Apoptin基因诱导体外培养的人眼球筋膜囊成纤维细胞凋亡的初步研究

Acceleration of Apoptosis by Transfection of Apoptin Gene in Human Tenon’s Capsule Fibroblasts in Vitro

目的观察基因Apoptin对人眼球筋膜囊成纤维细胞的杀伤效果,探讨其可能的机制。方法用脂质体将Apoptin基因导入人眼球筋膜囊成纤维细胞,通过RT-PCR法检测ApoptinmRNA的表达,同时用SABC免疫组化法分析Apoptin和p53的表达。采用细胞计数法检测细胞生长抑制率,TUNEL法标记凋亡细胞,流式细胞仪检测细胞周期的变化。结果转入Apoptin基因后,人眼球筋膜囊成纤维细胞的生长明显受抑制(P<0.05)。细胞周期分析可见凋亡峰,凋亡率35%。细胞中可见Apoptin阳性表达(P<0.05),p53表达无显著性差异(P>0.05)。凋亡细胞在荧光显微镜下形成凋亡小体。结论转入Apoptin基因可显著促进人眼球筋膜囊成纤维细胞的凋亡,Apoptin诱导的凋亡不依赖功能性p53的生成。此技术可能是抑制抗青光眼术后滤过道瘢痕化的新手段。

Objective To observe the effects of Apoptin gene on killing human Tenon＇s capsule fibroblasts in vitro and illustrates its mechanisms. Methods Apoptin gene was transfected into the cultured human Tenon＇s capsule fibroblasts by liposome. The mRNA of Apoptin were detected by RT-PCR. The protein of Apoptin and p53 were detected by SABC immunohistochemistry. The growth rate of the cultured human Tenon＇s capsule fibroblasts was studied by constructing the growth curve and TUNEL, cell apoptosis was observed by flow cytometry. Results The expression of Apoptin mRNA was positive in EJ/Apoptin cells （P〈0.05）. Apoptin protein expression of EJ/Apoptin cells was positive and p53 protein expression was nit increased （P〉0.05）. The growth of the cultured human Tenon＇s capsule fibroblasts was significantly inhibited after bak gene was transfected （P〈0.05）. Apoptosis cells increased significantly. The apoptosis rate was 35%. Conclusion Apoptin gene could inhibit the growth of the cultured human Tenon＇s capsule fibroblasts effectively. Its mechanisms was independent of p53 gene. The experiment may offer a new alternative to post-operational management of scar tissue fomation.