氯化锂对人Tenon囊成纤维细胞增生抑制作用的机制研究

Studies on the mechanism of inhibitory effect of lithium chloride on the proliferation of human Tenon capsule fibroblasts

目的探讨氯化锂对人Tenon囊成纤维细胞(HTFs)缝隙连接细胞间通讯(GJIC)的影响及其可能的作用机制。方法收集2019年4月于德州市人民医院眼科行斜视手术的1例患者的Tenon囊组织,剪成1 mmxl mmX1 mm的组织块,进行原代培养并传代,取第4代HTFs进行实验。将HTFs分为对照组和氯化锂处理组,对照组加入不含氯化锂的细胞培养基,氯化锂处理组加入含80 mmol/L氯化锂的细胞培养基,继续培养48 h。采用细胞划痕染料标记示踪法标记偶联指数,评估GJIC功能;采用细胞免疫荧光法检测HTFs中Cx43的表达和定位;采用实时荧光定量PCR和Western blot法检测HTFs中Cx43 mRNA及蛋白的表达水平。结果培养的细胞呈长梭形并呈单层放射状或涡旋状贴壁生长,细胞质vimentin染色呈阳性。细胞划痕染料示踪实验结果显示,氯化锂处理组细胞偶联指数为9.04±0,53,明显高于对照组的4.94±0,39,差异有统计学意义(t=-18.79,P<0.01)。免疫荧光染色结果显示,对照组可见Cx43荧光呈点状分布于细胞相连处的细胞膜上,氯化锂处理组Cx43染色明显增强。实时荧光定量PCR结果显示,对照组Cx43 mRNA相对表达量设为1,氯化锂处理组Cx43 mRNA相对表达量明显升高,为1.97±0,23,差异有统计学意义(t=-14.426,P<0.01)。Western blot检测结果显示,氯化锂处理组Cx43蛋白相对表达量为0.871±0,057,明显高于对照组的0.446±0.028,差异有统计学意义(t=-11.682,P<0.01)o结论氯化锂可上调HTFs中Cx43 mRNA及蛋白表达并增强HTFs间GJIC功能,提示氯化锂增强GJIC的功能可能是其抑制HTFs增生的机制之一。

Objective To investigate the effect of lithium chloride(LiCl)on the gap junctional intercellular communication(GJIC)in human Tenon capsule fibroblasts(HTFs)and its underlying mechanism.Methods The Tenon capsule tissue of a patient who underwent strabismus surgery in Dezhou People's Hospital in April 2019 was collected and cut into tissue blocks of dimensions 1 mmX 1 mmX 1 mm.Primary culture and subculture were carried out,and the 4th-generation HTFs were taken for experiment.HTFs were divided into the control group and LiCl treatment group and were cultured with cell medium without or with 80 mmol/L LiCl for another 48 hours according to grouping.The cell scratch and dye labeling technique were used to label the coupling index and evaluate the GJIC function.The expression and localization of Cx43 in HTFs were detected by immunofluorescence staining.The expression levels of Cx43 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot,respectively.The study protocol was approved by an Ethics Committee of Dezhou People's Hospital(No.2019-023).Written informed consent was obtained from the subject.Results The cultured spindle-shaped HTFs grew adhering to the wall showing radial monolayer or vortexlike,and the cytoplasm was vimentin positive.Results of dye tracer experiment of cell scratch showed that the cell coupling index of LiCl treatment group was 9.04±0.53,which was significantly higher than 4.94±0.39 of the control group(t=-18.79,P<0.01).Immunofluorescence staining showed that the Cx43 fluorescence was dotted in the cell membrane between adjacent cells in the control group,and Cx43 staining was obviously enhanced in the LiCl treatment group.The results of real-time fluorescence quantitative PCR showed that with relative expression level of Cx43 mRNA in the control group set to 1,the relative expression level of Cx43 in the LiCl treatment group was significantly increased to 1.97±0.23,showing a statistical significance between them(t=-14.426,P<0.01).Western blot showed that the relative expression level of Cx43 protein was 0.871±0.057 in the LiCl treatment group,which was significantly higher than 0.446±0.028 in the control group(t=-11.682,P<0.01).Conclusions LiCl can enhance the GJIC function between HTFs by upregulating the expression levels of Cx43 mRNA and protein,suggesting that the enhanced GJIC function by LiCl may be one of the mechanisms of its inhibition on HTFs proliferation.