大鼠骨髓间充质干细胞在心肌缺血微环境中Cav1.2的表达

The expression of Cav1.2 in rat allogeneic mesenchymal stem cells in the microenvironment of myocardial ischemia

目的观察大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)在心肌缺血微环境中向心肌细胞分化过程中Cav1.2的分化表达。方法采用左冠状动脉前降支(left anterior descending coronary artery,LAD)结扎术建立大鼠急性心肌梗死(myocardial infarction,MI)模型(n=52),并行心电图检查。将绿色荧光蛋白(green fluorescent protein,GFP)标记MSCs,贴壁培养,用心外膜下注射法将GFP标记的MSCs移植到心肌梗死周边区域;移植后7 d开胸取心脏组织,HE染色观心肌梗死区域病理变化。将移植后3,5,7 d的大鼠分为3 d组、5 d组、7 d组,每组10只动物,免疫荧光染色观察各组移植的MSCs上Cav1.2蛋白的表达;激光捕获显微切割技术分离各组心肌组织中GFP标记的MSCs群,应用real-time PCR测定各组心肌组织中Cav1.2及未移植大鼠MSCs mRNA的相对表达量;移植后9 d,对移植区域行TUNEL凋亡染色。结果免疫荧光染色观察3 d组、5 d组、7 d组心肌组织中移植的MSCs均不表达Cav1.2;real-time PCR测定显示,3 d组、5 d组、7 d组中Cav1.2相对表达量与未移植的MSCs相比均无明显变化(P>0.05),移植后9 d未观察到MSCs的表达,TUNEL凋亡染色显示移植的MSCs发生了凋亡。结论在大鼠心肌缺血微环境中移植的MSCs不表达Cav1.2且不能长期存活。

Objective To observe the expression of Cav1. 2 of allogeneic mesenchymal stem cells（ MSCs） in microenvironment of rat myocardial ischemia in vivo. Methods The myocardial infarction（ MI） rat models were established by ligating the left anterior descending coronary artery（ LAD）. The MSCs were labeled by green fluorescent protein（ GFP） and cultured using direct adherent culture methods. GFP-labeled MSCs were injected into the surrounding area of the epicardium myocardial infarction（ n = 52）. Then the gross specimens were observed and HE staining was performed on infarct areas after transplantation for 7 d. The rats were divided into three groups according to transplantation time： 3 d group,5 d group and 7 d group. The protein expression of Cav1. 2 in each group was observed by immunofluorescence staining,and the relative mRNA was acquired by laser capture microdissection and measured by realtime PCR. The transplanted areas were observed by TUNEL staining at day 9. Results The transplanted MSCs did not express Cav1. 2 at day 3,5,7. Compared with non-transplanted MSCs,the relative expression of Cav1. 2 showed no significant changes at day3,5,7 by real-time PCR in the transplanted MSCs（ P 〉0. 05）,and the expression of Cav1. 2 was not observed at day 9 by TUNEL staining,but there was apoptosis in the transplanted MSCs. Conclusion The transplanted allogeneic MSCs could not express Cav1. 2 in the microenvironment of myocardial ischemia,and could not effectively survive for a long time.