【摘要】：目的觀察microRNA-223(miR-223)對糖基化終產(chǎn)物(AGE)誘導(dǎo)人脂肪間充質(zhì)干細胞(h ADSC)凋亡和氧化應(yīng)激的影響。方法采用酶消化法分離培養(yǎng)h ADSC,并應(yīng)用流式細胞術(shù)對表面抗原CD14、CD34、CD45、CD90、CD105和人類白細胞抗原DR(HLA-DR)進行檢測。將h ADSC分為牛血清白蛋白(BSA)對照組、AGE修飾的牛血清白蛋白(AGE-BSA)作用組、miR-223模擬物轉(zhuǎn)染組、miR-223模擬物轉(zhuǎn)染+AGE-BSA組、miR-223抑制物轉(zhuǎn)染組和miR-223抑制物轉(zhuǎn)染+AGE-BSA組。應(yīng)用CCK-8法和TUNEL染色檢測各組細胞存活率和凋亡率,Western blot檢測Cleaved Caspase3蛋白表達水平,應(yīng)用雙氯熒光素(DCFH-DA)試劑盒檢測各組細胞活性氧(ROS)含量。結(jié)果流式細胞術(shù)檢測結(jié)果表明,原代培養(yǎng)的h ADSC高表達CD14、CD90和CD105,不表達CD34、CD45和HLADR。CCK-8法、TUNEL染色、Western blot和DCFH-DA法檢測結(jié)果表明,與BSA對照組相比,AGE-BSA作用組h ADSC凋亡率、miR-223表達水平、Cleaved Caspase3蛋白表達水平、ROS生成顯著升高,而細胞存活率下降(P0.05);與AGE-BSA作用組相比,miR-223模擬物轉(zhuǎn)染能夠進一步上調(diào)AGE-BSA引起的h ADSC凋亡率、Cleaved Caspase3蛋白表達水平和ROS生成增加,下調(diào)AGE-BSA引起的h ADSC存活率減少(P0.05);與AGE-BSA作用組相比,miR-223抑制物轉(zhuǎn)染能夠抑制h ADSC凋亡率、Cleaved Caspase3蛋白表達水平和ROS生成增加,拮抗AGEBSA引起的h ADSC存活率減少(P0.05)。結(jié)論 miR-223高表達能夠促進AGE-BSA引起的h ADSC細胞凋亡和ROS生成,其可能作為調(diào)控h ADSC促進糖尿病創(chuàng)傷愈合的新靶點。
[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.
【作者單位】： 中國醫(yī)科大學盛京醫(yī)院病理科;中國醫(yī)科大學細胞生物學衛(wèi)生部重點實驗室干細胞與再生醫(yī)學研究室;
【基金】：國家自然科學基金項目(81601692) 遼寧省教育廳科學研究項目(LK201602)
【分類號】：R587.2

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