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大鼠睫状神经节神经营养因子真核表达载体转染成肌细胞并促其分化 被引量：2

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摘要 目的应用DNA重组技术构建大鼠睫状神经节神经营养因子(CNTF)真核表达载体,再稳定转染肌源性干细胞,分析转染的肌源性干细胞能否稳定表达CNTF、并向神经元样细胞分化,为深入研究CNTF的功能及动物体内试验打下理论基础。方法用RT-PCR扩增大鼠带有Xho I和BamH I双酶切位点的CNTF cDNA片段,经双酶切获得的目的片段,再与双酶切过的pEGFP-C3质粒相连接,重组质粒转化E.coli DH5α感受态大肠杆菌,酶切方法鉴定阳性菌落。采用脂质体转染法,参照Lipofectamine2000试剂盒(LF2000I,nvitrogen公司)说明书,采用阳离子脂质体介导两组质粒(pEGFP-C3-CNTF和pEGFP-C3)转染成肌细胞。通过RT-PCR分析CNTF mRNA在成肌细胞中的表达,用神经元特异性蛋白(NES)免疫组化染色法鉴定是否有神经元样细胞分化,并于显微镜下计数阳性细胞占细胞总数的比例。结果通过PCR扩增成功获得了CNTF cDNA目的片段,大鼠CNTF真核表达载体构建成功,并成功转染成肌细胞,被转染的成肌细胞稳定表达CNTF并显著分化为神经元样细胞、实验组显著高于对照组(P<0.05),差异有统计学意义。结论大鼠CNTF真核表达载体pEGFP-C3-CNTF可以成功构建,并可转染大鼠成肌细胞,经转染的成肌细胞能有效表达大鼠的CNTF,且转染的成肌细胞显著分化为神经元样细胞,为研究大鼠CNTF在神经损伤修复方面的实验奠定了重要基础。
作者 段大波 张树鹰 王伟
机构地区 辽宁医学院 锦州市中心医院
出处 《中国医学工程》 2012年第3期154-155,157,共3页 China Medical Engineering
关键词 CNTF 成肌细胞 pEGFP-C3载体 基因转染
分类号 R329.28 [医药卫生—人体解剖和组织胚胎学]
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参考文献10

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共引文献18

同被引文献25

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中国医学工程
2012年 第3期

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