摘要
目的 脂质体介导TGF β1目的基因转染骨髓间充质干细胞 (MSC) ,研究对MSC成软骨分化的特异性细胞外基质的影响。方法 以梯度离心结合换液法获得并纯化骨髓间充质干细胞 (MSCs) ,采用脂质体介导法将重组真核表达质粒pcDNA3 TGF β1转染MSCs,RT PCR和Westernblot鉴定后 ,对转染后骨髓间充质干细胞的Ⅱ型胶原 (ColⅡ )、纤维连接蛋白 (FN)的表达行RT PCR和Westernblot检测。结果 获得纯度较高的成年中国小型猪骨髓间充质干细胞 (MSCs) ;脂质体为介导将全长人TGF β1目的基因导入MSC ,经RT PCR和Westernblot鉴定转染成功 ,转染后的MSC较未转染MSC成软骨分化特异性细胞外基质ColⅡ、FNmRNA和蛋白表达明显增强。结论 以脂质体介导法可以将TGF β1目的基因转染骨髓间充质干细胞 。
Purpose: To transfect mesenchymal stem cells (MSCs) by TGF- β1 gene with liprofectamine and investigate the influence on the specific extrancellular matrix (ECM) for chondrogenic differentiation. Methods: MSCs were obstained and purified by gradient centrifuge and medium changing. Recombinant plasmid pcDNA3-TGF-β1 was transferred into MSC with lipofectamine in vitro. RT-PCR and Western blot analysis were used for detecting TGF-β1 mRNA levels and peptides. After confirmation, Type II collagen (Col II) and fibronectin (FN) of transfected MSC were detected by RT-PCR and Western blot analysis. Results: Purified MSCs were obstained. TGF-β1 gene was introduced into MSCs with lipofectamine and was confirmed with RT-PCR and Western blot analysis. Compared with non-transfected MSC, mRNA and peptides of Coll II and FN, specific ECM components for chondrogenic differentiation, were expressed increasedly by the transfected MSC. Conclusions: The TGF-β1 gene transfection of MSC could be conducted with lipofectamine. The specific ECM for chondrogenic differentiation, Col II and FN, could be synthesized increasedly after gene transfection.
出处
《复旦学报（医学版）》
EI
CAS
CSCD
北大核心
2004年第4期343-346,F002,共5页
Fudan University Journal of Medical Sciences
基金
国家自然科学基金资助项目 (3 0 2 713 0 7)
复旦大学青年科学基金
