人神经营养因子-3基因的亚克隆及其基因工程细胞模型的建立

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (07): 805-808.

人神经营养因子-3基因的亚克隆及其基因工程细胞模型的建立

常红¹, 郭梦和¹, 郭坤元², 李永贺¹

1. 第一军医大学珠江医院耳鼻咽喉科, 广东, 广州, 510282;
2. 第一军医大学珠江医院血液科, 广东, 广州, 510282

出版日期:2004-07-20 发布日期:2004-07-20
基金资助:
收稿日期:2004-1-7。
基金项目:珠江医院科研创新院长基金(200401)
作者简介:常红(1963- ),女,硕士,主治医师,电话:020-61643388,E-mail:chang_hong278@msn.com

Subcloning of human neurotrophin-3 gene and construction of its genetically engineered cell model

CHANG Hong¹, GUO Meng-he¹, GUO Kun-yuan², LI Yong-he¹

1. 第一军医大学珠江医院耳鼻咽喉科, 广东, 广州, 510282;
2. 第一军医大学珠江医院血液科, 广东, 广州, 510282

Online:2004-07-20 Published:2004-07-20

摘要/Abstract

摘要： 目的亚克隆人神经营养因子-3(human neurotrophin-3,NT3)并转染至体外培养的人骨髓间充质干细胞(BM-MSCs),建立能够表达和分泌NT3的基因工程细胞模型。方法采用低糖DMEM培养液+10%胎牛血清体外培养BM-MSCs,流式细胞仪分析其表面标记;构建真核表达载体pcDNA3.1(+)/NT3并通过脂质体介导转染BM-MSCs;收集部分细胞提取其中的总RNA,经逆转录PCR检测NT3基因表达;利用培养上清液体外孵育毛细胞以鉴定其生物学活性。结果 BM-MSCs表达CD13、CD29、CD59,不表达CD11、CD14、CD31、CD34、CD45、CD80、CD86、CD117、HLA-DR;pcDNA3.1(+)/NT3转染BM-MSCs后,能够表达和分泌NT3,并具有生物学活性,可促进离体的豚鼠耳蜗毛细胞存活。结论在体外建立表达NT3基因并分泌NT3的基因工程细胞是可行的。

Abstract: Objective To subclone human neurotrophin-3 gene (NT3) and transfer this gene into human bone marrow mesenchymal stem cells (BM-MSCs) to construct genetically engineered cells that produce NT3 in vitro. Methods Human BM-MSCs were cultured in low-glucose DMEM supplemented with 10% fetal bovine serum and 10 ng/ml epidermal growth factor. Flow cytometry (FCM) was used to examine the phenotypes of the cells. The eukaryotic expression vector pcDNA3.1 (+)/NT3 was constructed and transferred into human BM-MSCs in vitro via liposomes. The genetically engineered BM-MSCs were selected several times with G418 and the clones were obtained and then amplified, followed by extraction of the RNA for detection of NT3 gene expression by reverse transcriptional (RT) PCR. The biological activity of the genetically engineered cells was examined by the collecting the supernatant of the culture medium for incubation of guinea pig cochlea hair cells. Results The cultured cells expressed CD13, CD29 and CD59, but no7 CD11, CD14, CD31, CD34, CD45, CD80, CD86, CD117 or HLA-DR. The BM-MSCs genetically modified with pcDNA3.1(+)/NT3 not only expressed and produced NT3, but also promoted the survival of the guinea pig cochlea hair cells in vitro. Conclusion It is possible to construct the genetically engineered BM-MSCs that excrete NT3 in vitro.

中图分类号:

Q343

常红¹, 郭梦和¹, 郭坤元², 李永贺¹. 人神经营养因子-3基因的亚克隆及其基因工程细胞模型的建立[J]. 南方医科大学学报, 2004, 24(07): 805-808.

CHANG Hong¹, GUO Meng-he¹, GUO Kun-yuan², LI Yong-he¹. Subcloning of human neurotrophin-3 gene and construction of its genetically engineered cell model[J]. Journal of Southern Medical University, 2004, 24(07): 805-808.

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