转化生长因子β1基因转染鼠脂肪间充质干细胞的可行性

背景:脂肪间充质干细胞在转化生长因子β_1等生长因子的调控下可被诱导分化为软骨细胞.然而,外源性转化生长因子β_1存在引起趋化性、激发炎症反应、造成局部损伤、有效成分容易被稀释或丢失、半衰期短、缺少理想的转运蛋白等缺陷.因此,利用基因重组技术,使种子细胞表达转化生长因子β_1,对软骨组织工程的进一步发展具有重要的指导意义.目的:探讨真核表达载体pcDNA3.1-转化生长因子β_1质粒的构建方法,并观察其转染脂肪间充质干细胞的可行性.方法:利用基因重组技术,将大鼠转化生长因子β_1全长基因的PCR产物与克隆载体pT7Blue连接,并用大肠杆菌筛选阳性重组体构建真核表达质粒,采用XboⅠ、Hind Ⅲ双酶切及测序方法对质粒进行鉴定;将已经纯化的pcDNA3.1-转化生长因子β_1质粒和空载体pcDNA3.1质粒分别采用阳离子脂质体试剂LipofectamineTM2000转染脂肪间充质干细胞,经G418抗性筛选后扩大培养,同时用pcDNA3.1-绿色荧光蛋白观察转染效率.结果与结论:重组质粒经XboⅠ、Hind Ⅲ双酶切后出现1.35 bp和5.4 kb 2条带,测序结果显示重组质粒所包含的转化生长因子β_1全长碱基序列完全正确,绿色荧光蛋白显示其转染脂肪干细胞的转染效率> 80%,且转染细胞后有转化生长因子β_1 mRNA和蛋白的表达上调.提示利用基因重组技术可成功构建能转染脂肪间充质干细胞的真核表达载体pcDNA3.1-转化生长因子β_1质粒.

Feasibility of transfection of transforming growth factor-beta 1 into rat dipose-derived mesenchymal stem cells

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) can be induced toward the chondrogenic lineages with chondrogenic medium contained transforming growth factor-β_1(TGF-β_1). However, it remains concerned about the disadvantage with use of TGF-β_1 in vitro, which can induce chemotaxis, activate inflammatory cells, cause local defect and want an ideal matrix to deliver proteins. Therefore, with advanced gene-delivery techniques, TGF-β_1 can be long-lasting expressed by transduced stem cells, which is very useful for Chondrogenic Tissue Engineering.OBJECTIVE: To master the method of construction and transfection of eukaryotic expression vector for rat transforming growth factor-β_1 and to study the possibility of gene transfection to ADSCs with this vector.METHODS: Recombining DNA techniques were applied to construct recombinant plasmid pcDNA3.1-TGF-β_1. And this plasmid was verified by restriction endonuclease mapping and DNA sequencing; Then the plasmid with TGF-β_1 gene or not was transfected into ADSCs by use of LipofectamineTM2000. After infection, transduced ADSCs were diluted and cultured with neomycin (G418). Gene transfer efficiency compared on the basis of green fluorescent protein expression was assessed.RESULTS AND CONCLUSION: Digestion of the plasmid with double restriction endo- nuclease XboⅠand Hind Ⅲ showed about two specific electrophoretic strips (1.35 bp and 5.4 kb), and the sequence of the rat TGF-β_1 gene in recombinant was concorded with that reported in Genbank. There were 80 percent of the cells which were transduced, and the expressions of mRNA and protein of TGF-β_1 in ADSCs were discovered positively. These indicated that the eukaryotic expression vector for rat TGF-β_1 (i.e. pcDNA3.1-TGF-β_1) , which is able to transfect the ADSCs, can be constructed through genetic recombination.