普罗布考对糖尿病小鼠脂肪间充质干细胞功能的影响
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辽宁省科技厅项目(2011225020);辽宁省科技厅项目(2012225014);沈阳市科技局项目(F11-262-9-52)


Effect of Probucol on Function of Diabetic Mice Adipose Tissue-derived Stem Cell
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    摘要:

    目的探讨普罗布考对糖尿病鼠脂肪来源的间充质干细胞(ADSC)功能的影响。方法体外分离培养ADSC,应用流式细胞术对3~5代细胞表面抗原CD34、CD45、CD90、CD105进行表型鉴定。将ADSC分为:①正常ADSC对照组;②正常ADSC+普罗布考组;③糖尿病ADSC对照组;④糖尿病ADSC+普罗布考组;⑤正常ADSC+高糖(30mmol/L)组;⑥正常ADSC+高糖+普罗布考组。应用WST法和Transwell迁移实验检测各组细胞增殖和迁移情况。应用ELISA和实时定量PCR检测各组细胞分泌血管内皮生长因子(VEGF)、肝细胞生长因子(HGF)、胰岛素样生长因子1(IGF-1)蛋白和mRNA表达水平。应用二氯醋酸荧光素探针检测各组细胞活性氧(ROS)含量。结果ADSC表达CD90和CD105,不表达CD34和CD45;与正常ADSC对照组相比,糖尿病ADSC增殖能力和迁移能力下降,VEGF、HGF、IGF-1蛋白和mRNA表达水平降低。与糖尿病ADSC对照组相比,糖尿病ADSC+普罗布考组增殖能力和迁移能力增加,VEGF、HGF、IGF-1蛋白和mRNA表达水平上升。结论糖尿病能损伤ADSC的生物学特性。普罗布考对糖尿病鼠ADSC增殖、迁移和分泌能力具有保护作用。

    Abstract:

    Aim To explore the effect of probucol on function of diatetic mice adipose tissue-derived stem cells (ADSC).Methods ADSC were isolated by mechanical separation and enzymatic digestion from nondiabetic and diabetic mice.The expression profiles of CD34,CD45,CD90 and CD105 were examined by flow cytometry.The ADSC were divided into 6 groups: control normal group,probucol normal group,control diabetic group,probucol diabetic group,high glucose normal group and probucol high glucose normal group.The proliferation and migration of ADSC was determined by WST-8 assay and transwell assay respectively.In addition,the mRNA and protein expression of vascular endothelial growth factor (VEGF),hepatocyte growth factor (HGF),and insulin-like growth factor-1(IGF-1) were determined by real-time PCR and ELISA analysis.The content of reactivated oxygen species (ROS) was also measured.Results The morphological feature of ADSC displayed fibroblast-like phenotype.The cells were positive for stem cells markers,including CD90 and CD105,and negative for CD34 and CD45,as shown by flow cytometry.Diabetic ADSC showed decreased proliferative potential and migration.In addition,the mRNA expression of VEGF,HGF and IGF-1 in diabetetic control group was obviously lower than that in nondiabetic control group.The contents of VEGF,HGF and IGF-1 on ADSC -conditioned medium in diabetic control group was also obviously lower than that in nondiabetic control group.Probucol promoted proliferation and migration of diabetic ADSC,and increase the mRNA expression of VEGF,HGF and IGF-1 in diabetetic ADSC.In addition,Probucol could increase contents of VEGF,HGF and IGF-1 in ADSC -conditioned medium in diabetic mice.Conclusion Our data indicate that diabetes alters ADSC intrinsic properties and impairs their function.Probucol can protect diabetic ADSC function of proliferation and migration,as well as releasing growth factors.

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王哲,刘晓玉,张殿宝,王秋实.普罗布考对糖尿病小鼠脂肪间充质干细胞功能的影响[J].中国动脉硬化杂志,2014,22(12):1207~1212.

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  • 收稿日期:2014-02-13
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