siRNA阻断B7H1在人胎盘间充质干细胞上表达对其黏附和迁移的影响

摘要

BACKGROUND: Human placenta mesenchymal stem cells (hPMSCs) express costimulatory molecules B7H1 which plays an important role in its immunosuppressive capabilities. However, the effect of B7H1 on the biological behavior of hPMSCs has not been reported.OBJECTIVE: To investigate the effect of B7H1 blockade on adhesion and migration of hPMSCs.METHODS: hPMSCs were isolated from mature placenta by the method of digestion. Then in vitro hPMSCs were cultured,expanded and were used in test after third passage. Flow cytometry and RT-PCR analysis were used to investigate the expression of B7H1 on hPMSCs. Specific siRNAs of B7H1 were transfected into hPMSCs via cathodolyte liposome transfection method. RT-PCR was used to detect the changes of B7H1 gene expression. Then the adhesion of hPMSCs was determined by cell count, and the migration of hPMSCs was evaluated using Transwell method.RESULTS AND CONCLUSION: siRNA could effectively block the expression of B7H1 which highly expressed on hPMSCs. After seeded for half an hour, the cell adhesion rate had no significantly difference in the blockade group and the control group. For 1 hour or 3 hours, however, the adhesion rate of hPMSCs was significantly higher in the blockade group than in the control group (P ＜ 0.05). Transwell assay indicated that the migration numbers of hPMSCs in the blockade group were significantly less compared with the control group under the culture conditions of SDF-1α, DMEM-LG complete medium or hPMSCs culture supernatant (P ＜ 0.01). The results showed that the adhesion ability of hPMSCs was enhanced while the ability of migration was inhibited after B7H1 blockade and suggested that B7H1 plays an important role in the adhesion and migration of hPMSCs.%背景:人胎盘间充质干细胞表达的共刺激分子B7H1在其负性免疫调节作用中发挥着重要作用,但B7H1对人胎盘间充质干细胞生物学行为的影响尚未见报道.目的:观察阻断B7H1对人胎盘间充质干细胞黏附和迁移的影响.方法:采用消化法分离、培养人胎盘间充质干细胞,体外扩增培养3代后用于实验.用流式细胞仪及RT-PCR检测人胎盘间充质干细胞对B7H1的表达.用阳离子脂质体法将针对B7H1的siRNA转染入细胞,应用RT-PCR法检测B7H1基因在mRNA水平的变化.然后应用细胞计数法检测人胎盘间充质干细胞的黏附能力,Transwell法检测迁移能力.结果与结论:人胎盘间充质干细胞高表达B7H1,siRNA转染能有效阻断B7H1的表达;接种后0.5 h阻断组人胎盘间充质干细胞的黏附率与对照组相比差异无显著性意义,但在接种后1,3 h阻断组细胞黏附率均明显高于对照组(P < 0.05);Transwell检测结果显示,在SDF-1α单独存在条件下、DMEM-LG完全培养基或者人胎盘间充质干细胞培养上清条件下,阻断组细胞的迁移数量均比对照组显著减少(P < 0.01).结果表明阻断B7H1的表达后,人胎盘间充质干细胞的黏附能力增强,而迁移能力受到抑制.提示B7H1在人胎盘间充质干细胞的黏附和迁移中发挥重要作用.