腺病毒介导转染人生长转化因子β1的脂肪干细胞复合多孔β-磷酸三钙支架体外培养的研究

目的 观察腺病毒介导转染人生长转化因子β1(Ad-hTGF-β1)的脂肪干细胞(ADSCs)与多孔β-磷酸三钙(β-TCP)支架材料在体外复合培养,探讨它们之间的相容性以及其构建组织工程软骨的可行性.方法 含增加型绿色荧光蛋白(EGFP)基因的腺病毒表达载体pAd-hTGF-β1,经包装、转染兔脂肪干细胞,筛选出成功转染Ad-hTGF-β1的ADSCs,然后将携Ad-hTGF-β1的ADSCs(密度1×106个/ml)与多孔β-磷酸三钙支架中行三维立体复合培养,定期扫描电镜观察细胞黏附能力、增殖活力以及形态学改变,并通过逆转录-聚合酶链反应(RT-PCR)、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(Collagen Ⅱ)的表达.结果 多孔β-TCP支架孔径在300～450μm之间,孔隙率65%;扫描电镜显示Ad-hTGF-β1的ADSCs与β-TCP支架材料在体外培养期间支架无塌陷及形变,且在支架上黏附、增殖良好,并能分泌软骨细胞外基质collagen Ⅱ,其分泌随时间呈增加的趋势.结论 多孔β-TCP支架具有适宜的微孔结构、良好的生物相容性和软骨诱导作用,可以作为软骨组织工程较理想的支架材料.且其与转染Ad-hTGF-β1的ADSCs相结合有望构建组织工程软骨.

Abstract：

Objective To observe the biological behaviors of cultured adipose-derived stem cells (ADSCs) transfected by adenoviral vector-mediated human transforming growth factor-β1 (Ad-hTGF-β1) combined with β-tricalcium phosphate (β-TCP) scaffold and investigate the feasibility of them for cartilage tissue engineering. Methods The adenoviral plasmid vector containing hTGF-β1 gene was transfected into ADSCs. The ADSCs were seeded onto the β-TCP scaffold and the effects of adhesion and morphological changes were observed under the phase-contrast microscopy and scanning electron microscopy (SEM). Reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were applied to detect the expression of collagen Ⅱ in the β-TCP scaffold. Results The SEM results showed the porous β-TCP scaffold had macro- and micro-porous strutures with the porosity being 65%. The ADSCs transfected by Ad-hTGF-β1 were successfully cultured in vitro. The induced cells adhered to the surface of the β-TCP scaffold and proliferated well. RT-PCR, Western blotting and immunohistochemistry revealed that Collagen Ⅱ was detected after mixed culture. Conclusion The porous β-TCP scaffold with excellent property may be a good matrix for ADSCs transfected by Ad-hTGF-β1, and can be used for cartilage engineering.