极度低氧条件下基质细胞衍生因子-1α过表达对间充质干细胞迁移的影响

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摘要目的研究极度低氧条件下基质细胞衍生因子-1α（SDF-1α）过表达对间充质干细胞（MSCs）迁移的影响。方法将来源于两只普通级雄性日本大耳白兔[2周龄，体重（250±30）g]的MSCs分为空白组（不进行干预），AMD3100组[加入SDF-1α受体（CXCR4）特异性抑制剂AMD3100]，SDF-1α腺病毒（Ad-SDF-1α）组（进行Ad-SDF-1α转染）和Ad-SDF-1α+AMD3100组（进行Ad-SDF-1α转染并添加AMD3100）。四组在极度低氧（1%）条件下培养48 h，通过Transwell实验检测其迁移能力，采用实时荧光定量PCR及Western blot检测迁移信号轴缺氧诱导因子-1（HIF-1α）、血管内皮生长因子（VEGF）、CXCR4的mRNA及蛋白表达情况。结果 MSCs贴壁后为梭形、多角形，呈克隆式生长，Ad-SDF-1α转染后可观察到均一且明亮的绿色荧光。AMD3100组光密度（OD）值低于空白组，Ad-SDF-1α组OD值高于空白组，Ad-SDF-1α+AMD3100组OD值高于AMD3100组且低于Ad-SDF-1α组，差异均有统计学意义（P ＜ 0.05）。AMD3100组VEGF、CXCR4 mRNA表达低于空白组，Ad-SDF-1α组HIF-1α、VEGF、CXCR4 mRNA表达均高于空白组，差异有统计学意义（P ＜ 0.05或P ＜ 0.01）。Ad-SDF-1α+AMD3100组VEGF、CXCR4 mRNA表达均高于AMD3100组，HIF-1α、VEGF、CXCR4 mRNA表达均低于Ad-SDF-1α组，差异有统计学意义（P ＜ 0.05或P ＜ 0.01）。AMD3100组HIF-1α、VEGF、CXCR4蛋白表达低于空白组，Ad-SDF-1α组HIF-1α、VEGF、CXCR4蛋白表达均高于空白组，Ad-SDF-1α+AMD3100组HIF-1α、VEGF、CXCR4蛋白表达均高于AMD3100组且均低于Ad-SDF-1α组，差异有统计学意义（P ＜ 0.05或P ＜ 0.01）。结论极度低氧条件下SDF-1α过表达仍可促进MSCs迁移，并一定程度上可逆转AMD3100的抑制效应。

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	王辉1 徐亮亮2 孙大明2 孟一2 张成龙1 许海甲1 宁宇3 李章华1

关键词 ：极度低氧, 间充质干细胞, 基质细胞衍生因子-1&alpha, 迁移

Abstract：Objective To investigate the effects of stromal cell-derived factor-1α (SDF-1α) overexpression on migration of mesenchymal stem cells (MSCs) under extreme hypoxia. Methods MSCs derived from two normal male Japanese white rabbits (two weeks of age, body weight [250±30] g) were divided into blank group (no intervention), AMD3100 group (added CXC subfamily receptor 4 [CXCR4] specific inhibitor AMD3100), SDF-1α adenovirus (Ad-SDF-1α) group (Ad-SDF-1α transfected), and Ad-SDF-1α+AMD3100 group (Ad-SDF-1α transfected and added AMD3100). The four groups were cultured under extreme hypoxia (1%) for 48 h, and the changes of migration ability were detected by Transwell experiment. The mRNA and protein expressions of HIF-1α, vascular endothelial growth factor (VEGF), and CXCR4 were detected by real-time quantitative PCR and Western blot. Results MSCs adhered to the wall in spindle and polygonal shape, and showed clonal growth. Uniform and bright green fluorescence could be observed after transfection with Ad-SDF-1α. The optical density (OD) value of AMD3100 group was lower than that of blank group, OD value of Ad-SDF-1α group was higher than that of blank group, OD value of Ad-SDF-1α+AMD3100 group was higher than that of AMD3100 group and lower than that of Ad-SDF-1α group, the differences were statistically significant (P ＜ 0.05). The mRNA expressions of VEGF and CXCR4 in AMD3100 group were lower than those in blank group, while the mRNA expressions of HIF-1α, VEGF, and CXCR4 in Ad-SDF-1α group were higher than those in blank group, the differences were statistically significant (P ＜ 0.05 or P ＜ 0.01). The mRNA expressions of VEGF and CXCR4 in Ad-SDF-1α+AMD3100 group were higher than those in AMD3100 group, while the mRNA expressions of HIF-1α, VEGF, and CXCR4 were lower than those in Ad-SDF-1α group, the differences were statistically significant (P ＜ 0.05 or P ＜ 0.01). The protein expressions of HIF-1α, VEGF, and CXCR4 in AMD3100 group were lower than those in blank group, while the protein expressions of HIF-1α, VEGF, and CXCR4 in Ad-SDF-1α group were higher than those in blank group, the protein expressions of HIF-1α, VEGF, and CXCR4 in Ad-SDF-1α+AMD3100 group were higher than those in AMD3100 group and lower than those in Ad-SDF-1α group, the differences were statistically significant (P ＜ 0.05 or P ＜ 0.01). Conclusion SDF-1α overexpression can still promote MSCs migration under extreme hypoxia, and reverse the inhibitory effect of AMD3100 to some extent.

Key words： Extreme hypoxia Mesenchymal stem cells Stromal cell-derived factor-1α Migration

基金资助:国家自然科学基金资助项目（81472103）；
湖北省自然科学基金项目（2020CFB369）；
湖北省卫生健康委面上项目（WJ2021M010）；
湖北省武汉市医学科研项目（WX18 M01、WX20D91）；
湖北省武汉市科技局应用基础前沿项目（2019020701011471）；
湖北省襄阳市科技局医疗卫生领域科技计划项目（2020YL25）。

通讯作者: 李章华（1973.7-），男，博士，教授，主任医师，硕士生导师；研究方向：股骨头坏死的发生机制与基因治疗。

作者简介: 王辉（1995.8-），男，武汉大学第二临床学院2019级骨科专业在读硕士研究生；研究方向：股骨头坏死的发生机制与基因治疗。

引用本文:

王辉1 徐亮亮2 孙大明2 孟一2 张成龙1 许海甲1 宁宇3 李章华1. 极度低氧条件下基质细胞衍生因子-1α过表达对间充质干细胞迁移的影响[J]. 中国医药导报, 2022, 19(16): 5-10.
WANG Hui1 XU Liangliang2 SUN Daming2 MENG Yi2 ZHANG Chenglong1 XU Haijia1 NING Yu3 LI Zhanghua1. Effects of stromal cell-derived factor-1α overexpression on migration of mesenchymal stem cells under extreme hypoxia. 中国医药导报, 2022, 19(16): 5-10.

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