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

Adipose-derived stem cells transfected by adenoviral vector mediated human transforming growth factor-β1 gene with β-tricalcium phosphate scaffold in vitro

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1×10^6个／m1）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型胶原蛋向（CollagenⅡ）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。

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-triealcium 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 transfeeted 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 immunohistoehemistry revealed that Collagen H 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.