过表达基质细胞衍生因子1基因促进骨髓间充质干细胞增殖和迁移

Overexpression of stromal cell-derived factor-1 promotes the proliferation and migration of bone marrow mesenchymal stem cells in vitro

背景:基质细胞衍生因子1(stromal cell derived factor,SDF-1)/CXCR4轴不仅能够促进骨髓间充质干细胞向损伤组织迁移,而且具有抑制骨髓间充质干细胞凋亡、增加骨髓间充质干细胞存活率及增殖活性等作用。目的:建立慢病毒介导的稳定过表达SDF-1α的骨髓间充质干细胞系,在体外观察其对骨髓间充质干细胞增殖和迁移的影响。方法:构建过表达SDF-1α重组慢病毒载体(pNL-SDF-1α-IRES_2-EGFP),并以空载体质粒pNL-IRES_2-EGFP、基因沉默质粒GV-118-SDF-1α-siRNA作为实验对照,分别转染293T细胞和骨髓间充质干细胞,建立稳定过表达SDF-1α的骨髓间充质干细胞系:SDF-1α-BMSCs组、null-BMSCs组和siRNA-BMSCs组;采用RT-PCR、Western Blot方法检测SDF-1αmRNA和蛋白的表达水平;MTT法检测骨髓间充质干细胞的增殖能力;Transwell迁移实验检测SDF-1α对骨髓间充质干细胞迁移能力的影响。结果与结论:(1)成功构建pNL-SDF-1α-IRES_2-EGFP质粒,经测序结果表明pNL-SDF-1α-IRES_2-EGFP重组质粒构建成功;(2)慢病毒转染48 h后可见293T细胞和各组骨髓间充质干细胞强烈表达EGFP;(3)SDF-1α-BMSCs组高效表达SDF-1α,siRNA-BMSCs组SDF-1α表达明显被抑制;(4)SDF-1α-BMSCs组的细胞增殖能力增强,SDF-1α可明显促进骨髓间充质干细胞的跨膜迁移。在抗SDF-1α多抗作用后各组细胞迁移指数明显下降;(5)结果可见,慢病毒载体可介导外源性基因SDF-1α在大鼠骨髓间充质干细胞中高效表达,并促进骨髓间充质干细胞的增殖和迁移。

BACKGROUND:The stromal cell-derived factor-1(SDF-1)/C-X-C chemokine receptor type 4(CXCR4)signaling pathway cannot only improve the migration ability of bone marrow mesenchymal stem cells(BMSCs),but also restrain BMSCs apoptosis,increase BMSCs survival and improve the proliferation activity of BMSCs.OBJECTIVE:To construct a rat BMSCs line with SDF-1αoverexpression and to explore its influence on the proliferation and migration of BMSCs in vitro.METHODS:The SDF-1αoverexpression vector(pNL-SDF-1α-IRES2-EGFP)was constructed.The lentivirus particles were packaged by transferring pNL-SDF-1α-IRES2-EGFP,pNL-IRES2-EGFP and GV-118-SDF-1α-siRNA into 293T cells.The BMSCs lines with SDF-1αoverexpression in SDF-1α-BMSCs group,null-BMSCs group and siRNA-BMSCs group were established by transfecting SDF-1α-lentiviru,null-lentivirus and siRNA-lentivirus into BMSCs respectively.The expression of SDF-1αat mRNA and protein levels in BMSCs was evaluated by RT-PCR and western blot assay,respectively.The influence of SDF-1αon proliferation and migration of BMSCs were evaluated by MTT and Transwell migration experiment respectively.RESULTS AND CONCLUSION:The pNL-SDF-1α-IRES2-EGFP recombinant plasmid was successfully constructed,which was proved by sequencing results.EGFP was strongly expressed in 293T cells and BMSCs in all groups after 48 hours in lentivirus transfection.SDF-1αat mRNA and protein levels were highly expressed in the SDF-1α-BMSCs group,but the expression was significantly inhibited in the siRNA-BMSCs group.The proliferative ability of BMSCs was strengthened in the SDF-1α-BMSCs group,and SDF-1αwas found to significantly promote the transmembrane migration of BMSCs.The migration index of BMSCs incubated with anti-SDF-1αmulti-antibodies was restrained markedly.To conclude,the lentivirus vector cannot only infect BMSCs efficiently but also make SDF-1 expresse stably in BMSCs.The overexpression of SDF-1αcan improve the proliferation and migration abilities of BMSCs.