摘要
背景:研究证明,低氧微环境下局部内皮祖细胞通过释放基质细胞衍生因子1等细胞因子促进内皮祖细胞的动员、归巢。目的:分析不同低氧时间点对内皮祖细胞基质细胞衍生因子1蛋白表达的影响,及对内皮祖细胞迁移影响,为治疗缺血性疾病提供新的思路。方法:(1)取SD大鼠的四肢长骨骨髓,分离、培养内皮祖细胞并鉴定;(2)将分离培养的细胞分4组置于常氧和低氧条件下:常氧对照组、低氧6 h组、低氧12 h组、低氧24 h组;(3)采用ELISA法、免疫荧光法检测不同氧气浓度和时间条件下内皮祖细胞上清液基质细胞衍生因子1的表达;RT-PCR及Western Blot检测不同条件下基质细胞衍生因子1 mR NA和蛋白的表达;Transwell检测低氧对内皮祖细胞迁移的影响。结果与结论:(1)实验分离培养的细胞经鉴定为内皮祖细胞;(2)ELISA和免疫荧光检测结果均显示:与常氧对照组比较,培养上清液中基质细胞衍生因子1的表达在低氧6 h开始增强,12 h表达最强,24 h又有所减弱;(4)RT-PCR及Western Blot检测结果显示:与常氧对照组相比,低氧诱导基质细胞衍生因子1的mR NA和蛋白相对表达量显著上调(P<0.01),其中,低氧12 h条件下基质细胞衍生因子1的表达最强,依次为低氧24 h和低氧6 h;(5)与常氧对照组比较,低氧组24 h穿膜细胞数显著增加(P<0.05),低氧12 h组显著多于低氧6 h组和低氧24 h组;(6)结果表明,低氧能够促进基质细胞衍生因子1的分泌,并诱导内皮祖细胞迁移。
BACKGROUND:Local endothelial progenitor cells(EPCs)have been reported to promote migration and homing of EPCs by releasing stromal cell-derived factor-1(SDF-1)under hypoxia environment.OBJECTIVE:To investigate the effect of different hypoxia periods on the expression of SDF-1 protein in EPCs and the migration of EPCs,thus providing new ideas for the treatment of ischemic diseases.METHODS:The bone marrow of the long bone was removed from Sprague-Dawley rats used for isolate EPCs.After culture and identification,EPCs were divided into four groups:control(normoxia),6-,12-and 24-hour hypoxia groups.The expression level of SDF-1 in the supernatant of EPCs in each group was detected by ELISA and immunofluorescence.The mRNA and protein levels of SDF-1 were detected by RT-PCR and western blot assay.The effect of hypoxia on the migration of EPCs was tested by Transwell assay.RESULTS AND CONCLUSION:The obtained cells were identified as EPCs.ELISA and immunofluorescence results showed that compared with the control group,the expression level of SDF-1 began to increase at 6 hours of hypoxia,peaked at 12 hours of hypoxia,and then decreased at 24 hours of hypoxia.RT-PCR and western blot assay results found that the mRNA and protein expression levels of SDF-1 were the highest in the 12-hour hypoxia group,followed by 6-and 24-hour hypoxia groups,and the lowest in the control group(P<0.01).The number of EPCs penetrating the membrane in the 12-hour hypoxia group was significantly more than that in the 6-and 24-hour hypoxia groups,and the number in the 24-hour hypoxia group was significantly more than that in the control group(P<0.05).In summary,hypoxia can promote the secretion of SDF-1 and induce the migration of EPCs.
作者
赵辉
郜玉忠
Zhao Hui;Gao Yu-zhong(the First Affiliated Hospital of Jinzhou Medical University,Jinzhou 121000,Liaoning Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2018年第12期1902-1908,共7页
Chinese Journal of Tissue Engineering Research
基金
辽宁省自然科学基金项目资助(2014022010)~~
