抑制囊泡转运对大鼠内皮祖细胞增殖及钙库操纵性钙内流的影响  

Influences of vesicular transport inhibition on proliferation and storeoperated calcium entry in rat endothelial progenitor cells

作　　者：张弢[1] 王彦伟[1] 杨杰[1] 袁方正圆 张继航[1] 黄岚[1] 

机构地区：[1]第三军医大学新桥医院全军心血管病研究所,重庆400037

出　　处：《中国病理生理杂志》2016年第4期591-596,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81370211)

摘　　要：目的:研究囊泡转运在大鼠内皮祖细胞(EPCs)增殖及钙库操纵性钙内流(SOCE)调控中的作用。方法:密度梯度离心法分离获取、并用ac LDL-Di I和FITC-UEA-I荧光双染鉴定脾源性EPCs。布雷菲德菌素A(BFA)抑制囊泡转运,CCK-8法和实时无标记细胞功能分析仪观察EPCs增殖变化,流式细胞术检测凋亡情况,并检测囊泡转运关键蛋白ADP核糖基化因子GTP酶活化蛋白1(ARFGAP1)的表达。激光共聚焦显微镜观察囊泡转运抑制后SOCE并用Western blot检测SOCC复合体蛋白表达,进一步采用RNA干扰的方式观察囊泡转运对瞬时受体电位通道1(TRPC1)蛋白表达及SOCE的影响。结果:双染鉴定大鼠脾源性EPCs阳性率为82.53%±6.12%。BFA显著抑制EPCs的增殖但对凋亡无明显影响,同时ARFGAP1表达也明显降低,说明EPCs的囊泡转运受到抑制。抑制EPCs囊泡转运显著下调TRPC1的表达,并降低SOCE。si TRPC1使EPCs的SOCE下降,但siTRPC1预处理并抑制囊泡转运并没有使EPCs的SOCE进一步降低。结论:抑制EPCs的囊泡转运可以抑制EPCs的增殖并通过下调TRPC1的表达降低SOCE水平。AIM： To investigate the effects of vesicular transport inhibition on the proliferation and regulation of store-operated calcium entry（ SOCE） in rat endothelial progenitor cells（ EPCs）. METHODS： EPCs were isolated from the rats with density-gradient centrifugation and confirmed via double fluorescence staining with ac LDL-Di I and FITC-UEAI. After inhibition of vesicular transport with brefeldin A（ BFA）,the proliferation of EPCs was measured by CCK-8 assay and real-time cell analyzer instrument,apoptosis was analyzed by flow cytometry,and the expression of ADP-ribosylation factor GTPase-activating protein 1（ ARFGAP1）,a key protein to vesicular transport,was also detected. SOCE was observed under laser scanning confocal microscope after the vesicular transport was inhibited,and the protein expression of SOCC complex was determined by Western blot. Furthermore,the influences of vesicular transport inhibition on the expression of transient receptor potential channel 1（ TRPC1） and SOCE were examined with a RNA interference method. RESULTS： The ac LDL-Di I and FITC-UEA-I double positive rate of the cells was 82. 53% ± 6. 12%. BFA insult significantly inhibited the proliferation of EPCs and down-regulated the expression of ARFGAP1,and no influence on the apoptosis of the EPCs was observed,suggesting that vesicular transport of EPCs was inhibited. Vesicular transport inhibition remarkably down-regulated the expression of TRPC1 and decreased SOCE level. No evident difference in the level of SOCE between si TRPC1 group and si TRPC1 ＋ BFA group,in which the cells were pretreated with si TRPC1 before BFA addition,was observed. CONCLUSION： Vesicular transport inhibition in EPCs reduces the proliferation of EPCs and decreases SOCE level through down-regulation of TRPC1.

关 键 词：内皮祖细胞 囊泡转运 瞬时受体电位通道1 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学]