转染CX3CR1基因对小鼠骨髓间充质干细胞向Fractalkine趋化能力的影响

Effects of transfection of CX3CR1 gene on migration of mouse bone marrow-derived mesenchymal stem cells by fractlkine in vitro

摘要:

目的：研究在Transwell共培养体系中，转染CX3CR1基因对小鼠骨髓间充质干细胞（mesenchymal stem cells，MSCs）向趋化因子Fractalkine（FKN）趋化能力的影响。方法：采用差速贴壁法分离纯化小鼠MSCs，体外培养、传代扩增，流式细胞术检测细胞表面抗原标志和细胞周期；将携带有趋化因子受体CX3CR1基因及绿色荧光蛋白（green fluorescent protein，GFP）基因的慢病毒（LV-CX3CR1-EGFP）及空载体病毒（LV-CON-EGFP）转染小鼠MSCs细胞，RT-PCR法分别检测空白对照组、实验组、阴性对照组细胞中CX3CR1 mRNA表达情况，Western blot印记法检测CX3CR1蛋白的表达水平，并通过Transwell共培养体系观察不同转染组细胞向趋化因子FKN的迁移情况。结果：成功获得了细胞形态均一，生长状态良好的MSCs；细胞CD29、CD44、CD34有阳性表达；阴性对照组和空白对照组小鼠MSCs不表达CX3CR1基因；构建的慢病毒过表达载体pUbi-MSC-CX3CR1-EGFP能成功转染至小鼠MSCs中，并使转染后的MSCs表达CX3CR1 mRNA及蛋白。在Transwell共培养体系中，转染CX3CR1基因的小鼠MSCs向趋化因子FKN趋化能力明显强于各对照组（P=0.000），其中阴性对照组浸润至下室的细胞数为：（13.80±2.17）个每高倍镜视野（n=5）。实验组浸润至下室的细胞数为：（35.80±3.34）个每高倍镜视野（n=5）。FKN抗体阻断组浸润至下室的细胞数为（14.80±1.92）个每高倍镜视野（n=5）。结论：转染CX3CR1基因能有效增加小鼠MSCs向趋化因子FKN趋化的能力。

Abstract:

Objective：To investigate the roles of Fractalkine in triggering the migration of the mesenchymal stem cells（MSCs） in the Transwell co-culture system. Methods：MSCs were isolated and cultured through the whole bone marrow method and were amplified by the different-speed adherent method. The morphology and biological characteristics of MSCs were examined by flow cytometry. And then the recombinant adenovirus containing CX3CR1 and green fluorescent protein（GFP）（LV-CX3CR1-EGFP） or empty vector（LV-CON-EGFP） were transfected into MSCs. The expression of CX3CR1 mRNA in MSCs was examined by RT-PCR and the pro－tein expression was detected by Western blot in LV-CX3CR1-EGFP infection group，LV-CON-EGFP infection group and non-infec－tion group. The migration of MSCs was detected with Transwell co-culture system. Results：Obtained MSCs were homogeneous in cell morphology with good living condition and the cells showed positive with CD29，CD44，CD34 .There was no expression of CX3CR1 in LV-CON-EGFP group and non-infection group but CX3CR1 can be expressed in LV-CX3CR1-EGFP group. Results of Transwell assays suggested that the migration level of MCSs was higher in LV-CX3CR1-EGFP group than in other groups（P=0.000）. The migrated MSCs were 13.80±2.17/HP，35.80±3.34/HP and 14.80±1.92/HP in LV-CON-EGFP group，LV-CX3CR1-EGFP group and non-infection group respectively. Conclusion：The transfection of CX3CR1 gene could improve the level of migration of MSCs by FKN in vitro.1