CXCR4基因修饰对骨髓间充质干细胞向急性肾损伤微环境定向迁移的放大效应及机制

Magnification effect for CXCR4 gene transfection on bone marrow-derived mesenchymal stem cells migration toward the acute kidney injury microenvironment and its mechanism

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。

Objective：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/reoxygenation 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 superuatant, andpAKT 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 CXCRd-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.