摘要
目的探讨血管生成素样蛋白3（ANGPTL3）对足细胞骨架重排的影响，及ANGPTL3引起的足细胞骨架重排是否主要通过整合素B3信号通路。方法体外培养永生化小鼠足细胞，分为6组：正常足细胞（WT）组；空质粒转染（MOCK）组；MOCK＋阿霉素（ADR）组；ANGPTL3质粒转染（ANGPTL3-cDNA）组；敲低ANGPTL3表达（miRNA）组；miRNA＋ADR组。用鬼笔环肽抗F肌动蛋白（F-actin）单克隆抗体行足细胞骨架蛋白F-actin染色，激光共聚焦显微镜下观察骨架重排和F-actin蛋白相对表达量变化。整合素B3单克隆抗体（CD61）特异性封闭足细胞表面整合素p3，观察足细胞骨架重排改变。Western印迹法检测足细胞整合素p3信号通路因子黏着斑激酶（FAK）和p-FAK表达改变。结果（1）激光共聚焦显微镜下观察结果显示，正常足细胞骨架肌纤维排列有序，荧光显示清晰；ADR组足细胞突触结构消失，骨架排列紊乱，荧光明显模糊减弱，组间比较差异有统计学意义（P〈0．01）。与MOCK组比较，miRNA组细胞骨架结构及F-actin相对表达量的差异无统计学意义；ANGPTL3-cDNA组细胞骨架明显重排、F-aetin表达量下降（P〈0．01）。与ADR组比较，ADR＋miRNA组细胞骨架重排的细胞比例和F-actin荧光强度均明显得到改善（均P〈0．01）。（2）CD61封闭足细胞整合素B3后，再给予ANGPTL3质粒转染，足细胞骨架重排比率较未封闭组明显改善（P〈0．01）。（3）高表达ANGPTL3组足细胞p-FAK的表达量较MOCK组明显升高（P〈0．01）。结论ANGPTL3是阿霉素诱导的足细胞骨架重排的关键分子，整合素B3是ANGPTL3诱导足细胞损伤的主要信号通路。
Objective To explore whether Angiopoietin-like protein 3 （ANGPTL3） is involved in podoeyte aetin rearrangement, and to analyze whether integrin β3 signal pathway is a key in ANGPTL3 inducing aetin rearrangement. Methods The cultured podocytes were divided into six groups： wild type, ADR treated, ADR＋Dex, MOCK, ANGPTL3- eDNA, miRNA, and AD ＋miRNA group. （1） We observed actin cytoskeleton using Invitrogen reagents with confocal microscopy; （2） Actin cytoskeleton after blocking 133 on podocytes was; （3） The expression of total FAK and p- FAK was through Western blotting. Results （1） The wild type podocyte＇s cytoskeleton is arranged orderly. After ADR treatment, podoeyte＇s actin are rearranged and weaken （P 〈 0.05）. There was no significant difference in actin arrangement between knock-down and MOCK group. In ANGPTL3-cDNA group the podocyte actin was also significantly rearranged; on the contrary, in miRNA ＋ ADR group, the actin rearrangement never obviously happened （P 〈 0.05）. （2） Over- expression of ANGPTL3 podocytes blocked integrin β3 did not happen actin rearrangement. （3） The expression of p- FAK significantly increased in over- expression ANGPTL3 podocytes. Conclusion ANGPTL3 is a key in inducing actin rearrangement. Intergrin β3 maybe a central pathway in ANGPTL3＂s role with podocytes.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2017年第1期43-47,共5页
Chinese Journal of Nephrology
基金
国家自然科学基金（81360114）
