Effect of Nrf2 overexpression on anti-hypoxia and anti-apoptotic ability of mesenchymal stem cells
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摘要:
目的 研究核因子E2相关因子2（Nrf2）对间充质干细胞（MSCs）的抗缺氧及抗凋亡能力的影响。 方法 将过表达Nrf2的重组质粒及辅助质粒转染人胚肾细胞（293FT），获得过表达Nrf2的高滴度慢病毒。MSCs分别转染过表达Nrf2慢病毒以及空载体慢病毒，构建稳定过表达Nrf2的Nrf2-MSCs（Nrf2过表达组）以及绿色荧光蛋白（GFP）-MSCs（对照组）。采用荧光显微镜观察两组细胞的绿色荧光表达情况。采用蛋白免疫印迹（Western Blot）法检测两组细胞中Nrf2蛋白的表达水平。采用光学显微镜观察两组细胞的抗缺氧能力，采用流式细胞术检测两组细胞的抗凋亡能力。 结果 成功构建了稳定过表达Nrf2的Nrf2-MSCs，Western Blot法检测结果显示Nrf2过表达组细胞的Nrf2蛋白表达水平明显高于对照组（P < 0.01）。缺氧处理15 h后，Nrf2过表达组的细胞活力明显高于对照组。流式细胞术检测表明Nrf2过表达组细胞的凋亡率为（30.9±1.4）%，明显低于对照组细胞的（61.3±1.3）%（P < 0.05）。 结论 稳定过表达Nrf2的Nrf2-MSCs在低氧环境中具有一定的抗缺氧、抗凋亡能力。 -
关键词:
- 核因子E2相关因子2（Nrf2） /
- 间充质干细胞（MSCs） /
- 慢病毒载体 /
- 转染 /
- 质粒 /
- 凋亡 /
- 缺氧 /
- 流式细胞术 /
- 人胚肾细胞 /
- 缺血-再灌注损伤（IRI）
Abstract:Objective To investigate the effect of nuclear factor erythroid-2-related factor 2(Nrf2) on the antihypoxia and anti-apoptotic ability of mesenchymal stem cells(MSCs). Methods Human embryonic kidney cells(293FT) were transfected with recombinant plasmid which overexpressed Nrf2 and helper plasmid. High-titer lentivirus which overexpressed Nrf2 were obtained. MSCs were transfected with lentivirus with Nrf2 overexpression and empty lentiviral vector to establish Nrf2-MSCs which stably overexpressed Nrf2 (Nrf2 overexpression group) and green fluorescent protein (GFP)-MSCs(control group). The expression of green fluorescent in 2 groups was observed by fluorescence microscope. The expression level of Nrf2 protein in 2 groups was measured by Western Blot. The anti-hypoxia ability of 2 groups was observed by light microscope. The anti-apoptotic ability of 2 groups was measured by flow cytometry. Results Nrf2-MSCs which stably overexpressed Nrf2 were successfully established. Western Blot analysis revealed that the expression level of Nrf2 protein in the Nrf2 overexpression group was significantly higher than that in the control group(P < 0.01). After 15 h hypoxia treatment, the cell activity in the Nrf2 overexpression group was significantly higher than that in the control group. Flow cytometry showed that the apoptosis rate in the Nrf2 overexpression group was (30.9±1.4)%, significantly lower than (61.3±1.3)% in the control group(P < 0.05). Conclusions Nrf2-MSCs which can stably overexpress Nrf2 possess certain anti-hypoxia and anti-apoptotic ability in hypoxia environment. -
图 1 重组质粒阳性克隆菌液的PCR产物
Figure 1. PCR products of recombinant plasmid positive clone bacteria solution
图 2 荧光显微镜下两组细胞的绿色荧光表达（×200）
A图为Nrf2过表达组细胞；B图为对照组细胞
Figure 2. The green fluorescent expression of cells in two groups by fluorescence microscope
图 3 两组细胞Nrf2蛋白的表达水平比较
与对照组比较，aP<0.05
Figure 3. Comparison of the expression level of Nrf2 protein of cells between two groups
图 4 两组细胞抗缺氧能力的比较（×100）
A图为Nrf2过表达组细胞；B图为对照组细胞
Figure 4. Comparison of the anti-hypoxia ability of cells between two groups
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