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目的:构建人PIG11的miRNA表达载体pcDNATM6.2-GW/EmGFPmiR,建立PIG11蛋白稳定低表达的HepG2细胞株,联合PIG11蛋白稳定高表达HepG2细胞株,探讨PIG11基因表达对HepG2细胞凋亡的影响。方法:构建miRNA干扰载体,通过脂质体转染HepG2细胞,建立PIG11蛋白稳定低表达的HepG2细胞,与前期已建立的PIG11蛋白稳定高表达的HepG2细胞模型作对比,利用PI染色流式细胞仪检测细胞的凋亡情况,蛋白质印迹法检测Caspase-3蛋白、Caspase-9蛋白的表达情况。结果:成功构建人PIG11的miRNA表达载体pcDNATM6.2-GW/EmGFPmiR,转染后获得PIG11基因沉默的HepG2细胞株。PI染色流式细胞仪检测结果显示,HepG2细胞、miR-PIG11-HepG2细胞、miR-HepG2细胞、pLXSN-PIG11-HepG2细胞和pLXSN-HepG2细胞的凋亡率分别为(5.72±0.81)%、(1.34±0.71)%、(5.10±0.40)%、(34.83±2.29)%和(5.34±0.60)%。PIG11高表达的pLXSN-PIG-11-HepG2细胞组凋亡率比其他组增高(P<0.01),PIG11低表达的miR-PIG11-HepG2细胞组凋亡率降低(P<0.01)。蛋白质印迹法检测结果显示,PIG11高表达的HepG2细胞Caspase-3及Caspase-9蛋白表达均上调(P<0.01),PIG11低表达的HepG2细胞Caspase-3及Caspase-9蛋白的表达下调(P<0.01)。结论:构建人PIG11的干扰载体成功获得PIG11蛋白稳定低表达的HepG2细胞株,PI染色流式细胞仪检测及Caspase-3的检测证实PIG11对HepG2细胞凋亡起负调控作用,Caspase-9检测显示PIG11诱导HepG2细胞凋亡可能通过线粒体途径。
OBJECTIVE: To construct the miRNA expression vector pcDNATM6.2-GW / EmGFPmiR of human PIG11 and to establish HepG2 cell line with stable low expression of PIG11 protein. HepG2 cell line was stably overexpressed with PIG11 protein and the effect of PIG11 gene on HepG2 cell apoptosis was explored. Methods: HepG2 cells were constructed by transfecting HepG2 cells with lipofectamine 2000. HepG2 cells stably expressing low level of PIG11 protein were constructed. Compared with HepG2 cell lines with stable and high expression of PIG11, which had been established previously, HepG2 cells were stained with PI staining flow cytometry The apoptosis of cells was detected. The expression of Caspase-3 protein and Caspase-9 protein was detected by Western blotting. Results: The miRNA expression vector pcDNATM6.2-GW / EmGFPmiR of human PIG11 was successfully constructed, and the HepG2 cell line with PIG11 gene silencing was obtained after transfection. PI staining showed that the apoptotic rate of HepG2 cells, miR-PIG11-HepG2 cells, miR-HepG2 cells, pLXSN-PIG11-HepG2 cells and pLXSN-HepG2 cells were 5.72 ± 0.81% 1.34 ± 0.71%, 5.10 ± 0.40%, 34.83 ± 2.29% and 5.34 ± 0.60%, respectively. The apoptosis rate of PIG11-overexpressed pLXSN-PIG-11-HepG2 cells was higher than that of other groups (P <0.01), and the apoptosis rate of PIG11-overexpressing miR-PIG11-HepG2 cells was decreased (P <0.01). The results of Western blotting showed that the expression of Caspase-3 and Caspase-9 in HepG2 cells with high PIG11 expression were up-regulated (P <0.01), while the expression of Caspase-3 and Caspase-9 in HepG2 cells with PIG11 low expression (P < 0.01). Conclusion: The construction of human PIG11 interference vector successfully obtained HepG2 cell line stably low expression of PIG11 protein. PI staining flow cytometry and Caspase-3 test confirmed that PIG11 negatively regulates HepG2 cell apoptosis, Caspase-9 test showed PIG11-induced HepG2 cell apoptosis may be through the mitochondrial pathway.