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目的观察EPO基因修饰对H2O2诱导的小鼠骨髓间充质干细胞(MSCs)凋亡及相关细胞信号传导通路的影响。方法体外培养小鼠MSCs,制作慢病毒载体并转染EPO基因至第6代MSCs,进行细胞鉴定,ELISA法测定细胞上清液中EPO浓度,Western blot方法检测基因修饰前后PI3K/Akt及NF-κB通路蛋白表达,流式细胞术测定细胞凋亡情况。结果成功地制作慢病毒载体,并将EPO基因转染至小鼠MSCs,转染前后细胞表面标志未发生明显变化,转染后细胞上清液中EPO浓度显著升高(P<0.01)。基因修饰可使小鼠MSCs的PI3K/Akt磷酸化水平及总的NF-κB水平升高,但对MSCs的NF-κB磷酸化水平和总的Akt水平无明显影响。转染组凋亡细胞所占百分率低于对照组(P<0.01)。结论 EPO基因修饰后提高了MSCs的分泌EPO能力,并且具有抑制H2O2诱导的MSCs凋亡的作用。其抗凋亡作用可能与PI3K/Akt及NF-κB信号通路有关。
Objective To observe the effect of EPO gene modification on H2O2-induced apoptosis of bone marrow mesenchymal stem cells (MSCs) and related cell signaling pathways. Methods Mouse MSCs were cultured in vitro and lentiviral vector was transfected into MSO of 6th generation to transfect EPO gene for cell identification. The concentration of EPO in supernatants was determined by ELISA. The levels of PI3K / Akt and NF- κB pathway protein expression, flow cytometry determination of apoptosis. Results The lentiviral vector was successfully constructed and the EPO gene was transfected into MSCs. The cell surface markers did not change significantly before and after transfection. The concentration of EPO in the cell supernatant after transfection was significantly increased (P <0.01). Gene modification could increase the phosphorylation of PI3K / Akt and total NF-κB in MSCs, but had no significant effect on the level of NF-κB phosphorylation and total Akt in MSCs. The percentage of apoptotic cells in transfection group was lower than that in control group (P <0.01). Conclusion EPO gene modification can improve the ability of MSCs to secrete EPO and inhibit the apoptosis of MSCs induced by H2O2. Its anti-apoptotic effect may be related to PI3K / Akt and NF-κB signaling pathway.