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Nrf2基因沉默对体外模拟炎性环境的骨髓间充质干细胞凋亡的影响
The Effect of Nrf2 Gene Silencing on Apoptosis of Bone Marrow Mesenchymal Stem Cells in Simulated Inflammatory Environment in Vitro
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DOI:
作者:
龙仙萍,邓文文,赵然尊,王冬梅,石蓓
LONG Xian-ping,DENG Wen-wen,ZHAO Ran-zun ,WANG Dong-mei,SHI Bei
作者单位:
贵州遵义医学院附属医院心内科
Department of Cardiology, the First Affiliated Hospital of Zunyi Medical Universtiy,
关键词:
心肌梗死;NF-E2相关因子2;siRNA;骨髓间充质干细胞;细胞凋亡
myocardial infarction; NF-E2 related factor 2; siRNA;bone mesenchymal stem cell; cell apoptosis
摘要:
目的 探讨Nrf2基因对缺氧预处理骨髓间充质干细胞(MSCs)凋亡的影响,为干细胞移植治疗心肌梗死提供较为优良的种子细胞。方法 用沉默(siRNA)核因子E2相关因子2(Nrf2)的慢病毒载体(LV-GFP-siRNA-Nrf2)转染入大鼠MSCs(即GFP-LV-MSCsNrf2-/-,简称MSCsNrf2-/-),采用蛋白印迹法(Western blot)检测Nrf2基因RNA干扰(RNAi)前后的蛋白表达变化。实验细胞经缺氧预处理后,实验随机分为沉默Nrf2的MSCs(简称MSCsNrf2-/缺氧组)和常态Nrf2的MSCs(简称MSCsNrf2+/-缺氧组)两个缺氧组,以常氧MSCs(简称MSCsNrf2+/-常氧组)作为对照组。采用Annexin V /7-AAD染色结合流式细胞术检测细胞凋亡,细胞免疫荧光检测凋亡蛋白caspase3的表达情况,(蛋白印迹)Western blot法检测Nrf2及其下游靶点血红素加氧酶-1(HO-1)蛋白表达水平和凋亡相关蛋白的表达情况。 结果 与MSCsNrf2+/-组相比, Nrf2被小的干涉RNA(siRNA)干扰后可在蛋白水平有效抑制Nrf2表达转录。流式细胞术结果显示:与常氧组比较,缺氧组细胞凋亡率显著增高(P值<0.05);而在缺氧环境中,MSCsNrf2-/-组细胞凋亡率较MSCsNrf2+/-组又显著增高(P值<0.05),且MSCsNrf2-/-组中凋亡蛋白Caspase3表达也明显增加(P值<0.05);Western bolt检测结果也显示Nrf2核蛋白及其下游靶基因HO-1蛋白表达水平在缺氧环境下明显升高(P值<0.05),然而,MSCsNrf2-/-缺氧组上述基因的表达则显著降低(P值<0.05),明显低于MSCsNrf2+/-缺氧组和MSCsNrf2+/-常氧组,(P值<0.05)。除此外,MSCsNrf2-/-缺氧组抗凋亡蛋白Bcl-2表达显著下降,而促凋亡蛋白Bax表达显著增加(P值<0.05)。结论 沉默细胞中的Nrf2基因可显著降低干细胞对缺氧的耐受能力,进一步加速细胞凋亡。
Objective To investigate the impact of Nrf2 gene silencing on apoptosis of bone marrow mesenchymal stem cells in hypoxic preconditioning , so as to provide better seed cells for stem cells transplantation in the treatment of myocardial infarction. Methods Nrf2 gene-specific siRNA was transfected into the rat bone marrow mesenchymal stem cells by taking the lentivirus as the vector. The changes in the protein expression before and after the Nrf2 gene RNAi were detected using Western blot. The hypoxic-ischemic cell models were prepared to simulate the myocardial infarction (MI) inflammatory environment, and the experiment was divided into four groups: GFP-LV-MSCsNrf2+/- normoxia group (referred to as MSCsNrf2+/- normoxia group), GFP-LV-MSCsNrf2-/- normoxia group (referred to as MSCsNrf2-/- normoxia group), GFP-LV-MSCsNrf2+/- hypoxic group (referred to as MSCsNrf2+/- hypoxic group), and GFP-LV-MSCsNrf2-/- hypoxic group (referred to as MSCsNrf2-/- hypoxic group). The apoptosis was detected using Annexin V /7-AAD staining combined with flow cytometry (FCM), the expression of apoptin caspase3 was detected using immunofluorescence, and the protein expression of Nrf2 and its downstream target HO-1 was detected using Western blot. Results Compared with MSCsNrf2+/- group, siRNA that targets to Nrf2 could effectively inhibit the expression of Nrf2 at the protein level. FCM results showed that the apoptosis rate in hypoxic groups was significantly higher than that of normoxia groups, while in the hypoxic environment, the apoptosis rate in MSCsNrf2-/ was significantly enhanced compared with that of MSCsNrf2+/-, suggesting that after Nrf2 gene silencing, the tolerance of mesenchymal stem cells to hypoxic-ischemic environment was weakened. Immunofluorescence results showed that the expression of apoptin caspase3 increased in MSCsNrf2-/- in hypoxic environment when compared with that in MSCsNrf2+/-. The expression level of Nrf2 nucleoprotein and HO-1 protein was detected using Western blot. The results showed that the protein expression level of Nrf2 and HO-1 was significantly increased in MSCsNrf2+/- hypoxic group compared with that in MSCsNrf2+/- normoxia group (P<0.05); the protein expression level of Nrf2 and HO-1 was significantly decreased in MSCsNrf2-/- hypoxic group compared with that in MSCsNrf2+/- hypoxic group (P<0.05); no significant difference was observed in the comparison between MSCsNrf2+/- normoxia group and MSCsNrf2-/- normoxia group (P>0.05). Conclusion Nrf2 gene silencing significantly decrease the tolerance of mesenchymal stem cells of hypoxia tolerance ability, futher accelerate apoptosis.
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