过表达MyoD1基因山羊胎儿成纤维细胞系的建立及其成肌诱导分化

doi:10.3864/j.issn.0578-1752.2013.14.019

摘要/Abstract

摘要： 【目的】通过建立过表达MyoD1基因山羊胎儿成纤维细胞系研究MyoD1基因的异位表达研究其在成肌分化中的生物作用。【方法】采用RT-PCR从激活的骨骼肌卫星细胞中克隆MyoD1基因，并将其cDNA终止密码子TGA定向突变为GGA，定向克隆至带有增强型水母绿色荧光蛋白（ehanced green fluorescent protein，eGFP）报告基因的真核表达载体pEGFP-N1中，构建融合蛋白表达载体pEGFP-MyoD1，经过酶切、测序鉴定后，采用LipofectiminTM LTX转染山羊胎儿成纤维细胞（goat embryonic fibroblast，GEF）以建立MyoD1异位表达细胞株并采用成肌诱导分化培养液进行成肌诱导分化，探究MyoD1在成肌过程中的生物学功能。【结果】成功克隆山羊MyoD1基因，并在MyoD1 的开放阅读框（ORF）两端引入XhoⅠ/EcoRⅠ酶切位点，将其终止密码子TGA定点突变为GGA，定向克隆至pEGFP-N1真核表达载体，获得融合蛋白表达载体pEGFP-MyoD1；经G418（400 μg•mL-1）筛选2周后，获得MyoD1异位表达的GEF细胞株；间接免疫荧光（indirect immunofluorescence assay，IFA）检测结果显示该细胞株能够表达Myf-5等成肌相关免疫学标志；采用成肌分化培养基分化培养处理2—3 d可见少量肌管产生，并表达MyoG、Desmin和MyHC等早期成肌分化标志，处理5 d可见大量肌管形成。【结论】成功克隆出山羊MyoD1基因，构建了pEGFP-MyoD1真核表达载体并建立过表达MyoD1 GEF细胞系，过表达MyoD1 GEF系能够在成肌诱导培养液诱导形成肌管。

关键词: MyoD1基因 , 基因克隆 , 异位表达 , 成肌分化

Abstract: 【Objective】 MyoD1 ectopic expression goat fibroblast line was constructed to explore the bio-functions of MyoD1 in myogenesis. 【Method】 MyoD1 was cloned from the skeletal satellite cell via RT-PCR method. To visualize the MyoD1 protein, MyoD1 was reconstructed to pEGFP-N1 with the mutation of TGA (termination codon) into GGA (glycine) and cloned into pEGFP-N1, a fusion protein expression vector. The MyoD1 expressed goat embryonic fibroblast (GEF) was founded to explore the function of MyoD1 during the myogenesis, via transfecting the pEGFP-MyoD vector into GEF with the help of LipofectiminTM LTX. MyoD1 expressed GEF was induced to undergo the myogenesis by the differentiation media. The myogenesis of this cell line was ditermined by morphology and indirect immunofluorescence assay (IFA). 【Result】 The sequencing results indicated that MyoD1 was successfully reconstructed into pEGFP-N1 with mutation of TGA into GGA, and MyoD1 was fused with eGFP. MyoD1 expressed GEF line was obtained through two weeks of continuous G418 resistance screening and expressed Myf5 (detected by indirect immunofluorescence assay (IFA)). A few signs of myotube were observed 2 days after differentiation treatment, which expressed the differentiation marker of myoblast (MyoG, Desmin, MyHC), while lots of myotube were obtained with contractility. 【Conclusion】MyoD1 gene was cloned and reconstructed with pEGFG-N1 to obtain pEGFP-MyoD1 expressed GEF line, successfully. The expression of MyoD1 would drive the GEF into myoblast with differentiation ability of myogenesis.

Key words: MyoD1 gene , gene clone , ectopic expression , myogenesis

李伟, 郑蒙蒙, 张亚妮, 朱才业, 邱峰龙, 韦光辉, 李碧春. 过表达MyoD1基因山羊胎儿成纤维细胞系的建立及其成肌诱导分化[J]. 中国农业科学, 2013, 46(14): 3032-3039.

LI Wei, ZHENG Meng-Meng, ZHANG Ya-Ni, ZHU Cai-Ye, QIU Feng-Long, WEI Guang-Hui, LI Bi-Chun. Construction of MyoD1 Expressed Goat Embryonic Fibroblast Line and the Myogenesis of This Cell[J]. Scientia Agricultura Sinica, 2013, 46(14): 3032-3039.

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