siRNA抑制Survivin基因对大鼠肝星状细胞HSC-T6凋亡的影响

发布时间：2018-08-07 14:39

【摘要】：目的：利用小分子RNA（siRNA）干扰技术，抑制survivin基因的表达，研究其对HSC-T6细胞株凋亡的影响及可能的机制。 方法：实验分为空白组、阴性对照组（pGPU6/GFP/Neo-shNC）、siRNA组（pGPU6/GFP/Neo-siRNA）。脂质体介导survivin siRNA转染HSC-T6细胞24小时后,倒置荧光显微镜下观察转染效率。Survivin siRNA转染HSC-T6细胞48小时后，利用RT-PCR、western-blot检测survivin mRNA及蛋白表达的改变,倒置显微镜下观察细胞形态学的改变，PI单染流式细胞仪测定细胞凋亡率，western-blot检测caspase-3蛋白的表达。 结果：survivin siRNA转染HSC24小时后，于倒置荧光显微镜下观察可见：siRNA组细胞内可见到清晰的绿色荧光，转染效率均达到75%；转染作用48小时后,RT-PCR及werstern-blot结果显示：空白组、pGPU6/GFP/Neo-shNC组survivinmRNA及蛋白表达最丰富，pGPU6/GFP/Neo-siRNA组survivin mRNA及蛋白表达较少，空白组与pGPU6/GFP/Neo-shNC组比较，无统计学意义（p0.05）；空白组、pGPU6/GFP/Neo-shNC组与pGPU6/GFP/Neo-siRNA组比较，有统计学意义（p0.05）。在倒置显微镜下观察，发现pGPU6/GFP/Neo-siRNA组细胞凋亡明显；PI单染流式细胞仪检测显示：与空白组、pGPU6/GFP/Neo-shNC组比较，pGPU6/GFP/Neo-siRNA组细胞凋亡率明显提高，有统计学意义（p0.05）；空白组与pGPU6/GFP/Neo-shNC组比较，无统计学意义（p0.05）。werstern-blot结果显示：空白组、pGPU6/GFP/Neo-shNC组caspase-3蛋白表达较少，pGPU6/GFP/Neo-siRNA组caspase-3蛋白表达丰富，空白组与pGPU6/GFP/Neo-shNC组比较，无统计学意义（p0.05），空白组、pGPU6/GFP/Neo-shNC组与pGPU6/GFP/Neo-siRNA组比较，，有统计学意义（p0.05）； 结论：1、Survivin siRNA能抑制survivin基因及蛋白的表达； 2、Survivin siRNA可能通过降低对caspase-3抑制作用而诱导HSC-T6细胞的凋亡。
[Abstract]:Aim: to inhibit the expression of survivin gene by using small molecule RNA (siRNA) interference technique, and to study its effect on apoptosis of HSC-T6 cell line and its possible mechanism. Methods: the experiment was divided into blank group, negative control group (pGPU6/GFP/Neo-shNC) and siRNA group (pGPU6/GFP/Neo-siRNA). After transfection of HSC-T6 cells with liposome mediated survivin siRNA for 24 hours, the transfection efficiency of survivin siRNA was observed under inverted fluorescence microscope for 48 hours. The changes of survivin mRNA and protein expression were detected by RT-PCR western-blot. The morphological changes of cells were observed under inverted microscope. The apoptosis rate was measured by Pi single staining flow cytometry and the expression of caspase-3 protein was detected by western-blot. Results the clear green fluorescence was observed under inverted fluorescence microscope, and the transfection efficiency was 75%. After 48 hours of transfection, the results of RT-PCR and werstern-blot showed that the expression of survivinmRNA and protein in pGPU6 / GFP / Neo-shNC group was the most abundant in the blank group, and the expression of survivin mRNA and protein in the pGPU6 / GFP / Neo-siRNA group was less than that in the control group (p0.05), but there was no significant difference between the blank group and the pGPU6/GFP/Neo-shNC group (p0.05), while the pGPU6 / GFP-Neo-shNC group in the blank group had statistical significance compared with the pGPU6/GFP/Neo-siRNA group (p0.05). Under inverted microscope, we found that the apoptosis rate of pGPU6/GFP/Neo-siRNA group was significantly higher than that of pGPU 6 / GFP / Neo-shNC group (p 0.05), and it was significantly higher in pGPU6/GFP/Neo-siRNA group than that in pGPU6/GFP/Neo-shNC group (p0.05), compared with the control group (pGPU 6 / GFP / Neo-shNC group), the apoptotic rate of pGPU 6 / GFP / Neo-shNC group was significantly higher than that of pGPU6/GFP/Neo-shNC group (p0.05). No significant (p0.05) .werstern-blot results showed that the expression of caspase-3 protein in the blank group was less than that in the control group (pGPU6 / GFP / Neo-shNC group). The caspase-3 protein expression in the blank group was significantly higher than that in the pGPU6/GFP/Neo-shNC group (p0.05). The expression of caspase-3 protein in the blank group was significantly higher than that in the pGPU6/GFP/Neo-siRNA group (p0.05). Conclusion survivin siRNA can inhibit the expression of survivin gene and protein, while survivin siRNA may induce the apoptosis of HSC-T6 cells by decreasing the inhibitory effect on caspase-3.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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