TGF-β1基因转染大鼠脂肪干细胞向软骨细胞分化的实验研究

EXPERIMENTAL STUDY ON DIFFERENTIATING INTO CHONDROCYTES FROM RAT ADSCS TRANSFECTED WITH TGF-β1 GENE

目的TGF-β1基因转染大鼠的ADSCs(adipose stromal cells,ADSCs),诱导其向软骨细胞分化,为软骨组织工程学种子细胞提供新方法。方法分离、培养大鼠的ADSCs,免疫荧光法进行鉴定;TGF-β1基因转染ADSCs,对转染后细胞进行筛选;MTT法测定筛选后细胞的增殖活性;RT-PCR、Western blot对筛选后细胞的表达进行检测。结果成功分离、培养大鼠的ADSCs;细胞表面标志物CD29和CD44表达阳性,CD106和CD34表达阴性;基因转染后细胞的增殖力变强;转染TGF-β1的ADSCs在目的基因TGF-β1、SOX9、Aggrecan、Collagen II mRNA的表达和软骨特异性基质-Collagen II的分泌增强,明显高于对照组和转染空载体组;结果TGF-β1基因转染后ADSCs具有了软骨细胞的表型特征,可以用作软骨组织工程的种子细胞。

Objective To transfect rat ADSCs with TGF - β1 gene and induce them differrentiating into chondrocytes, providing a new method for the tissue-engineering cartilage seed cells. Methods ADSCs were isolated, cultured, and then identified by immunofluorescence assay. The cells were transfected with TGF-β1 gene, and selected the cells with transgene. MTr was used to detect the proliferation of the selected cells. Meanwhile, RT- PCR and Western blot were used to analyse the expression level of the selected cells. Results We succeeded in separating and culturing rat ADSCs . The cell surface markers CD29 and CD44 were positive, whereas CD106 and CD34 were negative. The prolifetation ability became stronger after transfection with TGF-β1 gene. In the transfected group, the destinct gene expression of TGF-β1, soxg, aggrecan and collagen Ⅱ mRNA and cartilage specific matrix-collagen Ⅱ secretion became stongeer, much higher than those of the control group and vector group. Conclusion Transfected ADSCs by TGF-β1 gene have the phenotypes of chondrocytes, and can be used as the seed sells for cartilage tissue-engineering.