共表达EGFP和CXCR4的大鼠骨髓间充质干细胞的构建及其迁移能力

Construction and migration of rat bone marrow mesenchymal stem cells coexpressing EGFP and CXCR4

目的构建携带增强绿色荧光蛋白(EGFP)及CXCR4慢病毒载体,并实现其在大鼠骨髓间充质干细胞(rMSCs)中的表达,观察转CXCR4基因后对rMSCs迁移能力的影响。方法 RT-PCR扩增大鼠CXCR4编码区片段,将其插入慢病毒载体质粒PNL-IRES2-EGFP,获得的PNL-CXCR4-IRES2-EGFP与包装及包膜质粒用脂质体法共转染293T细胞,包装生产慢病毒。所获慢病毒转染rMSCs后,用RT-PCR、Western blot、细胞免疫荧光组织化学和流式细胞术检测转CXCR4基因rMSCs组、转空载体rMSCs组中CXCR4表达情况。利用Transwell方法检测两组rMSCs在SDF-1作用下的迁移能力。结果双酶切和测序证实,PNL-CXCR4-IRES2-EGFP构建正确,转CXCR4基因rMSCs组CXCR4表达明显增多,在SDF-1α作用下迁移能力明显增强。结论成功构建带有EGFP和大鼠CXCR4的慢病毒载体,并获得CXCR4-rMSCs基因工程细胞,为深入研究SDF/CXCR4轴在rMSCs向损伤组织定向迁移中的作用奠定了基础。

Objective To construct rat mesenchymal stem cells（rMSCs） coexpressing CXCR4 and EGFP and to observe the effect of CXCR4 on rMSCs migration.Methods The total RNA was isolated from SD rat＇s liver with Trizol,the CXCR4 gene was amplified by RT-PCR.The CXCR4 was inserted into the transfer vector of lentivirus after the vector was digested with restriction endonuclease.The lentiviral particles were produced in 293T cells by transient cotransfection involving a three-plasmid expression system,then were harvested and concentrated.The rMSCs were transfected by obtained lentiviral particles or null lentiviral particles.The expression of CXCR4 gene in CXCR4-rMSCs or null-rMSCs was examined with RT-PCR,Western blot,cellular immunofluorescence and flow cytometry.The migration of transfected cells induced by SDF-1 was assessed in the transwell system.Results The full-length fragment of CXCR4 gene was successfully cloned into the transfer vector of lentivirus.The result of sequencing showed that the cloned CXCR4 gene was consistent with that reported in GenBank.The CXCR4 gene-modified rMSCs expressed both the CXCR4 and EGFP.The expression of CXCR4 gene in CXCR4-rMSCs group（experimental group） was significantly higher than that in null-rMSCs group（control group） at both mRNA and protein levels.The migration ratios of the CXCR4-rMSCs group was significantly higher than that of the null-rMSCs group.Conclusion The rMSCs coexpressing CXCR4 and EGFP was successfully constructed,this will greatly facilitate further research,such as the evaluation of the effect of SDF-1/CXCR4 on rMSCs＇ recruitment to damaged tissue.