microRNA-223对糖基化终产物诱导人脂肪间充质干细胞凋亡和氧化应激影响的体外研究
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( 1.中国医科大学盛京医院病理科,辽宁省沈阳市 110004;2.中国医科大学细胞生物学卫生部重点实验室 干细胞与再生医学研究室,辽宁省沈阳市 110001)

作者简介:

王哲,博士,主治医师,讲师,研究方向为干细胞与再生医学,E-mail为wz_cmu@126.com。

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国家自然科学基金项目(81601692);辽宁省教育厅科学研究项目(LK201602)


Effect of microRNA-223 on apoptosis and oxidative stress of human adipose tissue-derived mesenchymal stem cell induced by advanced glycosylation end product in vitro
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1.Department of Pathology, Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004, China;2.Institute of Stem Cells and Reproductive Medicine & Key Laboratory of Cell Biology, Ministry of Health, China Medical University, Shenyang, Liaoning 110001, China)

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    摘要:

    目的 观察microRNA-223(miR-223)对糖基化终产物(AGE)诱导人脂肪间充质干细胞(hADSC)凋亡和氧化应激的影响。方法 采用酶消化法分离培养hADSC,并应用流式细胞术对表面抗原CD14、CD34、CD45、CD90、CD105和人类白细胞抗原DR(HLA-DR)进行检测。将hADSC分为牛血清白蛋白(BSA)对照组、AGE修饰的牛血清白蛋白(AGE-BSA)作用组、miR-223模拟物转染组、miR-223模拟物转染+AGE-BSA组、miR-223抑制物转染组和miR-223抑制物转染+AGE-BSA组。应用CCK-8法和TUNEL染色检测各组细胞存活率和凋亡率,Western blot检测Cleaved Caspase3蛋白表达水平,应用双氯荧光素(DCFH-DA)试剂盒检测各组细胞活性氧(ROS)含量。结果 流式细胞术检测结果表明,原代培养的hADSC高表达CD14、CD90和CD105,不表达CD34、CD45和HLA-DR。CCK-8法、TUNEL染色、Western blot和DCFH-DA法检测结果表明,与BSA对照组相比,AGE-BSA作用组hADSC凋亡率、miR-223表达水平、Cleaved Caspase3蛋白表达水平、ROS生成显著升高,而细胞存活率下降(P<0.05);与AGE-BSA作用组相比,miR-223模拟物转染能够进一步上调AGE-BSA引起的hADSC凋亡率、Cleaved Caspase3蛋白表达水平和ROS生成增加,下调AGE-BSA引起的hADSC存活率减少(P<0.05);与AGE-BSA作用组相比,miR-223抑制物转染能够抑制hADSC凋亡率、Cleaved Caspase3蛋白表达水平和ROS生成增加,拮抗AGE-BSA引起的hADSC存活率减少(P<0.05)。结论 miR-223高表达能够促进AGE-BSA引起的hADSC细胞凋亡和ROS生成,其可能作为调控hADSC促进糖尿病创伤愈合的新靶点。

    Abstract:

    Aim To investigate the effect of microRNA-223 (miR-223) on apoptosis and oxidative stress of human adipose tissue-derived mesenchymal stem cell (hADSC) induced by advanced glycosylation end product (AGE). Methods The hADSCs were isolated and cultured by enzymatic digestion, and flow cytometry was used to detect the surface antigen (CD14, CD34, CD45, CD90, CD105 and HLA-DR) of hADSC. The hADSCs were divided into 6 groups:bovine serum albumin (BSA) group, AGE modified BSA (AGE-BSA) treatment group, miR-223 mimic transfection group, miR-223 mimic transfection+AGE-BSA group, miR-223 inhibitor transfection group, and miR-223 inhibitor transfection+AGE-BSA group. Cell viability and apoptosis rate were evaluated by CCK-8 assay and TUNEL assay respectively.Western blot was used to assess the expression of Cleaved Caspase3 protein in hADSC. The content of reactive oxygen species (ROS) in hADSC was determined by 2’,7’-dichlorfluorescein-diacetate (DCFH-DA) reagent kit. Results The hADSCs were positive for stem cell markers, including CD14, CD90 and CD105, and negative for CD34, CD45 and HLA-DR, as shown by flow cytometry. CCK-8 method, TUNEL staining, Western blot and DCFH-DA test results showed that:Compared with the BSA control group, the hADSC apoptosis rate, miR-223 expression, Cleaved Caspase3 protein expression and ROS production increased significantly in AGE-BSA treatment group, but hADSC survival rate decreased (P<0.05); Compared with AGE-BSA treatment group, miR-223 mimic transfection could further increase the hADSC apoptosis rate, Cleaved Caspase3 protein expression and ROS production induced by AGE-BSA, and could further reduce hADSC survival rate (P<0.05); Compared with AGE-BSA treatment group, miR-223 inhibitor transfection could inhibit the increases of hADSC apoptosis rate, Cleaved Caspase3 protein expression and ROS production, and could prevent the decrease of hADSC survival rate induced by AGE-BSA (P<0.05). Conclusion High expression of miR-223 can promote the hADSC apoptosis and ROS production, and miR-223 may serve as a new target for the regulation of hADSC in promoting the healing of diabetic wound.

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王哲,张殿宝,杨向红. microRNA-223对糖基化终产物诱导人脂肪间充质干细胞凋亡和氧化应激影响的体外研究[J].中国动脉硬化杂志,2017,25(1):1~6.

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  • 收稿日期:2016-06-17
  • 最后修改日期:2016-10-18
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  • 在线发布日期: 2017-02-08