its broad clinical applications, however, the resistance of cancer cells to L-OHP often culminates in chemotherapeutic failure. In order to explore its resistant mechanisms, a resistant cell line—HT29/L-OHP was established. Methods A resistant cell line—HT29/L-OHP was established by stepwise increasing dose of L-OHP and intermittent administration. The growth curves、multidrug resistance and resistance index of HT29/L-OHP cell line to anticancer agents were detected by MTT assay. The changes of its biological characteristics were determined by light microscope, electron microscope, flow cytometry etc. The expression of glutathione S-transferase-π（GST-π） and Bcl-2 etc were analyzed and compared by immunohistochemical technique. The expression of tsg101mRNA and TSG101 protein in HT29 and HT29/L-OHP cell line was detected by RT-PCR and Western-blot. Results HT29/L-OHP cell line was established after 3 moths with stable resistance to L-OHP and resistance index 10.6430; HT29/L-OHP cells exhibited cross resistance to many other chemotherapeutic agents. As compared with parental cells, the morphological and chromatosome number of HT29/L-OHP changed; its doubling time prolonged; and the number of cells in S phase and G₀/G₁ phase decreased while in G₂/M phase increased by cell cycle analysis. The expression of tsgmRNA and TSG101 protein was decreased in the HT29 cell line. The expression of GST-π, MRP, NF-kB, INOS, HSP70, MTP53, Bcl-2 and E-cd was enhanced, but the expression of Bax and AE1/AE3 protein kept stable in the HT29/L-OHP cell line. Conclusion HT29/L-OHP cell line showed the typical multidrug resistant phenotype and might to serve as an ideal model for studying the mechanisms of resistance to L-OHP and filtrating reversing drug. HT29/L-OHP cell line possessed the basic characteristics of resistant cells. The primary factors resulting in HT29/L-OHP resistance to L-OHP were higher expression of GST-π, MRP, NF-kB, INOS, HSP70, MTP53, Bcl-2 and TSG101. PART TWO Construction and effect of TSG101-siRNA eukaryotic expression vectors with oxaliplatin on proliferation of HT29/L-OHPObjective To construct the eukaryotic expression plasmids of TSG101-siRNA and investigate the effect of RNA interference targeting tsg101 gene with oxaliplatin on colon cancer resistant cell line HT29/L-OHP. Methods The targeting fragments specifically against TSG101 were designed according to the principle of small interfering RNA designation using computer software. The sequences were cloned into siRNA expression plasmids through DNA recombinant technology. The expression of tsg101mRNA after transfected TSG101-siRNA was detected by RT-PCR. The expression levels of TSG101 after transfected TSG101-siRNA was detected by Western blotting. MTT test were applied to measure the inhibition combined with oxaliplatin on proliferation of HT29/L-OHP strain. Distribution of cell cycle was analyzed using flow cytometry after RNA interference HT29/L-OHP. Results The expected fragments were obtained by digestion identification and further confirmed by DNA sequencing. These TSG101-siRNA inhibited the expression of tsg101mRNA , and the expression of TSG101 protein, and proliferation of HT29/L-OHP obviously when combined with oxaliplatin. The analysis of cell cycle indicated that cells of G₂/M phases reduced and of G₀/G₁ and S phases increased. Conclusion The eukaryotic expression vector of TSG101-siRNA combined with oxaliplatin inhibits the proliferation of HT29/L-OHP and increases the sensitivity to chemotherapy.更多还原