【摘要】： 目的 探討增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞的可行性,明確經(jīng)蛋白標(biāo)記后的間充質(zhì)干細(xì)胞(mesenchymal stem cell,MSC)是否可以穩(wěn)定生長并被誘導(dǎo)成為心肌細(xì)胞以及心肌微環(huán)境對轉(zhuǎn)染MSC在體外向心肌樣細(xì)胞分化的影響,為進(jìn)一步做標(biāo)記的MSC體內(nèi)移植研究奠定基礎(chǔ)。 方法 1、提取、鑒定pEGFP質(zhì)粒; 2、大鼠骨髓間充質(zhì)干細(xì)胞的的分離和培養(yǎng); 3、新生乳鼠心肌細(xì)胞的分離和培養(yǎng); 4、制備心肌細(xì)胞裂解液; 5、EGFP基因轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞; 6、篩選陽性轉(zhuǎn)染細(xì)胞后在熒光顯微鏡觀察,然后對比建系,并體外誘導(dǎo)其向心肌樣細(xì)胞轉(zhuǎn)化; 7、把誘導(dǎo)的細(xì)胞做免疫組化檢測心臟特異性肌鈣蛋白T(cTnT)、α-肌動蛋白、連接蛋白43及CD31的表達(dá)情況,通過計算機(jī)分析系統(tǒng)測定其陽性率。 結(jié)果 原代MSC多呈紡錘形或梭形,有聚集生長的傾向,3～5個細(xì)胞成為一個集落。傳代后,細(xì)胞變?yōu)樾螒B(tài)均一的排列有序的成纖維細(xì)胞樣,長梭形,胞漿突起較少,胞體輪廓不甚清晰,胞體也相對較大;在脂質(zhì)體介導(dǎo)下將EGFP基因?qū)隡SC,在熒光顯微鏡下觀察,細(xì)胞的生長和增殖不受影響。增強(qiáng)型綠色熒光蛋白在基因轉(zhuǎn)染12h后開始表達(dá),48～72h達(dá)高峰。在1周內(nèi)有較強(qiáng)的表達(dá)。1周后表達(dá)綠色熒光的細(xì)胞逐漸減少,到4周時仍可見少量散在的熒光。瞬時轉(zhuǎn)染率達(dá)20%～30%。質(zhì)粒和脂質(zhì)體的濃度比例在1:2到1:3轉(zhuǎn)染效率最高。 結(jié)論 綠色熒光蛋白可成功轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞;轉(zhuǎn)染效率與脂質(zhì)體和質(zhì)粒濃度有關(guān);轉(zhuǎn)染后骨髓間充質(zhì)干細(xì)胞生長良好,在體外可以成功誘導(dǎo)成為心肌樣細(xì)胞。
[Abstract]:Objective to investigate the feasibility of enhanced green fluorescent protein (enhanced green fluorescent protein,EGFP (enhanced green fluorescent protein (mesenchymal stem cell,) transfer into bone marrow mesenchymal stem cells (MSCs). Whether MSC) can grow stably and be induced into cardiomyocytes and the effect of myocardial microenvironment on the differentiation of MSC into cardiomyocytes in vitro lays a foundation for the further study of in vivo transplantation of labeled MSC. Methods 1, pEGFP plasmid was extracted and identified, 2, isolation and culture of rat bone marrow mesenchymal stem cells, 3, isolation and culture of neonatal rat cardiomyocytes, 2, isolation and culture of rat bone marrow mesenchymal stem cells, 3, isolation and culture of neonatal rat cardiomyocytes. 4. Cardiomyocyte lysate was prepared. 5, EGFP gene was transferred into bone marrow mesenchymal stem cells. 6. The positive cells were screened and observed by fluorescence microscope, and then the lines were compared and induced to transform into cardiac muscle-like cells in vitro. 7. The expression of cardiac specific troponin T (cTnT), 偽-actin, junction protein 43 and CD31 was detected by immunohistochemistry, and the positive rate of cardiac specific troponin CD31 was measured by computer analysis system. Results most of the primary MSC were fusiform or fusiform, with the tendency of aggregation and growth. 3 / 5 cells became a colony. After passage, the cells became homogeneously arranged fibroblasts, long fusiform, less cytoplasm protrusions, the outline of the cell body was not very clear, and the cell body was relatively large. The introduction of EGFP gene into MSC, mediated by liposomes was observed under fluorescence microscope, and the growth and proliferation of the cells were not affected. The expression of enhanced green fluorescent protein began 12 hours after gene transfer and reached the peak at 48 h and 72 h. There was strong expression within 1 week. After 1 week, the number of cells expressing green fluorescence decreased gradually, and a small amount of scattered fluorescence was still observed at 4 weeks. The instantaneous dye transfer rate was 20% 鈮，

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