摘要
目的观察大鼠同种异体骨髓间充质干细胞(MSCs)在体内心肌缺血微环境中,向心肌细胞分化过程中主要心肌特异性标志物的分化表达及存活。方法采用左冠状动脉前降支(LAD)结扎术建立大鼠急性心肌梗死(MI)模型(n=62),将绿色荧光蛋白(GFP)转染标记的MSCs,于心外膜下注射到心肌梗死周边区;免疫荧光染色观察移植后3、5、7、9d移植的MSCs的存活情况及其主要特异性标志物(MYH、Cx43、cTnI、α-actin)蛋白的表达;应用激光捕获显微切割技术分离心肌组织中GFP标记的MSCs群,应用Real-time PCR法测定其MYH、Cx43、cTnI、α-actin相对于未移植的MSCs的mRNA表达,对移植后9 d的移植区域行TUNEL凋亡染色。结果免疫荧光染色观察到移植的MSCs于移植后3 d开始持续表达MYH、Cx43,于移植后5 d开始持续表达cTnI,未观察到α-actin的表达;Real-time PCR法结果显示,移植的MSCs上MYH于移植后5、7 d的表达量较3 d的明显增多(P<0.05),Cx43、cTnI于移植后7 d的表达量较5 d的均增加(P<0.05),于移植后9 d MSCs几乎观察不到,TUNEL凋亡染色示移植的MSCs发生了凋亡。结论移植的MSCs在大鼠体内心肌缺血微环境下向心肌细胞分化的过程中能够表达部分主要心肌特异性标志物,但其不能长时间、有效的存活。
Objective To observe the differentiation and survival of the main myocardial specific markers of mesen-chymal stem cells （ MSCs） in microenvironment of rat myocardial ischemia in vivo. Methods The myocardial in-farction（ MI） models of rats were established by ligating the left anterior descending coronary artery. The MSCs la-beled by green fluorescent protein（ GFP） were injected into the surrounding area of the epicardium myocardial in-farction （n=62）;the protein expression of the main myocardial specific markers （cTnI, MYH, Cx43, α-actin） on the transplanted MSCs was observed by immunofluorescence staining at day 3 , day 5 , day 7 and day 9;the cor-responding mRNA related to the non-transplanted MSCs was acquired by laser capture microdissection and was measured by real-time PCR. Then, the transplanted area was observed by TUNEL staining. Results Immunofluo-rescence staining and real-time PCR indicated MYH were detected at day 3 , significantly lower than day 5 and day 7;Cx43 were indicated at day 3 , and at day 7 higher than day 5;cTnI was found at day 5 , decreased compared to day 7 significantly（ P〈0.05 ）; while α-actin was not found; the transplanted MSCs which shared morphological features known from apoptotic cells by TUNEL staining decreased gradually and disappeared at day 9 . Conclusion The transplanted MSCs could express some main myocardial specific markers in the microenvironment of myocar-dial infarction, but the differentiated cardiomyocytes were immature, and could not survive for a long time effective-ly.
出处
《安徽医科大学学报》
CAS
北大核心
2014年第3期300-304,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:30800455)
