靶向VEGF的siRNA诱导MCF-7凋亡及其相关机制的研究

发布时间：2018-02-12 15:22

本文关键词： RNA干扰 MCF-7细胞小干扰RNA 血管内皮生长因子 survivin　出处：《青岛大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：探讨靶向血管内皮生长因子(VEGF)的siRNA下调VEGF表达后对人乳腺癌MCF-7细胞凋亡及凋亡相关基因survivin的影响方法：体外设计并合成靶向VEGF mRNA的siRNA,采用LipofectamineTM2000转染人乳腺癌细胞MCF-7,实验设空白对照组、空脂质体组、阴性对照SCR组、3个siRNA浓度梯度组(50、100、200nnmol/L),转染24小时后,AnnexinV/PI双染结合流式细胞仪测定细胞凋亡；半定量RT-PCR、细胞免疫组化技术检测转染前后MCF-7细胞survivin基因表达的变化。结果：靶向VEGF基因的siRNA转染MCF-7后,与空白对照组比较,50nmol/L组细胞凋亡率增加(p0.05),100、200nmol/L组细胞凋亡率显著增加(p0.01),但空脂质体组、阴性对照SCR组与空白对照组比较无统计学意义(p0.05);survivin mRNA在各siRNA转染组表达较之空白对照组均下调(p均0.05,其中100nmol/L和200nmol/L转染组p均0.01),空脂质体组、阴性对照SCR组与空白对照组比较无统计学意义(p0.05)；survivin蛋白表达水平在各浓度转染组下调(p均0.05,其中100nmol/L和200nmol/L转染组p均0.01),而与未处理组比较,阴性对照SCR组与空脂质体组差异无统计学意义(p0.05)。结论：靶向VEGF基因的siRNA转染MCF-7后诱导细胞凋亡,且与转染剂量有一定依赖关系；其分子机制与凋亡相关基因survivin表达有关。
[Abstract]:Objective: to investigate the effect of siRNA targeting vascular endothelial growth factor (VEGF) on apoptosis and apoptosis-related gene survivin in human breast cancer MCF-7 cells after down-regulation of VEGF expression. Methods: siRNAs targeting VEGF mRNA were designed and synthesized in vitro. Human breast cancer cell line MCF-7 was transfected with LipofectamineTM2000. In the negative control SCR group, three siRNA concentration gradient groups were divided into three groups. After 24 hours of transfection, Annexin V / Pi double staining combined with flow cytometry was used to detect apoptosis, and semi-quantitative RT-PCR was used to detect the changes of survivin gene expression in MCF-7 cells before and after transfection. Results: after transfection of siRNA targeting VEGF gene into MCF-7, the apoptotic rate of 50 nmol / L group increased significantly compared with that of blank control group, but the apoptosis rate increased significantly in the 200nmol / L group, but in the empty liposome group. There was no significant difference between the negative control group and the blank control group in the expression of survivin mRNA in each siRNA transfection group. The expression of survivin mRNA in each siRNA transfection group was significantly lower than that in the blank control group (P < 0.05), including 100nmol / L and 200nmol / L transfection group (p < 0.01) and empty liposome group (P < 0.05). There was no significant difference between the negative control group and the blank control group in the expression of p0.05 SCR protein. The down-regulated expression level of the protein in each concentration of transfection group was 0.05, including 100nmol / L and 200nmol / L transfection group (p 0.01), but compared with the untreated group (P < 0.05). There was no significant difference between SCR group and empty liposome group in negative control group (p 0.05). Conclusion: siRNA targeting VEGF gene can induce apoptosis of MCF-7 cells in a dose-dependent manner, and its molecular mechanism is related to the expression of apoptosis-related gene survivin.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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