慢病毒载体介导PAX6沉默引起HLE-B3细胞凋亡

Lentiviral vector-mediated PAX6 silence induce human lens epithelial cell apoptosis

目的:观察PAX6基因沉默后人晶状体上皮细胞系(HLE-B3)增殖的改变。方法:分别将4组PAX6 shRNA慢病毒载体及对照组慢病毒(pGCL-GFP-shRP1,2,3,4,NC)感染B3细胞;感染96h后,利用Real-time PCR和Western-blot检测B3细胞PAX6表达以确定PAX6沉默;选取有效沉默PAX6基因的pGCL-GFP-shRP4感染B3细胞,MOI=10,观察细胞数量变化。感染48h后采用流式细胞仪检测B3细胞凋亡情况。结果:感染RNAi病毒组的B3细胞,随感染时间延长,细胞数量减少。流式细胞仪检测结果显示沉默PAX648h的B3细胞,G1/G0期前出现凋亡峰。结论:慢病毒介导的RNA干扰能有效沉默B3细胞的PAX6表达;PAX6基因沉默后B3细胞凋亡。

AIM： To observe human lens epithelial cells proliferation changes when the PAX6 gene knocked-down. （HLE-B3） has been METHODS： Four groups PAX6 shRNA lentiviral vectors and the control group lentiviral （ pGCL-GFP-shRP1,2, 3, 4, NC） infect B3 cells. Real Time PCR and Western blot analysis was used to determine PAX6 silence after infection 96 hours. Effective PAX6-silence pGCL-GFP- shRP4 was selected to infect B3 cells, MOI （ Multiplicity of infection） = 10 and the changes were observed in the number of the cells. The B3 cells apoptosis was detect by flow cytometry after 48 hours in infection. .RESULTS： RNAi-virus-infected B3 cells decreased in the number of cells with infection prolonged. The flow cytometry results showed that after knock- down the PAX6 for 48 hours, apoptotic peak appeared before the G1/G0 phase. CONCLUSION ： Lentivirus- mediated RNA interference can effectively silence PAX6 expression in B3 cells. There is HLE-B3 apoptosis after PAX6 aene knock-down.