安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
ACTA UNIVERSITY MEDICINALIS ANHUI
2014年
3期
300-303,304
,共5页
张静%魏峰%吴忠东%王亭忠%倪雅娟%马爱群
張靜%魏峰%吳忠東%王亭忠%倪雅娟%馬愛群
장정%위봉%오충동%왕정충%예아연%마애군
心肌梗死%骨髓间充质干细胞%移植%心肌特异性标志物%MYH%Cx43%cTnI%α-actin
心肌梗死%骨髓間充質榦細胞%移植%心肌特異性標誌物%MYH%Cx43%cTnI%α-actin
심기경사%골수간충질간세포%이식%심기특이성표지물%MYH%Cx43%cTnI%α-actin
myocardial infarction%mesenchymal stem cell%transplantation%myocardial specific markers%MYH%Cx43%cTnI%α-actin
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、9 d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。
目的觀察大鼠同種異體骨髓間充質榦細胞( MSCs)在體內心肌缺血微環境中,嚮心肌細胞分化過程中主要心肌特異性標誌物的分化錶達及存活。方法採用左冠狀動脈前降支( LAD)結扎術建立大鼠急性心肌梗死( MI)模型( n=62),將綠色熒光蛋白(GFP)轉染標記的MSCs,于心外膜下註射到心肌梗死週邊區；免疫熒光染色觀察移植後3、5、7、9 d移植的MSCs的存活情況及其主要特異性標誌物( MYH、Cx43、cTnI、α-actin)蛋白的錶達；應用激光捕穫顯微切割技術分離心肌組織中GFP標記的MSCs群,應用Real-time PCR法測定其MYH、Cx43、cTnI、α-actin相對于未移植的MSCs的mRNA錶達,對移植後9 d的移植區域行TUNEL凋亡染色。結果免疫熒光染色觀察到移植的MSCs于移植後3 d開始持續錶達MYH、Cx43,于移植後5 d開始持續錶達cTnI,未觀察到α-actin 的錶達；Real-time PCR 法結果顯示,移植的MSCs上MYH于移植後5、7 d的錶達量較3 d的明顯增多(P<0.05),Cx43、cTnI于移植後7 d的錶達量較5 d的均增加(P<0.05),于移植後9 d MSCs幾乎觀察不到,TUNEL凋亡染色示移植的MSCs髮生瞭凋亡。結論移植的MSCs在大鼠體內心肌缺血微環境下嚮心肌細胞分化的過程中能夠錶達部分主要心肌特異性標誌物,但其不能長時間、有效的存活。
목적관찰대서동충이체골수간충질간세포( MSCs)재체내심기결혈미배경중,향심기세포분화과정중주요심기특이성표지물적분화표체급존활。방법채용좌관상동맥전강지( LAD)결찰술건립대서급성심기경사( MI)모형( n=62),장록색형광단백(GFP)전염표기적MSCs,우심외막하주사도심기경사주변구；면역형광염색관찰이식후3、5、7、9 d이식적MSCs적존활정황급기주요특이성표지물( MYH、Cx43、cTnI、α-actin)단백적표체；응용격광포획현미절할기술분리심기조직중GFP표기적MSCs군,응용Real-time PCR법측정기MYH、Cx43、cTnI、α-actin상대우미이식적MSCs적mRNA표체,대이식후9 d적이식구역행TUNEL조망염색。결과면역형광염색관찰도이식적MSCs우이식후3 d개시지속표체MYH、Cx43,우이식후5 d개시지속표체cTnI,미관찰도α-actin 적표체；Real-time PCR 법결과현시,이식적MSCs상MYH우이식후5、7 d적표체량교3 d적명현증다(P<0.05),Cx43、cTnI우이식후7 d적표체량교5 d적균증가(P<0.05),우이식후9 d MSCs궤호관찰불도,TUNEL조망염색시이식적MSCs발생료조망。결론이식적MSCs재대서체내심기결혈미배경하향심기세포분화적과정중능구표체부분주요심기특이성표지물,단기불능장시간、유효적존활。
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.
