siRNA抑制Livin基因的表达对人膀胱癌EJ细胞生长及化疗敏感性的影响

发布时间：2018-04-10 17:52

本文选题：膀胱癌 + RNA干扰　；参考：《桂林医学院》2015年硕士论文

【摘要】：目的:Livin是IAP家族的最新成员。其在正常膀胱组织中不表达,而在人膀胱癌细胞中的高表达可刺激肿瘤细胞的增殖,并提高膀胱癌的术后复发率,更能使肿瘤细胞对化疗药物产生耐药性。因此,其可成为膀胱癌的治疗靶点及预后复发的监测指标。本研究通过观察siRNA抑制人膀胱癌EJ细胞中Livin基因的表达对细胞增殖、凋亡及化疗敏感性的影响,为膀胱癌发病机制的研究,以及对膀胱癌的基因和化学治疗提供理论基础和实验依据。方法:(1)观察通过不同条件转染过的EJ细胞,进行siRNA转染细胞条件的优化及效率的评价。(2)采用脂质体转染技术将人工合成的特异性Livin siRNA转入人膀胱癌EJ细胞中。(3)经Livin siRNA转染细胞24h后,半定量RT-PCR检测各组EJ细胞中Livin mRNA的表达。(4)Western Blot进一步验证转染48h后细胞中Livin蛋白的表达。(5)选用THP作用于转染和未转染的细胞,CCK-8法检测各组细胞的增殖抑制率。(6)流式细胞术检测各组细胞的凋亡情况。结果:(1)用FAM标记的Negative control siRNA转染6孔板中的人膀胱癌EJ细胞,观察到当细胞数量为3×105/孔,si RNA为100pmol/孔、Lipofectamine#174;2000试剂为5μl/孔时,有较高转染效率(70%)。当细胞密度为4×103/孔接种于96孔板中,siRNA为5pmol/孔、Lipofectamine#174;2000试剂为0.25μl/孔时,有较高转染效率(70%);(2)Livin特异性siRNA转染人膀胱癌EJ细胞24h后,干扰组(转染siRNA)中Livin mRNA的表达较空白对照组及阴性对照组显著降低(P0.05);(3)转染48h后,Livin在蛋白水平的表达,较空白对照组及阴性对照组明显下调(P0.05);(4)经CCK-8检测发现,Livin siRNA干扰组、THP化疗组及干扰+化疗的联合作用组均可抑制EJ细胞的增殖。且联合作用组对细胞的增殖抑制率显著高于单独干扰组及单独化疗作用组(P0.05);(5)流式细胞术的检测结果显示,与空白对照组比较,干扰组、化疗组及联合作用组均可促使膀胱癌EJ细胞的凋亡,且联合作用组的细胞凋亡率显著高于单独干扰组及单独化疗作用组(P0.05)。结论:(1)Livin基因在膀胱癌的发生发展过程中起到一定的促进作用;(2)本实验设计的Livin特异性siRNA能够有效抑制人膀胱癌EJ细胞中Livin基因的表达;(3)通过抑制Livin基因的表达,可有效抑制人膀胱癌EJ细胞的增殖、并促进其凋亡;(4)经Livin siRNA转染的EJ细胞加强了对化疗药物THP的敏感性。
[Abstract]:Objective: Livin is the newest member of the IAP family.It is not expressed in normal bladder tissue, but high expression in human bladder cancer cells can stimulate the proliferation of tumor cells, increase the recurrence rate of bladder cancer, and make the tumor cells become more resistant to chemotherapeutic drugs.Therefore, it can be used as a therapeutic target and a predictor of recurrence of bladder cancer.In this study, the effects of siRNA on the proliferation, apoptosis and chemosensitivity of human bladder cancer EJ cells were studied.And to provide theoretical and experimental basis for gene and chemotherapy of bladder cancer.Methods the EJ cells transfected with different conditions were observed.Optimization of siRNA transfection conditions and evaluation of its efficiency. 2) transfection of synthetic specific Livin siRNA into human bladder cancer EJ cells by liposome transfection with Livin siRNA for 24 h.Semi-quantitative RT-PCR Detection of Livin mRNA expression in EJ cells. Further verify the expression of Livin protein in EJ cells 48 h after transfection. Use THP on transfected and untransfected EJ cells to detect the inhibitory rate of proliferation of EJ cells in each group by CCK-8 method.The apoptosis of the cells in each group was detected by cytology.When the cell density was 4 脳 10 3 / well inoculated in 96 well plate, the siRNA was 5pmol/ Hole Lipofectamine #174A reagent was 0.25 渭 l / well, the transfection efficiency was higher than 70% Livin specific siRNA transfection to human bladder cancer EJ cells for 24 h.The expression of Livin mRNA in the interference group (transfected siRNAs) was significantly lower than that in the blank control group and the negative control group.Compared with the blank control group and the negative control group, the P0. 05 P0. 05 and P0. 05 were significantly down-regulated by CCK-8. The results of CCK-8 test showed that the proliferation of EJ cells could be inhibited in both the THP chemotherapy group and the combined treatment group.The inhibition rate of cell proliferation in the combined group was significantly higher than that in the control group and the chemotherapy alone group. The results of flow cytometry showed that compared with the blank control group, the inhibition rate of the cell proliferation in the combined group was significantly higher than that in the control group.The apoptosis rate of EJ cells in combination group and chemotherapy group was significantly higher than that in control group and chemotherapy alone group (P 0.05).Conclusion Livin specific siRNA can effectively inhibit the expression of Livin gene in human bladder cancer EJ cells by inhibiting the expression of Livin gene.EJ cells transfected with Livin siRNA enhanced the sensitivity to the chemotherapeutic drug THP.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.14

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