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 marrowderived mesenchymal stem cells by fractlkine in vitro

作　　者：郭晓书[1] 李法琦[1] 

机构地区：[1]重庆医科大学附属第一医院老年科,重庆400016

出　　处：《重庆医科大学学报》2014年第9期1280-1286,共7页Journal of Chongqing Medical University

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。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 protein expression was detected by Western blot in LV-CX3CR1-EGFP infection group,LV-CON-EGFP infection group and non-infection 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

关 键 词：骨髓间充质干细胞 趋化因子FRACTALKINE 趋化因子受体CX3CR1 慢病毒载体 基因转染 细胞迁移 

分 类 号：Q872[生物学]