Transwell侵袭小室技术的改良及其在人骨髓间充质干细胞诱导分化中的应用

Transwell chamber technique improvement and its appliance in the induction and proliferation of human mesenchymal stem cells

目的:分离、培养和鉴定人骨髓间充质干细胞(Mesenchymalstemcells,MSCs),应用改良的Transwell侵袭小室技术,探讨血管内皮生长因子(VEGF)及内皮细胞条件诱导液对其体外诱导分化中的作用。方法:采用Percoll(1·073g/ml)分离液分离骨髓单个核细胞,体外培养MSCs,流式细胞术分析鉴定MSCs的纯度,Transwell侵袭小室技术结合LSCM,实时监测MSCs在Matrigel与VEGF/内皮细胞条件诱导液构成的内皮细胞生长微环境中的运动迁移情况。结果:经Percoll分离、体外培养扩增的MSCs,细胞纯度可达95%左右;VEGF组迁移的深度虽高于对照组(P<0·05),但迁移至聚碳酸脂膜下的细胞与对照组相比,并无统计学意义(P>0·05)。内皮细胞条件诱导液促进MSCs的迁移,在Matrigel内迁移的深度及迁移至聚碳酸脂膜下的细胞均明显多于对照组(P<0·05)。结论:共聚焦激光扫描显微术与Transwell侵袭小室技术的结合,能够从时间和空间上对后者进行观察,使该实验得到改良;利用内皮细胞条件诱导液与Matrigel模拟体外内皮细胞生长的微环境,并从空间上观测了MSCs穿越人工基底膜的情况,为MSCs向内皮细胞体外诱导开辟了新的思路。

Objective：To isolate, culture and identify human mesenchymal stem cells（hMSCs） and with developed transweU chamber technology to investigate the role of vascular endothelial growth factor（VEGF） and endothelium conditioned medium in the differentiation of MSCs. Methods：MSCs were separated from bone marrow with percoll reagent and expanded in culture medium. The surface antigens were detected, the labelled cells were analysed on a FACS can flow cytometer. Transwell chamber combined with LSCM observing MSCs, moving and migration in VEGF and conditioned medium. Results：Cultured MSCs were expanded as undifferentiated cells and the purity reached 95%. Endothelium conditioned medium improved MSCs, moving and migration and the depth and cell number in matrigel much more than control（ P 〈 0. 05）. Although in the tendency of VEGF, the depth of MSCs, migration was deeper than control （P 〈 0. 05）, the cells number migrated to Polycarbonate membrane had no statistical significance between VEGF group and control （P 〉 0. 05）. Conclusion：MSCs can be cultured in the tendency of endothelium conditioned medium and matrigel resembling environment in vivo. The developed transweU chamber technology can observed MSCs moving and migration to artificial fundus membrane from both time and space,which was benefit to experiments and bring perfect prospect in MSCs inducing in vitro.