【摘要】 构建一种高表达基质细胞衍生因子受体(CXCR4)的大鼠骨髓间充质干细胞(MSCs),考察CXCR4对细胞定向迁移能力的影响。实验构建了pMSCV-CXCR4-IRES-GFP基因质粒,并通过逆转录病毒将其转入预先培养的MSCs中。分别采用流式细胞分析仪、反转录聚合酶链反应(RT-PCR)和免疫荧光染色法检测CXCR4在MSCs中的表达,并利用Transwell方法检测基质细胞衍生因子(SDF-1)对MSCs体外定向迁移的影响。流式细胞仪分析显示,转基因MSCs表面CXCR4的阳性率为46%,绿色荧光蛋白(GFP)阳性率为57%。RT-PCR进一步证实了CXCR4的表达。细胞迁移实验结果表明,CXCR4基因修饰促进了MSCs向SDF-1的定向迁移,当SDF-1浓度为50 ng/ml时,这种定向迁移能力较之对照组提高了近5倍。通过以上结果可以初步证实,MSCs表面CXCR4表达量在SDF-1/CXCR4介导的细胞迁移通路中发挥着重要作用,这为进一步研究MSCs的体内迁移和归巢提供了依据。更多还原

【Abstract】 This study was amied to construct CXCR4 gene modified bone marrow mesenchymal stem cells(MSCs),and investigate the effect of CXCR4 expression on MSCs migration.The retrovirus vector pMSCV-CXCR4-IRES-GFP that expresses human CXCR4 gene was cloned,the MSCs were transduced by the virus,and the expression of CXCR4 was analyzed by FACS,RT-PCR and immunofluorescence staining.The migration assay was performed using Transwell method in the presence of SDF-1.FACS results showed that 46% of the transduced MSCs were CXCR4 positive,and 57% were GFP positive.The expression of CXCR4 in MSCs was also confirmed by RT-PCR and immunostaining.The migration of MSCs was induced by SDF-1 and was strongly dependent on CXCR4 expression.The concentration of SDF-1 had effect on the migration and the transmigration rate of CXCR4 modified;the amount of MSCs was 5-fold higher than that of untransduced MSCs when the optimal concentration rose to 50 ng/ml.These data indicate that SDF-1/CXCR4 plays an important role in MSCs migration,and the CXCR4 genetic modification approach could be applied to enhance cell homing,and engraftment in MSCs therapy.更多还原