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
发布时间：2018-10-18 09:32
【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(h ADSC)凋亡和氧化应激的影响。方法采用酶消化法分离培养h ADSC,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h ADSC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h ADSC高表达CD14、CD90和CD105,不表达CD34、CD45和HLADR。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h ADSC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(P0.05);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h ADSC凋亡率、Cleaved Caspase3蛋白表达水平和ROS生成增加,下调AGE-BSA引起的h ADSC存活率减少(P0.05);与AGE-BSA作用组相比,miR-223抑制物转染能够抑制h ADSC凋亡率、Cleaved Caspase3蛋白表达水平和ROS生成增加,拮抗AGEBSA引起的h ADSC存活率减少(P0.05)。结论 miR-223高表达能够促进AGE-BSA引起的h ADSC细胞凋亡和ROS生成,其可能作为调控h ADSC促进糖尿病创伤愈合的新靶点。
[Abstract]:Objective to observe the effect of microRNA-223 (miR-223) on (h ADSC) apoptosis and oxidative stress induced by (AGE). Methods ADSC, was isolated and cultured by enzyme digestion, and the surface antigen CD14,CD34,CD45,CD90,CD105 and human leukocyte antigen DR (HLA-DR) were detected by flow cytometry. H ADSC were divided into three groups: bovine serum albumin (BSA) (BSA) control group, AGE modified bovine serum albumin (AGE-BSA) group, miR-223 mimic transfection AGE-BSA group, miR-223 inhibitor transfection group and miR-223 inhibitor transfection AGE-BSA group. CCK-8 and TUNEL staining were used to detect the cell survival rate and apoptosis rate. The expression of Cleaved Caspase3 protein was detected by, Western blot, and the content of reactive oxygen (ROS) was detected by DCFH-DA kit. Results the results of flow cytometry showed that the h ADSC in primary culture did not express CD34,CD45 and HLADR.CCK-8 by high expression of CD14,CD90 and CD105, while TUNEL staining with, Western blot and DCFH-DA showed that h ADSC could not express CD34,CD45 and HLADR.CCK-8. Compared with BSA control group, h ADSC apoptosis rate, miR-223 expression, Cleaved Caspase3 protein expression and ROS production were significantly increased in AGE-BSA treated group. Compared with AGE-BSA group, the transfection of miR-223 mimics further up-regulated the apoptosis rate of h ADSC induced by AGE-BSA, the expression of Cleaved Caspase3 protein and the production of ROS. Compared with AGE-BSA group, miR-223 inhibitor transfection could inhibit the apoptosis rate of h ADSC, increase the expression of Cleaved Caspase3 protein and ROS production, and antagonize the survival rate of h ADSC induced by AGEBSA (P0.05). Conclusion the high expression of miR-223 can promote the apoptosis and ROS production of h ADSC cells induced by AGE-BSA, which may be a new target for regulating h ADSC to promote diabetic wound healing.
【作者单位】： 中国医科大学盛京医院病理科;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;
【基金】：国家自然科学基金项目(81601692) 辽宁省教育厅科学研究项目(LK201602)
【分类号】：R587.2
本文编号：2278713
[Abstract]:Objective to observe the effect of microRNA-223 (miR-223) on (h ADSC) apoptosis and oxidative stress induced by (AGE). Methods ADSC, was isolated and cultured by enzyme digestion, and the surface antigen CD14,CD34,CD45,CD90,CD105 and human leukocyte antigen DR (HLA-DR) were detected by flow cytometry. H ADSC were divided into three groups: bovine serum albumin (BSA) (BSA) control group, AGE modified bovine serum albumin (AGE-BSA) group, miR-223 mimic transfection AGE-BSA group, miR-223 inhibitor transfection group and miR-223 inhibitor transfection AGE-BSA group. CCK-8 and TUNEL staining were used to detect the cell survival rate and apoptosis rate. The expression of Cleaved Caspase3 protein was detected by, Western blot, and the content of reactive oxygen (ROS) was detected by DCFH-DA kit. Results the results of flow cytometry showed that the h ADSC in primary culture did not express CD34,CD45 and HLADR.CCK-8 by high expression of CD14,CD90 and CD105, while TUNEL staining with, Western blot and DCFH-DA showed that h ADSC could not express CD34,CD45 and HLADR.CCK-8. Compared with BSA control group, h ADSC apoptosis rate, miR-223 expression, Cleaved Caspase3 protein expression and ROS production were significantly increased in AGE-BSA treated group. Compared with AGE-BSA group, the transfection of miR-223 mimics further up-regulated the apoptosis rate of h ADSC induced by AGE-BSA, the expression of Cleaved Caspase3 protein and the production of ROS. Compared with AGE-BSA group, miR-223 inhibitor transfection could inhibit the apoptosis rate of h ADSC, increase the expression of Cleaved Caspase3 protein and ROS production, and antagonize the survival rate of h ADSC induced by AGEBSA (P0.05). Conclusion the high expression of miR-223 can promote the apoptosis and ROS production of h ADSC cells induced by AGE-BSA, which may be a new target for regulating h ADSC to promote diabetic wound healing.
【作者单位】： 中国医科大学盛京医院病理科;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;
【基金】：国家自然科学基金项目(81601692) 辽宁省教育厅科学研究项目(LK201602)
【分类号】：R587.2
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