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綠色熒光蛋白和成肌調節因子在骨髓基質干細胞中的共表達
作者：李美山，張成，陳松林，于美娟，張雅妮，王淑輝，熊符【關鍵詞】 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）對骨髓基質干細胞（MSCs）的轉染，以及綠色熒光蛋白（GFP）和成肌調節因子MyoD和Myogenin在MSCs中的表達. 方法：用差速貼壁法體外培養大鼠MSCs，并用流式細胞儀（FCM）檢測其表面標志；對構建有綠色熒光蛋白的重組腺病毒（AdGFP）進行擴增和鑒定，并轉染MSCs；用RTPCR檢測MyoD和Myogenin在轉染后MSCs中的表達；將MSCs與C2C12成肌細胞共培養，誘導前者的分化，并用免疫熒光檢測MyoD的表達. 結果：FCM檢測證實，在MSCs的表面標志中CD11b和CD45陰性，而CD29和CD44陽性；隨著AdGFP感染復數（MOI）的增加，轉染效率也相應提高，但在MOI大于100后，MSCs開始出現病變；RTPCR結果提示MyoD和Myogenin在轉染后的MSCs中有表達；免疫熒光檢測證實誘導后的MSCs可以表達MyoD蛋白. 結論：MSCs可以被AdGFP有效轉染而表達GFP；同時內源性成肌調節因子的激活，可以促進MSCs的成肌分化.
【關鍵詞】 基質干細胞；腺病毒；綠色熒光蛋白；C2C12；MyoD；Myogenin
0引言
骨髓基質干細胞（mesenchymal stem cells, MSCs）具有向成骨、脂肪、神經以及肌肉等多種類型細胞分化的潛能，其易于分離和擴增的特性，使其成為細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，對其進行外源性基因修飾和/或內源性基因的激活，成為目前亟待解決的問題. 我們通過構建有綠色熒光蛋白（green fluorescent protein, GFP）的５型重組腺病毒（AdGFP）轉染大鼠的MSCs，并與C2C12成肌細胞共培養，通過激活內源性成肌調節因子，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熒光標記小鼠抗大鼠抗體（PharMingen and Serotec公司）、人胚腎293細胞和AdGFP（中山大學黃文林教授惠贈）、C2C12細胞（中山大學潘秋
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