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目的:探讨龟版有效成分对无血清培养所致的表皮干细胞(epidermal stemcell,ESC)凋亡的药效作用和可能机制。方法:分离培养孕龄2周的Sprague-Dawley胎鼠背部皮肤制备ESC,分为正常组、模型对照组和龟版有效成分各组。采用含龟版有效成分的无血清达尔伯克改良伊格尔培养基培养24、48和72h后,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5-di methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide,MTT)法检测ESC的活力,筛选药物最佳作用时间;ESC用各药物培养48h后,异硫氰酸荧光素标记的膜联蛋白V和碘化丙锭双标后用流式细胞仪检测细胞凋亡率,蛋白质印迹法检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)和Bcl-2蛋白的表达水平。结果:MTT检测结果表明,在24、48和72h时各有效成分组ESC活力较模型对照组增强(P<0.01),且作用48h时乙酸乙酯部位提取物组ESC活力高于十八酸乙酯、十四酸甾醇酯、4-甾酮各组(P<0.05,P<0.01)。作用48h时,乙酸乙酯部位提取物抗凋亡效果最强,4-甾酮抗凋亡效果最弱。蛋白免疫印迹结果表明,乙酸乙酯部位提取物、十八酸乙酯、十四酸甾醇酯、4-甾酮均能显著上调Bcl-2和下调caspase-3的表达,有抗凋亡作用。结论:乙酸乙酯部位提取物有较强的抗ESC凋亡作用,优于它的各组分单独作用,调控Bcl-2与caspase-3之间的表达平衡可能是其抗ESC凋亡的机制之一。
OBJECTIVE: To investigate the pharmacological effects and possible mechanisms of the active components of turtle on the apoptosis of epidermal stem cells (ESC) induced by serum-free culture. METHODS: ESC was isolated from the skin of the back of Sprague-Dawley fetus at gestational age 2 weeks and divided into normal group, model control group and active ingredient of turtle version. After 24, 48 and 72 h incubation with serum-free Dulbecco’s modified Eagle’s medium containing turtle version of the active ingredient, the cells were treated with 3- (4,5-dimethylthiazol-2) -2,5-diphenyltetrakis The activity of ESC was detected by MTT assay, and the optimal time of drug administration was screened. After 48 hours of ESC incubation with different drugs, Fluorescein-labeled annexin V and propidium iodide double-labeled by flow cytometry to detect apoptosis rate, Western blotting detection of caspase-3 (caspase-3) and Bcl-2 protein expression levels. Results: The results of MTT assay showed that the activity of ESC in each active ingredient group was higher than that of the model control group at 24, 48 and 72 hours (P <0.01), and the activity of ESC in the ethyl acetate extract group was higher than that of the control Ester, myristate, and 4-ketosteroids (P <0.05, P <0.01). After 48 hours, the extract of ethyl acetate had the strongest anti-apoptotic effect and the anti-apoptotic effect of 4-sterone was the weakest. Results of Western blotting showed that ethyl acetate extract, ethyl octadecanoate, sterol ester of myristate and 4-sterol could significantly up-regulate Bcl-2 and down-regulate the expression of caspase-3, and had anti-apoptotic effects. CONCLUSION: The ethyl acetate extract has stronger anti-ESC apoptosis effect than its components alone, and the regulation of the expression balance between Bcl-2 and caspase-3 may be the mechanism of anti-ESC apoptosis one.