To investigate the effect of CXCR4 gene transfection on BMSCs directional migration and its possible mechanism. Methodology; With Gateway technique, plasmid containing CXCR4 target gene was constructed ( eGFP as the tracer gene) , and blank control carrier ( only carrying eGFP) was constructed at the same time. CXCR4-BMSCs and null-BMSCs were constructed by transfecting BMSCs with lentiviral vector that carried the above plasmid. Cell viability, differentiation potential and CXCR4 expression were detected. RTECs were cultured in hypoxia/re-oxygenation condition for 12h,respectively. BMSCs, CXCR4-BMSCs and null-BMSCs were co-cultured with HR-RTECs, respectively, and to co-culture BMSCs with RTECs was set as the control. Four experimental groups were included. After 48h co-culturing, the number of migrating BMSCs was detected. SDF-1 levels in the RTECs culture supernatant, and pAKT and pMAPK in BMSCs were measured. AKI mice were randomly divided into BMSCs transplantation group, CXCR4-BMSCs transplantation group and null-BMSCs transplantation group. The transplanted cells were all marked by BrdU. 7d later, the mice were sacrificed and distribution of BMSCs in the nephridial tissue was measured by immunohistochemistry. Results: Cell viability and differentiation potential were not changed for the both transfected BMSCs, and CXCR4 expression was enhanced in CXCR4-BMSCs. Co-culturing with HR-RTECs enhanced BMSCs migration. Blank carrier transfection showed no additional influence. While for CXCR4-BMSCs, the migrating number was increased significantly. SDF-1 level in the HR-RTECs culture supernatant was increased with corresponding increased levels of pAKT and pMAPK in all the three kinds of co-cultured BMSCs, the highest for CXCR4-BMSCs. CXCR4 gene modification increased the proportion of BrdU+ cells ( BMSCs) in the AKI nephridial tissue. Conclusion: AKI microenvironment has obvious chemotaxis effect on BMSCs. CXCR4 gene modification can further enhance the directional migration of BMSCs. SDF-1/CXCR4 axle with its downstream AKT and MAPK signaling pathway is the possible mechanism in achieving this function. CXCR4-BMSCs transplantation is expected to be a novel and effective approach for AKI repair.%目的:低氧/复氧预处理肾小管上皮细胞(HR-RTECs)体外模拟急性肾损伤(AKI),与CXCR4基因修饰的骨髓间充质干细胞(BMSCs)共培养,观察该共培养环境下CXCR4基因修饰对BMSCs定向迁移力的影响,探讨其可能机制,并以动物模型进一步验证. 方法:Gateway技术构建含CXCR4目的基因的质粒[绿色荧光蛋白(eGFP)为示踪基因)],同时构建仅含eGFP的对照载体,以携带上述质粒的慢病毒为载体转染BMSCs合成CXCR4-BMSCs和null-BMSCs,检测转染细胞活力、分化潜能及CXCR4在转染细胞中的表达.RTECs于HR环境中各培养12h获得HR-RETCs.实验共分四组,将BMSCs、CXCR4-BMSCs、null-BMSCs分别与HR-RTECs共培养,BMSCs与RTECs共培养作为对照.培养48h后检测各组BMSCs的定向迁移能力,ELISA检测RTECs培养上清中基质细胞衍生因子1(SDF-1)浓度,Western印迹检测BMSCs内pAKT、pMAPK水平.构建AKI小鼠模型,随机分为BMSCs移植组、CXCR4-BMSCs移植组和null-BMSCs移植组,移植细胞均采用5溴脱氧尿嘧啶核苷(BrdU)标记,移植7d后处死,免疫组化法检测移植细胞在肾组织的分布. 结果:成功转染的BMSCs活力及分化潜能均无变化,CXCR4表达在CXCR4-BMSCs中明显增加.HR-RTECs模拟的AKI共培养环境可增强BMSCs的迁移能力,空载体转染对BMSCs的迁移能力并无额外影响,但CXCR4修饰的BMSCs向AKI微环境的迁移细胞数进一步显著增加.HR-RTECs培养上清中的SDF-1浓度增加,与其共培养的三种BMSCs内的pAKT、pMAPK水平也均增加,且以CXCR4-BMSCs的pAKT、pMAPK水平最强.CXCR4基因修饰可显著增加AKI肾组织中BrdU+细胞(BMSCs)的比例. 结论:AKI微环境对BMSCs有明显趋化作用,CXCR4基因修饰可进一步增强BMSCs的定向迁移能力,SDF-1/CXCR4轴为发挥作用的主要趋化因子/受体,其下游的AKT和MAPK通路为发挥作用的可能信号机制.CXCR4-BMSCs移植有望成为修复AKI的一种新的有效方法.
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