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

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，单纯雌二醇组与阴性对照组细胞增殖活性差异不明显（P值均大于0．05）。培养72h，与阴性对照组比较，单纯雌二醇组细胞增殖活性明显增高（P〈0．01）；Akt抑制剂组、ERK抑制剂组细胞增殖活性均明显低于前2组（P值均小于0．01）。（3）与阴性对照组的（51．6±1．1）％比较，单纯雌二醇组细胞PI明显升高[（58．5±0．8）％，P〈0．05]；Akt抑制剂组、ERK抑制剂组细胞P1分别为（34．9±0．8）％、（48．2±0．4）％，均明显低于前2组（P值均小于0．01）。（4）与阴性对照组的0．566±0．034比较，单纯雌二醇组细胞p-Akt蛋白水平明显升高（1．048±0．077，P〈0．01）；Akt抑制剂组和ERK抑制剂组细胞p-Akt蛋白水平分别为0．682±0．095、0．672±0．019，明显低于单纯雌二醇组（P值均小于0．01）。与阴性对照组的0．469±0．013比较，单纯雌二醇组、Akt抑制剂组、ERK抑制刊组细胞p-ERK蛋白水平均明显升高（1．064±0．089、1．010±0．038、0．778±0．065，P值均小于0．01）。与单纯雌二醇组比较，ERK抑制剂组细胞p-ERK蛋白水平明显降低（P〈0．01）。与阴性对照组的0．386±0．053比较，单纯雌二醇组细胞的PCNA蛋白水平明显升高（0．743±0．043，P〈0．01）；Akt抑制剂组和ERK抑制剂组细胞PCNA蛋白水平分别为0．264±0．019、0．223±0．065，明显低于前2组（P值均小于0．01）。结论雌激素缺乏时，小鼠表皮发育能力减弱。雌激素（17β-雌二醇）可通过激活ERK／Akt信号通路上调PCNA表达，以促进HaCaT细胞的增殖。

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 growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 dishes in each group. At PCH 72, the pro- tein levels of phosphorylated Akt （p-Akt） , phosphorylated ERK （p-ERK） , and PCNA were determined with Western blotting. The cell experiments were repeated for 3 times. Data were processed with t test, one- way analysis of variance, analysis of variance of factorial design, and LSD test. Results （ 1 ） The epider- mis thickness of mice in ovariectomized group was （33.5 ± 3.0） p,m, which was obviously thinner than that in estrus group [（51.4±3.1） μm, t =20.7, P 〈0.011. The PCNA-positive cells mainly aggregated in the basal layer of epidermis of mice in two groups. The number of PCNA-positive cells in epidermis of mice in ovarieetomized group was 37 ± 12 per 200 fold visual field, obviously fewer than that in estrus group （96 ±15 per 200 fold visual field, t = 15.3, P 〈 0.01 ）. （2） During PCH 0 to 48, there were no significant differences in the proliferation activity of ceils between group PE and group NC （ with P values above 0.05 ）. At PCH 72, compared with that in group NC, the proliferation activity of cells in group PE was obviously in- creased （ P 〈 0. 01 ）. The proliferation activity of cells in groups AI and EI was obviously lower than that in the previous two groups （with P values below 0.01）. （3） Compared with that in group NC [ （51.6 ± 1. 1）% 1, the PI of cells in group PEwas obviously increased [（58.5±0.8）%,P 〈0.051. The PI values of cells in groups Ai and EI were （34.9± 0.8） % and （48.2 ± 0.4） % respectively, both obviously lower than those in the previous two groups （ with P values below 0.01 ）. （4） Compared with that of group NC （0. 566 ±0. 034） , the protein level of p-Akt in cells of group PE was significantly increased （ 1. 048 ±0. 077, P 〈 0.01 ）. Compared with that of group PE, the protein level of p-Akt was obviously decreased in cells of groups AI and EI （ respectively 0. 682 ± 0. 095 and 0. 672± 0. 019, with P values below 0.01 ）. Compared with that of group NC （0. 469 ± 0. 013） , the protein level of p-ERK obviously increased in cells of groups PE, AI, and EI （ respectively 1. 064±0. 089, 1. 010± 0. 038, 0. 778±0. 065, with P values be- low 0.01 ）. The protein level of p-ERK in cells of group EI was obviously lower than that in group PE （ P 〈 0.01）. Compared with that of group NC （0. 386 ± 0. 053）, the protein level of PCNA was obviously in- creased in cells of group PE （0. 743 ± 0. 043, P 〈 0.01 ）. The protein levels of PCNA in cells of groups AI and EI were 0. 264±0. 019 and 0. 223 ± 0. 065 respectively, both obviously lower than those in the previous two groups （ with P values below 0.01 ）. Conclusions Lack of estrogen damages the growth ability of epi- dermis of mice. Estrogen （17 β-estradiol） can promote the proliferation of HaCaT cells by increasing the ex- pression of PCNA via activating ERK/Akt signaling pathway.