HSV-tk/GCV系统对人Tenon囊成纤维细胞的旁观者效应及其机制研究

Bystander effect of HSV-tk/GCV system on human Tenon's capsule fibroblasts and its mechanism

目的观察HSV-tk/GCV系统对人Tenon囊成纤维细胞(HTFs)的旁观者效应并研究其作用机制。方法将转HSV-tk基因HTFs(HTFs-tk)与正常HTFs于两种接种密度下以不同比例混合,加入5μg/m l更昔洛韦作用5 d,MTT法检测对HTFs的杀伤作用;染料传输法观察HTFs细胞间隙连接细胞间通讯(G JIC)的能力;免疫细胞化学检测Cx43分布;RT-PCR及W estern b lot检测Cx43表达。结果旁观者效应随HTFs-tk/HTFs比例增加而增强,高细胞接种密度组明显强于低密度组;染料通过G JIC传输6级以上;Cx43主要分布于HTFs细胞膜并检测到其mRNA与蛋白表达。结论旁观者效应需要细胞间密切接触,Cx43介导G JIC可能是HSV-TK/GCV系统对HTFs产生旁观者效应的主要机制,HTFs表达Cx43可能在结膜伤口愈合反应中发挥重要作用。

Objective Suicide gene/prodrug system has been used to treating proliferative ocular disease,and bystander effect is one of key mechanisms, The present paper was focused to bystander effect of herpes simplex virus thymidine kinase/ ganciclovir system（HSV-tk/GCV） on human Tenon＇ s capsule fibroblasts, Methods Human Tenon＇ s capsule fibroblasts transferred by HSV-tk gene （HTFs-tk） and normal HTFs cells were mixed at a different ratio and seeded at two cell densities,and these cells were exposed to 5 μg/ml ganciclovir （GCV） for 5 days, Bystander effect was measured by MTT methods. The function of gap junction intercellular communication （GJIC） of HTFs cells was detected by scrape-loading lucifer yellow dye transfer method. The distribution and expression of connexin43 （Cx43） was assessed by immunocytochemistry,RT-PCR and Western blot separately. Results Bystander effect was increased with the increasing ratio of HTFs-tk to HTFs,and nearly 80% of HTFs cells were killed when ratio of HTFs-tk to HTFs was 60% in the lower cells density group and all HTFs cells were killed in the higher cells density group with the ratio of 40%, The transporting degree of lucifer yellow was more than 6 grades through GJIC in HTFs cells. Cx43 was stained into brown and mainly distributed on cell membrane by immunocytochemistry. The mRNA and protein expressions of Cx43 were also detected, Conclusion Bystander effect needs close intercellular contact. Cx43-mediated GJIC may be the important mechanism of bystander effect of HSV-tk/GCV system on HTFs cells in vitro. The expression of Cx43 in HTFs cells may play an important role during the wound healing process of conjunctiva. It could be a novel approach to improvement of filtering surgery by modulating Cx43 expression in vivo.