Survivin-shRNA对视网膜母细胞瘤HXO-RB44细胞自噬及凋亡的影响

目的:研究Survivin-shRNA对视网膜母细胞瘤HXO-RB44细胞自噬及凋亡的影响。方法:培养视网膜母细胞瘤HXO-RB44细胞，构建Survivin-shRNA载体。按照处理不同分为Survivin-shRNA组、GFP组和CON组。分别采用流式细胞术检测Survivin-shRNA对HXO-RB44细胞凋亡和细胞周期的影响，应用MTT法检测Survivin-shRNA对HXO-RB44细胞活性的影响，Western blot法检测Survivin-shRNA对HXO-RB44 细胞自噬相关蛋白LC3、p62 与mTOR 表达的影响。结果:流式细胞术结果表明，与Control组和GFP组相比，Survivin-shRNA组细胞凋亡率增加，差异有统计学意义（P＜0.05）；随着作用时间的延长，S期出现阻滞，48 h阻滞最强，显著高于对照组（P＜0.05）。MTT结果发现Survivin-shRNA可抑制HXO-RB44细胞增殖活性，与Control组和GFP组相比，差异有统计学意义（P＜0.05）；Western blot结果发现，与对照组相比，LC3表达量显著增加（P＜0.01）；而mTOR表达量降低（P＜0.01）。结论:Survivin-shRNA可促进视网膜母细胞瘤HXO-RB44细胞的凋亡和自噬水平，进而特异性杀伤肿瘤细胞。

Effects of Survivin-shRNA on autophagy and apoptosis of retinoblastoma HXO-RB44 cell

Objective:To investigate the effects of survivin-shRNA on the autophagy and apoptosis of HXO-RB44 cell.Methods:HXO-RB44 cells were cultured in DMEM medium and recombinant adenovirus vector Survivin-shRNA and negative control rAd5-GFP were transfected into HXO-RB44 cells.The clone in which the rAd5-Survivin-shRNA virus vectors transfected was named as Survivin-shRNA group.The negative control vectors transfected was named as GFP group and HXO-RB44 cells were named as CON group.Flow cytometry was used to detect cell apoptosis and cell cycle.MTT assay was used to detect cell proliferation inhibition rate.Western blot assay was used to determine the protein expression level of LC3,p62 and mTOR.Results:After Survivin-shRNA was transfected into HXO-RB44 cells,it was found that knockdown of Survivin markedly increased the apoptotic index of HXO-RB44 cells compared with GFP group and CON group indicated by flow cytometry.Flow cytometry analysis showed that the cell cycle of HXO-RB44 cells was blocked at S phase,which was most significant at 48 h and significantly higher than that in control group（P＜0.05）.Knockdown of Survivin could significantly reduce the proliferative activities of cells compared with GFP group and CON group.Western blot assays performed that the protein expression level of mTOR was decreased,while LC3 expression was increased in treated group compared with the CON group.Conclusion:Survivin-shRNA could promote the autophagy and apoptosis of HXO-RB44 cell and then specifically kill tumor cells.