【摘要】：目的研究三甲氧芐嗪(TMZ)對脂肪來源間充質(zhì)干細(xì)胞(ADSCs)氧化應(yīng)激凋亡的影響及其機制。方法使用雙酶消化獲得SD大鼠脂肪組織的ADSCs,進行流式細(xì)胞學(xué)鑒定和多向分化潛能評估;應(yīng)用過氧化氫(H_2O_2)誘導(dǎo)的ADSCs損傷凋亡模型,使用不同濃度的TMZ(250μmol/L、500μmol/L)干預(yù)氧化應(yīng)激損傷的過程;Hoechst 33342染色定性分析凋亡細(xì)胞形態(tài);JC-1線粒體膜電位染色和線粒體電鏡檢測觀察TMZ作用下線粒體損傷逆轉(zhuǎn)情況;Western blot檢測TMZ對線粒體凋亡通路蛋白的影響;通過檢測活性氧簇(ROS)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、丙二醛(MDA)水平評價TMZ對ADSCs抗氧化能力的作用。結(jié)果獲取的ADSCs,陽性表達CD29和CD90抗原,低或不表達CD34、CD45及CD31;ADSCs能夠成功被誘導(dǎo)分化為脂肪細(xì)胞和骨性細(xì)胞;H_2O_2處理ADSCs,細(xì)胞凋亡數(shù)量增加,線粒體膜電位降低,線粒體結(jié)構(gòu)破壞崩解,保護性凋亡蛋白(Bc12)表達下降,凋亡蛋白(Bax、Bad、Caspase3)表達上調(diào);胞內(nèi)ROS含量與MDA的質(zhì)量摩爾濃度上升,SOD的活性和GSH的質(zhì)量摩爾濃度降低。TMZ對ADSCs有濃度依賴性的保護作用,可降低H_2O_2誘導(dǎo)的細(xì)胞凋亡,逆轉(zhuǎn)線粒體低電位,減輕H_2O_2對線粒體結(jié)構(gòu)的損傷,提高Bc12的表達和下調(diào)Bax、Bad及Caspase3的表達,減低過量的ROS和MDA,恢復(fù)SOD和GSH的抗氧化系統(tǒng)功能。結(jié)論 TMZ通過調(diào)控保護性蛋白的表達和提高內(nèi)源性抗氧化能力,提高ADSCs的存活率,從而逆轉(zhuǎn)氧化應(yīng)激損傷。
[Abstract]:Objective to study the effect of trimethoxybenzidine (TMZ) on oxidative stress apoptosis of adipose derived mesenchymal stem cells (ADSCs) and its mechanism. Methods the ADSCs, of adipose tissue of SD rats was obtained by double enzyme digestion for flow cytometry identification and multidirectional differentiation potential evaluation, the apoptosis model induced by hydrogen peroxide (H_2O_2) was used to interfere with oxidative stress injury with different concentrations of TMZ (250 渭 mol / L, 500 渭 mol / L), and the morphology of apoptotic cells was qualitatively analyzed by Hoechst 33342 staining. JC-1 mitochondrial membrane potential staining and mitochondrial electron microscope were used to observe the effect of TMZ on mitochondrial apoptosis pathway protein induced by TMZ, and the effect of TMZ on antioxidant capacity of ADSCs was evaluated by detecting the level of malondialdehyde (MDA) of (ROS), Superoxide Dismutase (SOD), Glutathion (GSH), (MDA) in activated oxygen cluster (ROS),). Results ADSCs, positive expression of CD29 and CD90 antigens, low or no expression of CD34,CD45 and CD31;ADSCs could be successfully induced to differentiate into adipocytes and bone cells, the number of apoptosis in ADSCs, cells increased, the membrane potential of mitochondria decreased, the structure of mitochondria destroyed and disintegrated, the expression of protective apoptosis protein (Bc12) decreased, and the expression of apoptotic protein (Bax,Bad,Caspase3) upregulated. The intracellular ROS content and the mass molar concentration of MDA increased, but the activity of SOD and the mass molar concentration of GSH decreased. TMZ had a concentration-dependent protective effect on ADSCs, which could decrease the apoptosis induced by H_2O_2, reverse the low potential of mitochondria, reduce the damage of H_2O_2 to the structure of mitochondria, increase the expression of Bc12 and down-regulate the expression of Bax,Bad and Caspase3, and reduce the antioxidant system function of SOD and GSH restored by excessive ROS and MDA,. Conclusion TMZ can reverse oxidative stress injury by regulating the expression of protective proteins and increasing the antioxidant capacity of ADSCs.
【作者單位】： 四川大學(xué)華西醫(yī)院分子醫(yī)學(xué)研究中心;
【基金】：國家高技術(shù)研究發(fā)展計劃(863計劃)(No.2014aa0021604)資助
【分類號】：R96

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