腺病毒介导转染hTGF-β_1基因的兔脂肪干细胞向软骨细胞分化的实验研究

Effect of rabbit adipose-derived stem cells transfected by adenoviral vector mediated hTGF-β_1 gene on chondrocyte differentiation in vitro

目的探讨腺病毒介导转染hTGF-β_1(Ad-hTGF-β_1)基因的兔脂肪干细胞(ADSCs)在体外定向诱导分化成软骨细胞的可行性。方法含增加型绿色荧光蛋白基因(EGFP基因)的腺病毒表达载体pAd-hTGF-β_1,经293细胞包装重组腺病毒后,感染第3代培养的兔ADSCs。将转染与未转染hTGF-β_1的ADSCs置于诱导培养液(CMM液)中行单层培养和包埋于藻酸钠微珠支架中行三维立体培养,定期观察细胞的大体形态学变化、反转录-聚合酶链反应(RT-PCR)法检测目的基因hTGF-β_1的表达及RT-PCR法、Western blot法和免疫组织化学法检测软骨特异性细胞外基质蛋白Ⅱ型胶原蛋白(CollagenⅡ)的表达。结果转染hTGF-β_1组的ADSCs在单层培养中呈现形态规则、多角型细胞增多、增殖能力明显增强;转染后7d与21d在三维藻酸钠微珠支架中培养的ADSCs的hTGF-β_1基因表达均明显升高,其RT-PCR结果与未转染组比较差异有统计学意义(P<0.01)。同时转染组的兔ADSCs分泌CollagenⅡ明显增多,转染后7d与21d的CollagenⅡRT-PCR、Western blot和免疫组织化学结果均明显高于未转染组(P<0.01),并且转染组内其表达差异无统计学意义(P>0.05)。结论经Ad-hTGF-β_1基因修饰的兔ADSCs在体外不需外来生长因子诱导下能定向分化成软骨细胞,且其分化能力强,外来基因hTGF-β_1表达高、持续时间长,是理想的软骨组织工程的种子细胞,这也为转基因干细胞治疗软骨缺损提供理论依据。

Objective To investigate the proliferation of rabbit adipose-derived stem cell （ADSCs） transfected by adenoviral vector mediated hTGF-β1 gene and its chondrocyte differentiation potential. Methods The Ad-hTGF-β1 plasmid vetor which had the hTGF-β1 gene was developed and transfected the ADSCs. The experimental group was the hTGF-β1 transfected group. The cells enclosed by alginate were cultured in complete chondrogenic medium （CMM）. The morphology of the cells were observed, and RT-PCR was used to measure hTGF-β1 and collagen Ⅱ expression, at the same time western blot and immunohistochemistry were applied to detect the expression of collagen Ⅱ in ADSCs before and after transfected with hTGF-β1 gene. Results The hTGF-β1 transfected ADSCs became the polygon and it proliferated well. The RT-PCR result of hTGF-β1 on the transfected group was better than the control after transtected for 7 day and 21 day. The difference between the two groups was significant （P〈0.01 or P〈0.05）. The RT-PCR, western blot and immuno- histoehemistry of collagen Ⅱ had the same results, and these was a significant （P〈0.01 or P〈0.05） difference between the two groups. Conclusion ADSCs transfected by the adenoviral vector mediated hTGF-β1 gene have strong potential for chondrocytes differentiation. They therefore can serve as seed cells for cartilage tissue engineering.