毒胡萝卜素对小鼠肺成纤维细胞内钙离子水平及caspase-3蛋白表达的影响

Effects of Thapsigargin on Intracellular Ca^(2+) and Caspase-3 Protein Expression in Mouse Lung Fibroblast

目的探讨钙泵抑制剂毒胡萝卜素(thapsigargin,TG)对体外小鼠肺成纤维细胞内钙离子(Ca^(2+))水平及凋亡相关蛋白含半胱氨酸的天冬氨酸蛋白水解酶-3(caspase-3)表达的影响。方法小鼠肺成纤维细胞(L929)分为3组:空白对照组(不做处理)、转化生长因子β_1(TGF-β_1)组、TG组,后两组均用5ng/mL TGF-β_1诱导24h,TG组在诱导后再用4μmol/L TG作用24h。细胞处理48h后,分别收集各组细胞,采用透射电子显微镜观察细胞超微结构变化,激光共聚焦显微镜观察各组细胞内Ca^(2+)水平,免疫细胞化学方法检测caspase-3蛋白表达。结果透射电子显微镜示,空白对照组细胞凋亡较少,TGF-β_1组无明显凋亡细胞,TG组有大量成纤维细胞凋亡。TGF-β_1组Ca^(2+)水平和caspase-3蛋白水平均低于空白对照组(P<0.05),TG组细胞内Ca^(2+)水平和caspase-3蛋白水平均较TGF-β_1组、空白对照组升高(P<0.05)。结论TG可使小鼠肺成纤维细胞细胞内钙稳态失衡,增加caspase-3蛋白表达,促进细胞凋亡。

Objective To explore the effects of calcium pump inhibitor thapsigargin（TG）on intracellular Ca^2＋ and the expression of caspase-3 protein in mouse lung fibroblast.Methods Mice lung fibroblast cells were divided into three groups：the blank control group,transforming growth factor-β1（TGF-β1）group and TG treated group.The cells were induced by 5 ng/mL TGF-β1 for 24 hin TGF-β1 group and TG group,4μmol/L TG was added in TG group for 24 h.After 48 h,the cells were collected,and the cell structure was observed by transmission electron microscope,intracellular Ca^2＋ level was detected with laser confocal microscope,the protein expression of caspase-3 was examined using immunohistochemistry method.Results Transmission electron microscopy showed that the cells in the blank control group had obvious nucleolus,complete organelles and less apoptosis.In TGF-β1 group,the cell morphology was intact,chromatin was evenly distributed,and no apoptotic cells were found.In TG group,there were a large number of apoptosis of fibroblasts,chromatin clumps in nuclei and a small amount of collagen fibers.The level of Ca^2＋ in TGF-β1 group was significantly lower than that in control group（P〈0.05）,and which in TG group was significantly higher（P〈0.05）.The protein expression of caspase-3 in TGF-β1 group were significantly lower than that in blank control group（P〈0.05）,which in TG group increased obviously（P〈0.05）.Conclusion TG could cause intracellular calcium dysregulation in mouse lung fibroblasts,increase caspase-3 protein expression and promote cell apoptosis.