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.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
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）
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
Bone marrow- derived mesenchymal stem cells
Hypoxia/re- oxygenation
Renal tubular epithelial cells
CXCR4
Repair
