雌激素在小鼠表皮发育与人表皮细胞株HaCaT增殖中的作用与机制  

Effects of estrogen on epidermis growth of mice and proliferation of human epidermal cell line HaCaT and its mechanism

作　　者：周涛[1] 陈婧[1] 黄宗伟[2] 方利[1] 陈渝[1] 陈雅洁[1] 彭毅志[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆400038 [2]广西医科大学第一附属医院烧伤整形科

出　　处：《中华烧伤杂志》2016年第5期299-304,共6页Chinese Journal of Burns

摘　　要：目的观察雌激素在小鼠表皮发育和Kc（人表皮细胞株HaCaT）增殖中的作用并探讨其机制。方法（1）通过阴道脱落细胞检查法选取5只处于动情期成年C57BL／6小鼠设为动情期组，另将性发育前行卵巢切除的5只成年C57BL／6小鼠设为卵巢切除组。取2组小鼠尾根部全层皮肤，HE染色观测表皮厚度，免疫组织化学染色观察表皮中增殖细胞核抗原（PCNA）阳性细胞分布并计数。（2）取对数生长期HaCaT细胞，用含体积分数10％FBS的RPMI1640培养液培养，按随机数字表法分为阴性对照组、单纯雌二醇组、蛋白激酶B（Akt）抑制剂组、细胞外信号调节激酶（ERK）抑制剂组，每组20孔。各组培养液中，阴性对照组加入1nL二甲基亚砜；单纯雌二醇组加入100nmol／L17 β-雌二醇1 μL；Akt抑制剂组和ERK抑制剂组均加入同前剂量与体积17 β-雌二醇，另分别加入10 μmol／L LY294002和30 μmol／LPD98059各1 μL。分别于培养0（即刻）、24、48、72h，每组取5孔细胞，用细胞计数试制盒8与酶标仪检测细胞增殖活性。（3）取对数生长期HaCaT细胞，同前分组处理，每组3孔。培养72h，用流式细胞仪检测细胞周期分布，计算细胞增殖指数（PI）。（4）取对数生长期HaCaT细胞，同前分组处理，每组3皿。培养72h，蛋白质印迹法检测细胞中磷酸化Akt（p-Akt）、磷酸化ERK（p-ERK）和PCNA蛋白水平。细胞实验均重复3次。对数据行t检验、单因素方差分析、析因设计方差分析、LSD检验。结果（1）卵巢切除组小鼠表皮厚度为（33．5±3．0） μm，明显薄于动情期组的（51．4±3．1） μm（t=20．7，P〈0．01）。2组小鼠PCNA阳性细胞主要集中于表皮基底层；卵巢切除组小鼠表皮中PCNA阳性细胞数为每200倍视野下（37±12）个，明显少于动情期组的每200倍视野下（96±15）个（t：15．3，P〈0．01）。（2）培养0～48h，单纯雌二醇组�Objective To observe the effects of estrogen on epidermis growth of mice and prolifera- tion of keratinocytes （human epidermal cell line HaCaT） , and to explore its mechanism. Methods （1） Five adult C57BL/6 mice in estrus cycle were identified by vaginal exfoliative cytology diagnosis and set as estrus group, while another 5 adult C57BL/6 mice with ovary resected before sexual development were set as ovariectomized group. The full-thickness skin from the tail root of mice in two groups were collected. The thickness of epidermis was observed and measured after HE staining. The distribution of proliferating cell nu- clear antigen （PCNA）-positive ceils in epidermis was observed by immunohistochemical staining, the num- ber of which was counted. （2） HaCaT cells in logarithmic growth phase were cultured with RPMI 1640 nu- trient solution containing 10% fetal bovine serum, and they were divided into negative control group （NC） , pure estradiol group （PE） , protein kinase B （Akt） inhibitor group （AI） , and extraeellular signal-regulated kinase （ERK） inhibitor group （EI） according to the random number table, with 20 wells in each group. To nutrient solution of each group, 1 p,L dimethyl sulfoxide, 1 p,L 17 β-estradiol （100 nmol/L）, 1 μL LY294002 （10 μ mol/L） , and 1 μL PD98059 （30 μmol/L） were added in group NC, group PE, group AI, and group EI respectively, and the last two groups were added with 1 μL 17[3-estradiol （ 100 nmol/L） in ad- dition. At post culture hour （PCH） 0 （immediately after culture） , 24, 48, 72, 5 wells of cells from each group were collected to detect the proliferation activity of cells by cell counting kit 8 and microplate reader. （3） HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 wells in each group. At PCH 72, cell cycle distribution was detected by flow eytometer to calculate proliferation index （PI） of ceils. （4） HaCaT cells in logarithmic growt

关 键 词：雌激素类 表皮 增殖细胞核抗原 细胞外信号调节MAP激酶类 蛋白激酶类 HACAT细胞 

分 类 号：R641[医药卫生—外科学]