本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：基因轉(zhuǎn)染　出處：《中國(guó)矯形外科雜志》2013年21期 　論文類(lèi)型：期刊論文

【摘要】：[目的]研究慢病毒載體(lentiviral vectors,LVs)介導(dǎo)增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,eGFP)標(biāo)記大鼠骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)的可行性及對(duì)細(xì)胞生物學(xué)特性的影響。[方法]采用密度梯度離心法分離培養(yǎng)大鼠BMSCs,經(jīng)Lenti-emGFP/hygro慢病毒載體轉(zhuǎn)染和潮霉素藥篩,鏡下觀察細(xì)胞形態(tài)和eGFP表達(dá)情況,MTT法檢查轉(zhuǎn)染前后細(xì)胞增殖活力,流式細(xì)胞儀檢測(cè)細(xì)胞表面標(biāo)記物(CD34、CD44、CD45、CD90)和eGFP,檢測(cè)轉(zhuǎn)染后細(xì)胞的成骨、成脂分化潛能并分別行茜素紅染、油紅O染色鑒定。[結(jié)果]大鼠BMSCs呈成纖維細(xì)胞樣形態(tài),漩渦樣狀排列,轉(zhuǎn)染后細(xì)胞形態(tài)不變,并高效表達(dá)eGFP,兩組細(xì)胞增殖活力一致,流式細(xì)胞儀檢測(cè)顯示,轉(zhuǎn)染后的P3代大鼠BMSCs-eGFP的eGFP陽(yáng)性率為99.97%,轉(zhuǎn)染前后,兩組細(xì)胞CD34和CD45陽(yáng)性率低于2%,而CD44和CD90陽(yáng)性率高于99%。轉(zhuǎn)染后的P3代大鼠BMSCs-eGFP成骨誘導(dǎo)后,茜素紅染色,可見(jiàn)呈桔紅色的塊狀鈣結(jié)節(jié),成脂誘導(dǎo)后,油紅O染色顯示有大量紅色脂質(zhì)沉淀,成骨成脂誘導(dǎo)過(guò)程中,eGFP持續(xù)表達(dá)。[結(jié)論]Lenti-emGFP/hygro轉(zhuǎn)染大鼠BMSCs并經(jīng)潮霉素藥篩后,eGFP可穩(wěn)定高效表達(dá),對(duì)細(xì)胞的生物學(xué)特性無(wú)明顯影響,是一種較為理想的標(biāo)記方法。
[Abstract]:[Objective] to study the lentiviral vector (lentiviral, vectors, LVs) - mediated enhanced green fluorescent protein (enhanced green fluorescent protein, eGFP) labeled rat bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs)]. The feasibility of the method and effect of the biological characteristics of the cells isolated from rat BMSCs by density gradient centrifugation with Lenti-emGFP/hygro lentiviral vector transfection and hygromycin drug screening, microscopic observation of the cell morphology and the expression of eGFP, MTT before and after transfection method were used to examine the cell proliferation, cell surface markers were detected by flow cytometry (CD34, CD44, CD45, CD90 and eGFP), detection of osteoblasts after transfection the cells of the adipogenic differentiation potential and were stained by alizarin red staining, oil red O staining. The BMSCs rats showed fibroblast like morphology, the vortex like like arrangement, cell morphology did not change after transfection, and high expression of eGFP, two groups of fine Cell proliferation, flow cytometry showed that the positive rate of eGFP P3 in rat BMSCs-eGFP after transfection was 99.97%, two groups of cells before and after transfection, CD34 and CD45 positive rate of less than 2%, while the positive rate of CD44 was higher than that of 99%. and CD90 after transfection of P3 rats BMSCs-eGFP after osteogenic induction, alizarin red staining, there were small orange red calcium nodules, after adipogenic induction, oil red O staining showed a large number of red lipid deposition, osteogenic and adipogenic induction process, eGFP continued to express. Conclusion]Lenti-emGFP/hygro transfected rat BMSCs and hygromycin drug screening, eGFP stable high expression, no obvious effect the biological characteristics of the cells, is an ideal marker method.

【作者單位】： 第三軍醫(yī)大學(xué)附屬新橋醫(yī)院骨科;
【基金】：國(guó)家自然基金青年項(xiàng)目(編號(hào):31000438) 國(guó)家自然科學(xué)基金面上項(xiàng)目(編號(hào):81271362) 軍隊(duì)“十二五”重點(diǎn)項(xiàng)目(編號(hào):BWS11C040)
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王繼東;杜之渝;蘭芬;;慢病毒載體介導(dǎo)增強(qiáng)型綠色熒光蛋白轉(zhuǎn)導(dǎo)兔角膜上皮細(xì)胞的體外實(shí)驗(yàn)研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2010年13期

2 程明;戴閩;祝蔭;程細(xì)高;范紅先;劉虎誠(chéng);;人骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)及體外誘導(dǎo)分化為成骨細(xì)胞[J];中國(guó)矯形外科雜志;2012年01期

【共引文獻(xiàn)】

相關(guān)期刊論文 前8條

1 蔣濤;任先軍;陰洪;周長(zhǎng)立;王開(kāi)見(jiàn);;大鼠骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)及多向分化研究的體外實(shí)驗(yàn)[J];脊柱外科雜志;2013年05期

2 王繼東;馬曉輝;蘭芬;王健;陳余;范原銘;杜之渝;;Lenti-eGFP體外轉(zhuǎn)染兔角膜上皮細(xì)胞的實(shí)驗(yàn)研究[J];重慶醫(yī)科大學(xué)學(xué)報(bào);2011年12期

3 李瑞琦;張國(guó)平;任立中;李亞麗;呂亞軍;;骨髓間充質(zhì)干細(xì)胞治療股骨頭壞死的評(píng)價(jià)[J];中國(guó)組織工程研究;2013年35期

4 婁向新;袁卉華;包敏;張彥中;;模擬干細(xì)胞生長(zhǎng)微環(huán)境以促進(jìn)間充質(zhì)干細(xì)胞的擴(kuò)增[J];中國(guó)細(xì)胞生物學(xué)學(xué)報(bào);2013年11期

5 江祖p，

本文編號(hào)：1641415