BDM支架“人工羊膜”的制备及立体培养的初步实验研究

Preparation of BDM scaffold“Artificial Amniotic Membrane”and preliminary experimental study of Three-dimensional cultivation

目的利用羊膜间充质干细胞与生物基质体外制备“人工羊膜”,并对其活性进行验证,以期为优化人工羊膜材料和后续实验积累经验。方法分离、培养人羊膜间充质干细胞,经鉴定后,用混合法按体积1∶1比例将干细胞悬液与BD-基质胶(BD-matrigel,BDM)结合,制成底面积为2 cm 2,厚约3~5 mm,浓度为3×10^5/mL的圆形薄膜,即“人工羊膜”,将其置于三维培养体系中培养(实验组);同期将3×10^5/mL浓度,不含BD基质胶的羊膜间充质干细胞置于相同培养体系中(对照组)。分别在1、4、7 d时,用普通光学显微镜观察两种不同培养方式下的细胞变化,同时运用流式细胞仪检测二者凋亡情况,应用ELISA检测其分泌血管内皮生长因子(Vascular endothelial growth factor,VEGF)的量。结果细胞形态学观察发现,对照组间充质干细胞在原代培养后48 h左右开始完全贴壁生长,7 d时融合度可达90%以上,传至第三代呈均一长梭形的间充质干细胞;实验组间充质干细胞则表现为聚集成球生长;两组细胞生长良好,漂浮细胞少见。经Annexin-V染色后,用流式细胞仪检测结果显示:实验组与对照组细胞凋亡率分别为2.91%、4.91%,差异有统计学意义(P<0.05)。ELISA结果显示实验组间充质干细胞VEGF表达量与对照组相比无统计学差异(P>0.05)。结论羊膜间充质干细胞在以BD基质胶支架的“人工羊膜”中可以存活,并具有良好的增殖、分泌能力。

Objective To develop“artificial amniotic membrane”in vitro using amniotic mesenchymal stem cells and biological matrices,and verify its ability of proliferation and apoptosis.Methods Human amnion derived mesenchymal stem cells were isolated and cultured.After identification,the stem cell suspension was combined with BD-matrigel(BD-MDM)in a volume ratio of 1∶1 by a mixing method,and a circular membrane with a bottom area of 2 cm 2,a thickness of about 3~5 mm and a concentration of 3×10^5/ml,which was called“artificial amniotic membrane”,was cultured in a three-dimensional culture system(experimental group).At the same time,the stem cells with a concentration of 3×10^5/ml without BD-MDM were placed in the same culture system(control group).At 1,4 and 7 days,the changes of cells in two different culture modes were observed by common light microscope,and the apoptosis of the cells was detected by flow cytometry.The vascular endothelial growth factor(VEGF)protein level was measured by ELISA.Results The morphological observations revealed that the mesenchymal stem cells in the control group began to attach about 48 hours after primary culture,the degree of fusion reached more than 90%at 7 days,and the shape was uniformly spindle-shaped when the cells spread to the third generation.However,the mesenchymal stem cells in the experimental group showed aggregated spheroid growth.Most cells in the two groups grew well and floating cells were rare.After Annexin-V staining,the results of flow cytometry showed that the apoptosis rate of the experimental group was 2.91%lower than 4.91%of the control group(P<0.05).ELISA results showed that there was no significant difference between the VEGF protein levels in mesenchymal stem cells of the two groups(P>0.05).Conclusion Amniotic mesenchymal stem cells can survive in the“Artificial Amniotic Membrane”with BD-MDM scaffold,and have good proliferation and secretion ability.