过表达CXCR4的骨髓间充质干细胞对共培养低氧／复氧肾小管上皮细胞的修复作用  

Effect of CXCR4-overexpressing bone marrow-derived mesenchymal stem cells on the repair of the co- cultured hypoxia/re- oxygenation renal tubular epithelial cells and its possible mechanism

作　　者：刘楠梅[1] 梅长林[1] 张金元[2] 田军[2] 程劲[2] 

机构地区：[1]第二军医大学附属长征医院肾内科解放军肾脏病研究所,上海200003 [2]解放军第四五五医院肾内科南京军区肾脏病研究所

出　　处：《中华肾脏病杂志》2013年第11期830-836,共7页Chinese Journal of Nephrology

基　　金：国家自然科学基金青年项目（81300568）;全军医学科技青年培育基金（13QNP050）;上海市青年科技启明星计划项目（12QA1405000）;上海市自然科学基金（11ZRl449600）;上海市基础研究重大项目（12DJ1400203）;上海市卫生系统优秀青年人才培养计划（XYQ2013088）

摘　　要：目的构建CXCR4质粒并转染小鼠骨髓间充质干细胞（BMSC），与低氧／复氧（hypoxia／re—oxygenation，HR）预处理的肾小管上皮细胞（RTEC）共培养，观察CXCR4-BMSC对HR—RTEC的修复效应并探讨其机制。方法采用基因转染技术获得CXCR4-BMSC（CXCR4-BMSC／eGFP，eGFP为示踪基因）和null—BMSC（BMSC／eGFP），检测转染细胞的CXCR4表达。RTEC于低氧／复氧环境中各培养12h获得HR．RETC，体外模拟急性肾损伤（AKI）细胞模型。BMSC与HR—RTEC共培养12h，免疫荧光法检测HR—RTEC的凋亡细胞比例，Western印迹法检测HR—RTEC内的凋亡相关蛋白cleavedCaspase-3和Bcl．2水平，结晶紫法计数迁移BMSC数量。以HR-RTEC上清液分别干预BMSC、CXCR4-BMSC和null—BMSC，免疫荧光法检测各组BMSC角蛋白18（CKl8）的表达，ELISA法检测BMSC上清中骨形态发生蛋白7（BMP-7）、肝细胞生长因子（HGF）和白细胞介素10（IL—lo）的浓度。结果成功转染的CXCR4．BMSC可高效表达CXCR4。HR—RTEC分别与BMSC、CXCR4-BMSC、null—BMSC共培养后，凋亡细胞比例均有下降，尤以与CXCR4．BMSC共培养组降低最为明显，并伴随着细胞内cleavedCaspase．3水平显著降低、Bcl-2表达升高（均P〈0．05）。结晶紫计数显示CXCR4-BMSC向HR—RTEC培养室的迁移数量最多。经HR—RTEC上清液干预后，BMSC、CXCR4-BMSC和null．BMSC均仅能微量表达CKl8，各组间差异无统计学意义。CXCR4过表达可显著增加BMSC的BMP-7、HGF和IL—10分泌。结论过表达CXCR4的BMSC对共培养HR—RTEC的修复效应增强，BMSC的定向迁移能力增加和迁移BMSC的分泌能力增强可能是CXCR4-BMSC的作用机制。Objective CXCR4-overexpressing bone marrow-derived mesenchymal stem cells （CXCR4-BMSC） were constructed and co-Cultured with hypoxia/re-oxygenation pretreated renal tubular epithelial cells （HR-RTEC）. Repair of HR-RTEC was detected and the possible mechanism was also discussed. Methods CXCR4-BMSC （CXCR4-BMSC/eGFP, eGFP as the tracer gene） and nnll-BMSC （BMSC/eGFP） were obtained by gene transfection technique, and the level of CXCR4 in the transfected cells was detected. RTEC was cultured under hypoxia/re-oxygenation condition for 12 h, respectively, to obtain HR-RTEC, which was used to simulate AKI in vitro. BMSC and HR-RTEC were co-cultured for 12 h, and the proportion of apoptotic cells among the HR- RTEC was assayed by immunofluorescence technique. Western blot was used to test the protein levels of cleaved Caspase-3 and Bcl- 2. The number of migrating BMSC was also assayed. After culturing with the HR- RTEC culture supernatant, the expression of cytokeratin 18 （CK18） in BMSC was tested by immunofluorescence staining. Cytokines including bone morphogenetic protein- 7 （BMP- 7）, hepatic growth factor （HGF） and interleukin- 10 （IL-10） in the BMSC culture supernatant were detected by ELISA method. Results Expression of CXCR4 was enhanced in CXCR4-BMSC. Proportions of the apoptotic cells among HR-RTEC after being co-cultured with BMSC, CXCR4-BMSC and null-BMSC were all decreased, especially in the C/H group. The decreased cleaved Caspase-3 and enhanced Bcl-2 were also observed in HR-RTEC. The number of migrating CXCR4-BMSC was the highest. Proportions of CK18 ~ cells in BMSC, CXCR4- BMSC and null- BMSC were all low and showed no difference. However, CXCR4 overexpression in BMSC stimulated secretions of BMP- 7, HGF and IL- 10. Conclusions CXCR4-overexpressing BMSC has more repair effect on the co-cultured HR-RTEC, the enhanced migration ability and secretion ability of CXCR4-BMSC are the possible mechanisms.

关 键 词：骨髓间充质干细胞 肾小管上皮细胞 修复 低氧 复氧 CXCR4 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]