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綠色熒光蛋白和成肌調(diào)節(jié)因子在骨髓基質(zhì)干細(xì)胞中的共表達(dá)
作者：李美山，張成，陳松林，于美娟，張雅妮，王淑輝，熊符【關(guān)鍵詞】 腺病毒
Coexpression of both green fluorescent protein and myogenic regulatory factors in bone marrowderived mesenchymal stem cells
【Abstract】 AIM: To investigate the transfection of bone marrowderived mesenchymal stem cells (MSCs) by recombinant adenovirus (Ad), and the expressions of both green fluorescent protein (GFP) and myogenic regulatory factors, which are MyoD and Myogenin. METHODS: Rat MSCs were separated, cultured and expanded in vitro, and taken for identification of surface antigens by flow cytometry (FCM). AdGFP was amplified and identified to transfect MSCs. Expressions of both MyoD and Myogenin were detected in transfected MSCs by RTPCR. Differentiation of MSCs was induced by cocultured with C2C12 myoblasts, and MyoD expression was identified by immunofluorescence (IF). RESULTS: FCM showed that CD11b and CD45 were negative, CD29 and CD44 were positive in MSCs surface antigens. With the increasing of AdGFP multiple of infection (MOI), transfection rate were also increased. When MOI was over 100, cytopathic effect appears on MSCs. Expressions of both MyoD and Myogenin in transfected MSCs were approved by RTPCR; and MyoD protein can be found in induced MSCs by IF. CONCLUSION: MSCs can be effectively transfected by AdGFP and express GFP; and activation of intrinsic myogenic regulatory factors will enhance the myogenic differentiation of MSCs.
【Keywords】 mesenchymal stem cells; adenovirus; green fluorescent protein; C2C12; MyoD; Myogenin
【摘要】 目的：研究重組腺病毒（Ad）對骨髓基質(zhì)干細(xì)胞（MSCs）的轉(zhuǎn)染，以及綠色熒光蛋白（GFP）和成肌調(diào)節(jié)因子MyoD和Myogenin在MSCs中的表達(dá). 方法：用差速貼壁法體外培養(yǎng)大鼠MSCs，并用流式細(xì)胞儀（FCM）檢測其表面標(biāo)志；對構(gòu)建有綠色熒光蛋白的重組腺病毒（AdGFP）進(jìn)行擴(kuò)增和鑒定，并轉(zhuǎn)染MSCs；用RTPCR檢測MyoD和Myogenin在轉(zhuǎn)染后MSCs中的表達(dá)；將MSCs與C2C12成肌細(xì)胞共培養(yǎng)，誘導(dǎo)前者的分化，并用免疫熒光檢測MyoD的表達(dá). 結(jié)果：FCM檢測證實(shí)，在MSCs的表面標(biāo)志中CD11b和CD45陰性，而CD29和CD44陽性；隨著AdGFP感染復(fù)數(shù)（MOI）的增加，轉(zhuǎn)染效率也相應(yīng)提高，但在MOI大于100后，MSCs開始出現(xiàn)病變；RTPCR結(jié)果提示MyoD和Myogenin在轉(zhuǎn)染后的MSCs中有表達(dá)；免疫熒光檢測證實(shí)誘導(dǎo)后的MSCs可以表達(dá)MyoD蛋白. 結(jié)論：MSCs可以被AdGFP有效轉(zhuǎn)染而表達(dá)GFP；同時內(nèi)源性成肌調(diào)節(jié)因子的激活，可以促進(jìn)MSCs的成肌分化.
【關(guān)鍵詞】 基質(zhì)干細(xì)胞；腺病毒；綠色熒光蛋白；C2C12；MyoD；Myogenin
0引言
骨髓基質(zhì)干細(xì)胞（mesenchymal stem cells, MSCs）具有向成骨、脂肪、神經(jīng)以及肌肉等多種類型細(xì)胞分化的潛能，其易于分離和擴(kuò)增的特性，使其成為細(xì)胞治療的一種很有前景的治療手段［1-3］. 為促使MSCs更好地定向分化，對其進(jìn)行外源性基因修飾和/或內(nèi)源性基因的激活，成為目前亟待解決的問題. 我們通過構(gòu)建有綠色熒光蛋白（green fluorescent protein, GFP）的５型重組腺病毒（AdGFP）轉(zhuǎn)染大鼠的MSCs，并與C2C12成肌細(xì)胞共培養(yǎng)，通過激活內(nèi)源性成肌調(diào)節(jié)因子，啟動MSCs的成肌分化.
1材料和方法
1.1材料DMEM和1640培養(yǎng)基（Gibco公司）、胎牛血清（杭州四季青公司）、新生牛血清（Hayclone公司）、FITC熒光標(biāo)記小鼠抗大鼠抗體（PharMingen and Serotec公司）、人胚腎293細(xì)胞和AdGFP（中山大學(xué)黃文林教授惠贈）、C2C12細(xì)胞（中山大學(xué)潘秋
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