与羊膜上皮细胞共培养对羊膜间充质干细胞生物学特性的影响

Effect of co-culture with amniotic epithelial cells on biological characteristics of amniotic mesenchymal stem cells

目的:研究与羊膜上皮细胞(amniotic epithelial cells,AEC)共培养对羊膜间充质干细胞(amniotic mesenchymal stem cells,AMSC)生物学特性的影响,并探讨基质细胞衍生因子1(stromal cell derived factor-1,SDF-1)/CXCR4轴在AMSC归巢、迁移过程中的作用。方法:从人羊膜中分别分离、培养AMSC和AEC,进行扩增及鉴定。实验设AMSC与AEC共培养组、无血清AMSC培养组以及血清AMSC培养组,培养24、48、72 h后,通过CCK-8及锥虫蓝实验检测AMSC细胞增殖活性,免疫荧光流式细胞术和Real-time PCR检测CXCR4 m RNA的表达水平,细胞迁移实验检测AMSC细胞体外迁移能力。结果:48、72 h共培养组及血清培养组细胞增殖活性及增殖率均高于无血清培养组(P<0.05);共培养组与无血清培养组AMSC细胞的CXCR4 m RNA和蛋白表达明显高于血清培养组(P<0.05),两组AMSC细胞依赖迁移能力明显高于血清培养组(P<0.05)。结论:与AEC共培养的AMSC细胞仍具备间充质干细胞的基本生物学特性,且能保持良好的增殖活性,共培养能上调AMSC表面CXCR4,并在体外增强其迁移能力。

Objective： The aim of this study was to investigate the effect of co-culture with AEC（amniotic epithelial cell） on the biological characteristics of AMSC（amniotic mesenchymal stem cell）, and to investigate the roles of SDF-1/CXCR4 axis in the homing and migration of AMSC. Methods： AMSC and AEC were isolated from human amnion, and then cultured, amplified and identified, respectively. The AMSC were divided into three groups： AEC co-cultured group, serum-free cultured group and serum cultured group. After culture for 24 h, 48 h, and 72 h, the proliferation viability of AMSC was measured by CCK-8 assay and trypan blue staining; the expression of CXCR4 m RNA was analyzed by flow cytometry and Real-time RT-PCR, and the migration ability of AMSC in vitro was observed by migration assay. Results： Cell viability（48 h and 72 h） and survival rate in the co-culture and serum groups were higher than those in the serum-free group（all P〈0.05）. The m RNA and protein expressions of CXCR4 in AMSC of the co-culture and serum-free groups were significantly higher than those of the serum group（P〈0.05）. The migration ability of AMSC in the co-culture and serumfree groups, which increase with the SDF-1（stromal cell derived factor-1） concentration gradient, were higher than that in the serum group（P〈0.05）. Conclusion： AMSC co-cultured with AEC still have the basic biological characteristics of MSC, and showed good growth activity. Co-culture with AEC can up-regulate CXCR4 on AMSC surfaces and enhance the migration ability of AMSC in vitro.