龟版有效成分抗无血清损伤表皮干细胞凋亡的研究  

Active ingredients of Plastrum Testudinis inhibit epidermal stem cell apoptosis in serum-deprived culture

作　　者：陈兰[1] 黎晖[1] 李春[2] 曹佳会[1] 伍艺灵[1] 刘洋[1] 周健洪[1] 陈东风[1] 李伊为[2] 

机构地区：[1]广州中医药大学解剖学教研室,广东广州510006 [2]广州中医药大学护理教研室,广东广州510006

出　　处：《中西医结合学报》2011年第8期888-893,共6页Journal of Chinese Integrative Medicine

基　　金：广东省中医药局科研基金资助项目(No.2007085);广州中医药大学创新基金资助项目(No.2007C007)

摘　　要：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 effects of active ingredients of Plastrum Testudinis（PT）on serum deprivation-induced apoptosis of epidermal stem cells（ESCs）.Methods：ESCs were isolated from the back skin of fetal Sprague-Dawley rats with 2 weeks of gestational age and were divided into normal group（10% fetal bovine serum）,control group（serum-deprived culture）and groups treated with serum deprivation plus active ingredients of PT,including ethyl acetate extract（2B）,stearic acid ethyl ester（S6）,tetradecanoic acid sterol ester（S8）and（＋）-4-cholesten-3-one（S9）.The vitality of ESCs after 24,48 and 72 h of culture was measured with MTT method;apoptotic ESCs double-stained with Annexin V-FITC and propidium iodine were detected by flow cytometry（FCM）;Bcl-2 and caspase-3 expressions were measured by Western blotting.Results：MTT results indicated that the vitality of ESCs in the active ingredients of PT groups at 48 h was increased compared with the control group and 2B had better effects than the others.FCM results indicated that 2B had the most significant anti-apoptotic effect compared with the control as well as S6,S8 and S9.Western blot results indicated that 2B,S6,S8 and S9 up-regulated the expression of Bcl-2 protein and down-regulated the expression of caspase-3 protein compared with the control.Conclusion：Ethyl acetate extract of Plastrum Testudinis inhibits epidermal stem cell apoptosis in serum-deprived culture by regulating the expressions of Bcl-2 and caspase-3 proteins and has a stronger anti-apoptotic effect than its constituents S6,S8 and S9.

关 键 词：龟版 表皮干细胞 凋亡 BCL-2 半胱氨酸天冬氨酸蛋白酶3 

分 类 号：R285[医药卫生—中药学]