Objective: To construct mouse CXC chemokine receptor type 4 (Cxcr4) gene overexpressing lentiviral vector and to evaluate its biological effect on mouse mesenchymal stem cells (MSCs).
Methods: Cxcr4 gene was amplified and subcloned into pCR-Blunt vector. Cxcr4 gene and enhanced green fluorescent protein (EGFP) gene expressed bicistronic recombinant lentiviral vector LV-CXCR4-IRES-EGFP and control vector LV-IRES-EGFP were constructed, respectively. Both plasmids were co-transfected into 293FT packaging cell line with packaging plasmid pSPAX2 and enveloping plasmid pMD.2G using Lipofectamine 2000 to produce lentiviral virus, respectively. The recombinant viruses were harvested and the virus titer was determined by limiting dilution. Mouse MSCs were infected with viral supernatant. EGFP expression was visualized using fluorescence microscope and efficiency of infection was determined by flow cytometry (FCM). Cell counting kit-8 (CCK-8) was applied in mixed lymphocyte reaction (MLR) to evaluate the suppressive effect of MSCs on mice splenocyte proliferation in vitro. Wound healing ability of MSCs was measured by scratch experiment and migration capacity by a chemotaxis assay using a transwell assay.
Results: The Cxcr4 fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) and verified by DNA sequencing. The restriction enzyme digestion experiment demonstrated that the recombinant lentiviral vector LV-CXCR4-IRES-EGFP and the control vector LV-IRES-EGFP were successfully constructed. Expression of CXCR4 was detected by fluorescence microscopy, which indicated that the lentiviral particles expressing CXCR4 were packaged. Furthermore, expression of EGFP were detected by fluorescence microscopy in MSCs after infection and the expression of CXCR4 protein on MSCs surface in CXCR4-MSC group was significantly increased comparing to those in the control group(P < 0.05).CXCR4-MSCs group and the control group were (90.3 ± 3.37)% and (1.53 ± 0.34)%, respectively. Meanwhile, overexpression of CXCR4 had no effect on their capacity of immune regulation when co-cultured with splenocyte(P > 0.05). Moreover, overexpression of CXCR4 can not only accelerated the wound healing after scratch, but also enhanced the migration ability of cells in the transwell induced by high concentration of SDF-1 in a dose-dependent manner compared with the EGFP control group.
Conclusion: The CXCR4 expressing lentiviral vector LV-CXCR4-IRES-EGFP was successfully constructed. The lentiviral vector can not only efficiently infect mouse MSCs, but also stably express CXCR4 in MSCs. The MSCs modified with CXCR4 have biological characteristic in vitro.
目的 探讨慢病毒表达载体介导的小鼠CXC型趋化因子受体4 （CXCR4）基因体外对小鼠骨髓间充质干细胞（MSC）生物学特性的影响。方法 克隆小鼠CXCR4基因，构建携带CXCR4基因与增强型绿色荧光蛋白（EGFP）双顺反子结构的重组慢病毒表达载体LV-CXCR4-IRES-EGFP，同时构建对照载体LV-IRES-EGFP。脂质体法包装病毒，并测定滴度。通过优化慢病毒感染MSC条件，建立高表达CXCR4的MSC，荧光显微镜观察EGFP表达，流式细胞术检测细胞表面CXCR4表达，CCK-8法检测MSC对单向混合淋巴细胞培养体系中淋巴细胞增殖的影响，划痕实验和Transwell实验检测MSC体外迁移能力。结果 成功克隆小鼠CXCR4基因，并构建重组慢病毒载体LV-CXCR4-IRES-EGFP和对照载体LV-IRES-EGFP。制备共表达CXCR4和报告基因EGFP的高滴度慢病毒颗粒。优化感染条件后，LV-CXCR4-IRES-EGFP感染组MSC表面CXCR4的表达明显高于LV-IRES-EGFP对照组（P<0.05），分别为（90.3±3.37）%和（1.53±0.34）%。过表达CXCR4基因不影响MSC对T淋巴细胞增殖的抑制作用（P>0.05）。划痕实验和Transwell实验结果表明，过表达CXCR4可明显提高MSC对划痕损伤修复能力和向高浓度基质细胞衍生因子-1（SDF-1）的迁移能力，并且具有剂量依赖性。结论 成功构建重组慢病毒载体LV-CXCR4-IRES-EGFP，慢病毒载体系统可高效介导CXCR4基因在小鼠MSC表达，并具有生物学活性。