【作者】 臧凤琳；

【导师】 孙保存；

【作者基本信息】 天津医科大学 ， 病理与病理生理学， 2004， 硕士

【摘要】 目的： 大肠癌是胃肠道最常见的恶性肿瘤之一，目前各种治疗方法均不能有效地防止其转移和复发，其中最主要的原因是肿瘤组织的凋亡水平下调，从而逃避机体的免疫监视，免遭免疫系统的杀伤和攻击。因此进一步阐释肿瘤的免疫逃逸机制，寻找新的治疗靶点，已成为当今国内外肿瘤治疗的研究热点。本论文聚焦于Fas受体凋亡途径中的抑制蛋白——FLIP，采用RNA干扰技术，比较干扰前后体外培养的大肠癌细胞株凋亡敏感性的变化，阐明在大肠癌中是否存在由FLIP介导的免疫逃逸机制，并为肿瘤的基因治疗提供新的思路。 方法： 体外培养大肠癌细胞株，采用直接免疫荧光流式细胞学技术和半定量RT-PCR法筛选体外实验模型。利用电穿孔，将干扰性小RNA(siRNA)片段转染至HT-29细胞，以相对半定量PCR法计算干扰效率，确定FLIP基因被封阻情况。在激活型抗Fas抗体诱导下，采用Annexin V法和细胞周期分析共同判定转染前后HT-29细胞凋亡敏感性的变化。 结果： 1．综合Fas和FLIP两项指标的测定结果，筛选出HT-29细胞为适宜的体外实验模型(其Fas的表达水平为42.46％，c-FLIP_L mRNA的相对含量为83.36％)。 2．在摸索到的最佳电穿孔条件下(90mOsmol／kg低渗环境，电压500V，时程70μs，外源片段浓度100nM，室温)，将对应目的基因FLIP的siRNA转染入HT-29细胞，FLIP mRNA的相对含量由78.55％下降到27.48％，干扰效率达65.02％。 3．在激活型抗Fas抗体的刺激下，转染后的HT-29细胞凋亡比例由1.76％上升到29.5％，细胞周期分布图上出现“凋亡峰”。 天津医科大学硕士学位论文中文摘要结论:1.大肠癌细胞中存在一定水平的FLIP表达，经RNA干扰后，FUPm丑NA表 达水平下调，细胞表面Fas受体表达水平未产生明显变化，细胞对Fas介导 的凋亡敏感性上升，从而证实在大肠癌中存在着由FLIP介导的免疫逃逸机 制。2.随着FLIP表达水平的下降，在激活型抗Fas抗体的刺激下，大肠癌细胞中 凋亡细胞所占比例增加，提示FLIP作为受体凋亡通路的关键因子，可望成 为肿瘤基因治疗的新靶点。3.将对应目的基因FLIP的外源siRNA片段转染入体外培养细胞后，高效、特 异地降低了FLIP mRNA的表达，并进而阐明FLIP的生物学功能，提示RNA 干扰作为一种新型基因研究手段拥有较广阔的应用前景。更多还原

【Abstract】 Objective:Colorectal cancer is one of the most general intestinal tumors and none of therapies can effectually prevent it from metastasis and recurrence by now. The most possible reason about this phenomenon is that tumor cells reduce their apoptotic sensitivity, evade immune surveillance and survive upon many kinds of signals. Thus, how to exclude the mechanism of tumor evasion and find out new targets of tumor therapy has been the hotspot. Here, we focus on FLIP, one . of the inhibitory proteins of Fas-mediated apoptotic pathway. After RNA interfering, the mRNA expression level of FLIP is decreased and the apoptotic sensitivity is increased in the transfected colorectal cancer cells. We hope our study could exclude whether there is FLIP-associated apoptotic evasion in colorectal cancer cells and put a new way on tumor gene therapy. Methods:Four colorectal cancer cell lines were cultured in vitro. After direct immunofluoscent FACScan and semi-quantitative RT-PCR analysis, the optimal experimental model was selected. HT-29 was transfected with siRNA by electroporation. The inhibitory of FLIP mRNA was detected by relative semi-quantitative PCR. After that, the transfected cells were treated with agonistic anti-Fas antibody in order to induce apoptosis. We used Annexin V and cell cycle analysis to determine the apoptotic sensitivity of transfected HT-29. Results:1. Combining the levels of Fas and FLIP, HT-29 was selected as the best experimental model.2. After transfecting siRNA into HT-29, the expression level of FLIP mRNA wasdecreased from 78.55 % to 27.48 % and the interfering effectiveness is 65.02 %. 3. After treated with agonistic anti-Fas antibody, the apoptotic rate of transfected HT-29 was increased from 1.76 % to 29.5 % and we can find an "apoptotic peak" on the cell cycle analysis. Conclusions:1 . There is FLIP-associated immune evasion mechanism in colorectal cancer cell lines exactly.2. As an important factor in Fas-mediated apoptotic pathway, FLIP could be one of the effective targets in tumor gene therapy.3. Since the mRNA level of FLIP is effectually reduced after transfecting siRNA into HT-29 cell line, RNAi will possess expansive applicable prospect in gene silencing area.更多还原