【摘要】：目的：利用小分子RNA（siRNA）干擾技術，抑制survivin基因的表達，研究其對HSC-T6細胞株凋亡的影響及可能的機制。 方法：實驗分為空白組、陰性對照組（pGPU6/GFP/Neo-shNC）、siRNA組（pGPU6/GFP/Neo-siRNA）。脂質體介導survivin siRNA轉染HSC-T6細胞24小時后,倒置熒光顯微鏡下觀察轉染效率。Survivin siRNA轉染HSC-T6細胞48小時后，利用RT-PCR、western-blot檢測survivin mRNA及蛋白表達的改變,倒置顯微鏡下觀察細胞形態(tài)學的改變，PI單染流式細胞儀測定細胞凋亡率，western-blot檢測caspase-3蛋白的表達。 結果：survivin siRNA轉染HSC24小時后，于倒置熒光顯微鏡下觀察可見：siRNA組細胞內可見到清晰的綠色熒光，轉染效率均達到75%；轉染作用48小時后,RT-PCR及werstern-blot結果顯示：空白組、pGPU6/GFP/Neo-shNC組survivinmRNA及蛋白表達最豐富，pGPU6/GFP/Neo-siRNA組survivin mRNA及蛋白表達較少，空白組與pGPU6/GFP/Neo-shNC組比較，無統(tǒng)計學意義（p0.05）；空白組、pGPU6/GFP/Neo-shNC組與pGPU6/GFP/Neo-siRNA組比較，有統(tǒng)計學意義（p0.05）。在倒置顯微鏡下觀察，發(fā)現(xiàn)pGPU6/GFP/Neo-siRNA組細胞凋亡明顯；PI單染流式細胞儀檢測顯示：與空白組、pGPU6/GFP/Neo-shNC組比較，pGPU6/GFP/Neo-siRNA組細胞凋亡率明顯提高，有統(tǒng)計學意義（p0.05）；空白組與pGPU6/GFP/Neo-shNC組比較，無統(tǒng)計學意義（p0.05）。werstern-blot結果顯示：空白組、pGPU6/GFP/Neo-shNC組caspase-3蛋白表達較少，pGPU6/GFP/Neo-siRNA組caspase-3蛋白表達豐富，空白組與pGPU6/GFP/Neo-shNC組比較，無統(tǒng)計學意義（p0.05），空白組、pGPU6/GFP/Neo-shNC組與pGPU6/GFP/Neo-siRNA組比較，，有統(tǒng)計學意義（p0.05）； 結論：1、Survivin siRNA能抑制survivin基因及蛋白的表達； 2、Survivin siRNA可能通過降低對caspase-3抑制作用而誘導HSC-T6細胞的凋亡。
[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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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