TGF-β1基因真核表达载体的构建及在BMSCs中的表达

CONSTRUCTION OF EUKARYOTIC VECTOR CARRYING TGF-β1 GENE AND ITS EXPRESSION IN BONE MESENCHYMAL STEM CELLS

目的 研究真核表达载体pCDNA3 .1 (+) TGF- β1 在骨髓间充质干细胞(BMSCs) 中的表达。方法 基因克隆构建pCDNA3 .1 (+) TGF- β1 真核表达载体, 转染大鼠BMSCs, G418 筛选获得稳定转染的细胞, RT- PCR、ELISA和免疫细胞化学检测其表达, MTT 法检测其增殖活性。结果 成功构建含TGF- β1 基因的真核表达载体; RT- PCR、ELISA、免疫细胞化学证实了TGF- β1基因在BMSCs中至少可以表达一个月; MTT法提示转染TGF- β1基因可以促进BMSCs增殖。结论 pCDNA3. 1 (+) TGF -β1转染BMSCs可获得稳定表达。

Objective To construct the eukaryotic vector containing TGF-β1, which was transfected intobone mesenchymal stem cells and expressed. Methods Plasmid pCDNA3.1(+) containing human transforming growth factor β1 gene was constructed and then transfected into the primary cultured rBMSCs under the mediation of Lipofectamine. Positive clones were selected by G418; the expression was verifiedby using RT-PCR, ELISA, and immunocytochemical staining; and the proliferation of the transfected BMSCs was evaluated by MTT. Results RT-PCR, ELISA and immounhistochemical staining of post-transgenic BMSCs were TGF-β1positive. MTT colorimetric methodshowed that the transfected TGF-β1 had a positiveeffect on the proliferationof BMSCs. Conclusion Transfection ofpCDNA3.1(+)-TGF-β1can provide persistent expression in BMSCs.