人眼Tenon囊成纤维细胞缝隙连接细胞间通讯的检测

Detection of gap junctional intercellular communication in cultured human Tenon's capsule fibroblasts

目的:观察体外培养的人眼Tenon囊成纤维细胞(human Tenon's capsule fibroblasts,HTFs)缝隙连接细胞间通讯(gap junctional intercellular communication,GJIC)功能及其构成蛋白connexin43(Cx43)的表达及分布。方法:锐刀划痕罗氏黄(lucifer yellow)荧光染料传输法观察GJIC功能;RT-PCR及Western blot分别检测Cx43 mRNA及蛋白表达;免疫细胞化学方法检测Cx43的分布。结果:荧光染料自划痕线向邻近未损伤细胞传输达6级细胞以上,同时检测到Cx43 mRNA与蛋白的表达;免疫细胞化学法观察到Cx43分布于细胞膜。结论:培养的HTFs具有较强的GJIC功能,通过Cx43介导GJIC赋予细胞能够相互之间传递信息的能力,提示在创伤愈合过程中GJIC对成纤维细胞功能同步化方面发挥重要作用。

AIM：To explore gap junctional intercellular communication（GJIC） in cultured human Tenon＇s capsule fibroblasts（HTFs） and detect the expression and distribution of connexin43（Cx43）.METHODS：GJIC were demonstrated by scrape-loading and Lucifer yellow dye transfer methods.RT-PCR and Western blot were carried out to detect mRNA and protein expression of Cx43.Immunocytochemistry was performed to observe the distribution of Cx43.RESULTS：Cultured HTFs demonstrated functional GJIC in vitro.The transporting degree of Lucifer yellow dye was more than 6 grades.The mRNA and protein expression of Cx43 were also detected.Cx43 were located on cells＇ membrane by immunocytochemistry.CONCLUSION：Cultured HTFs have GJIC function which confer these cells the ability to establish and maintain intercellular communication mediated by Cx43.This property may be critical in maintaining function synchronization of fibroblasts during wound-healing process.