人淋巴细胞-NT3基因工程细胞的建立及鉴定

Construction and Assessment of the Model of NT3 Genetically Engineered Lymphocyte

麻晓峰;陈杰;徐琳;顾香芳;高下;

1:东南大学临床医学院

2:南京大学医学院附属鼓楼医院耳鼻咽喉头颈外科

3:东南大学临床医学院

4:南京大学医学院附属鼓楼医院科研部

摘要
目的建立能够存活较长时间并稳定表达神经营养素-3(neurotrophin-3,NT3)的人淋巴细胞株,为后续的动物和人体耳蜗内基因转染实验奠定基础。方法从正常人全血中通过Ficoll液提取人淋巴细胞,并在其完全血清培养基中加入白细胞介素-2(IL-2)使其良好生长。通过RT-PCR得到人胎肝组织的NT3cDNA,以T4DNA连接酶和真核表达载体pIRES-DsRed2相连接。以阳离子脂质体作为载体,将pIRES-DsRed2-NT3转染人淋巴细胞,进行RT-PCR及Western Blot分析鉴定。结果建立了一种能使体外培养淋巴细胞成活时间延长的新方法,成功转染NT3基因至淋巴细胞中,并能继续存活相对较长时间,Western Blot证明细胞上清液中有NT3,构建了稳定表达NT3的人基因工程淋巴细胞。结论构建重组质粒pIRES-DsRed2-NT3,利用淋巴细胞作为中介宿主,进而表达、分泌NT3,为进一步NT3基因转染致聋动物耳蜗实验奠定了基础,并有可能为人体基因转染治疗耳聋找到很好的中介细胞。
关键词
神经营养素-3;基因克隆;淋巴细胞;转染
基金项目(Foundation):
南京市医学科技重点项目(编号:ZKX0207)
作者
麻晓峰;陈杰;徐琳;顾香芳;高下;
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