黄芪甲苷对无血清及缺氧诱导MSCs凋亡的影响

Study on Astragaloside Pretreatment Inhibiting MSCs Apoptosis Induced by Free Serum and Hypoxia

目的观察黄芪甲苷预处理对无血清及缺氧诱导的骨髓间充质干细胞(MSCs)凋亡的影响,并探讨其作用机制。方法采用密度梯度离心法分离培养大鼠MSCs,流式细胞仪检测第3代MSCs表面抗原。分别以40,80,160μg.mL-1的黄芪甲苷预处理MSCs 24 h,再进行无血清及缺氧培养24 h,细胞核荧光染色观察细胞凋亡情况,用流式细胞仪检测细胞凋亡率及线粒体膜电位。结果密度梯度离心法可有效分离大鼠MSCs;细胞表面抗原CD29、CD44表达阳性,CD31、CD34表达阴性;Hoechst33342染色显示经黄芪甲苷预处理可减少细胞凋亡的形态学改变,Annexin V/PI双染法证实,80,160μg.mL-1黄芪甲苷预处理MSCs细胞凋亡率较缺血缺氧组降低,差异有统计学意义(P<0.05);JC-1荧光探针检测证实80,160μg.mL-1黄芪甲苷预处理MSCs线粒体膜电位比缺血缺氧组升高,差异有统计学意义(P<0.05)。结论黄芪甲苷可抑制无血清及缺氧诱导的MSCs凋亡,其作用可能是通过抑制线粒体膜电位降低实现的。

Objective To explore the effect and mechanism of astragaloside pretreatment on bone mesenchymal stem cells（MSCs） apoptosis induced by free serum and hypoxia. Methods Density gradient centrifugation method was used to isolate and cultivate rat MSCs. The cell surface antigens of the third generation of MSCs were detected with flow cytometry. MSCs were pretreated by astragaloside at 40, 80, 160 μg·mL^-1 respectively for 24 h, and then cultured under the conditions of free serum and hypoxia for another 24 h. Nucleus fluorescence staining was used to observe cell apoptosis. Flow cytometry was used to detect the apoptotic rate and mitochondrial membrane potential. Results The rat MSCs were isolated effectively by using density gradient centrifugation. The cell surface antigen CD29, CD44 expression was positive while CD31, CD33 expression was negative. The results of Hoeehst 33342 nucleus staining showed that astragaloside could relieve the morphological changes of apoptosis, and the results of Annexin V/PI double staining method proved that the MSCs pretreated by 80, 160 μg·mL^-1 astragaloside had lower apoptotic rate than the serum-free and hypoxia group（P 〈 0.05）. JC-1 fluorescence probe detection results showed that the MSCs pretreated by 80, 160 μg·mL^-1 astragaloside Conclusion Astragaloside may had higher membrane potential than the serum-free and hypoxia group （P 〈 0.05） inhibit MSCs apoptosis induced by free reduction of mitochondrial membrane potential. serum and hypoxia through inhibiting the