包裹骨形态发生蛋白-2基因转染干细胞的微囊化方法和蛋白释放效应

In vitro study Of BMP-2 gene transfected bone marrow derived mesenchymal stem cells in APA microcapsules

目的观察转基因骨髓间充质干细胞的微囊化包裹方法和骨形态发生蛋白-2（BMP-2）释放效应。方法利用高压微电场制备由海藻酸纳和多聚赖胺酸组成的微囊（细胞浓度为3×10^6个／ml，海藻酸钠浓度为1．5％）包被转B-gal（报告基因）或BMP-2基因的骨髓间充质干细胞，根据X-gal染色观察细胞在微囊内的存活情况，ELISA方法检测其所释放的BMP-2蛋白量，共培养方法检测其生物学活性。结果7、14、21、28d时微囊内转基因细胞X-gal染色均为阳性。包裹后30d，培养液上清中仍可检测到BMP-2蛋白。转BMP-2基因微囊化细胞与未包裹骨髓间充质干细胞共培养10d后，ALP活性定量检测结果表明，Adv-hBMP-2转染细胞组ALP活性为（52．537±1．774）U／ml，明显高于未转染细胞组的（42．336±0．857）U／ml，差异有统计学意义（P〈0．05）。结论微囊化转基因干细胞生长良好，能持续分泌BMP-2蛋白并促进干细胞向成骨细胞方向分化。

Objective To investigate the viability of gene transfer ceils encapsulated in microcapsule and the bioaetivity of secreted BMP-2. Methods An electrostatic droplet generator was employed to produce β-gal or BMP-2 gene transfected bone marrow derived mesenchymal stem ceils （MSCs） encapsulated in APA microcapsule at the 3 × 10^6/ml of ceil concentration, 1.5 g/ml of the alginate concentration. X-gal staining was used to indicate the viability of the encapsulated ceils. The BMP-2 proteins secreted from the encapsulated ceils were determined by the ELISA methods. ALP activities of the co-culture stem ceils were analyzed. Results The X-gal staining of the encapsulated cells were still positive till 2g days after encapsulation. The secreted BMP-2 proteins could be detected 30 days after encapsulation. The ALP activity of co-cultured MSCs was （52.537 ± 1.774） U/ml,was higher than （42.336 ±0. 857） U/ml in control group （P 〈 0.05）. Conclusion MSCs could be encapsulated in APA microcapsules and secreted active BMP-2 proteins.