线粒体钙单向转运体相关钙超载调控钙调蛋白对雨蛙肽诱导胰腺导管上皮细胞微丝骨架的影响

Effects of mitochondrial calcium uniporter associated calcium overload on actin cytoskeleton of caerulein-induced pancreatic ductal epithelial cells by regulating calmodulin

目的 通过体外细胞研究探讨线粒体钙单向转运体(MCU)相关钙超载通过调控钙调蛋白(CaM)对雨蛙肽诱导的胰腺导管上皮细胞微丝骨架的影响。方法 使用10^(-7)mol/L雨蛙肽及10^(-5)mol/L MCU抑制剂钌红(RR)干预人胰腺导管上皮细胞(HPDE6-C7)24 h,将细胞分为:Control组、雨蛙肽组、RR组、雨蛙肽+RR组。Western blot法检测细胞内MCU及CaM蛋白表达,荧光探针法检测细胞内游离Ca^(2+),免疫荧光双标法检测细胞内线粒体Ca^(2+),微丝染色法观察细胞微丝骨架结构。结果 雨蛙肽组与Control组比较,MCU及CaM蛋白表达、细胞质及线粒体内Ca^(2+)含量升高(P<0.05),细胞微丝骨架破坏;雨蛙肽+RR组与雨蛙肽组比较,MCU及CaM蛋白表达、胞质Ca^(2+)及线粒体内Ca^(2+)含量降低(P<0.05),细胞微丝骨架破坏减轻。结论 MCU相关钙超载可能通过激活CaM促进雨蛙肽诱导的胰腺导管上皮细胞微丝骨架的破坏。

Objective To investigate effects of mitochondrial calcium unidirectional transporter(MCU)associated calcium overload on actin cytoskeleton of caerulein(CAE)-induced pancreatic ductal epithelial cells by regulating calmodulin(CaM).Methods Human pancreatic duct epithelial cells(HPDE6-C7)were treated with CAE(10^(-7)mol/L)and ruthenium red(RR)(10^(-5)mol/L)for 24 h.The cells were divided into four groups:CAE,RR,CAE+RR and a control group.The expression of MCU and CaM was analyzed by Western blotting.Intracellular free calcium was detected by fluorescence probe.Mitochondrial calcium was evaluated by double immunofluorescent staining.Actin filaments stained with fluorescein isothiocyanate-phalloidin were observed.Results The expression of MCU and CaM,intracellular free calcium and mitochondrial calcium increased and microfilament cytoskeleton were impaired in the CAE group,compared with the control group(P<0.05).The expression of MCU and CaM,intracellular free calcium and mitochondrial calcium and the impairment of microfilament cytoskeleton were decreased in the CAE+RR group,compared with the CAE group(P<0.05).Conclusion MCU associated calcium overload may impair the actin cytoskeleton of CAE-induced pancreatic ductal epithelial cells by activating CaM.