蛋白激酶B／哺乳动物雷帕霉素靶蛋白信号通路关键蛋白在糖尿病大鼠皮肤组织和创面组织中的表达研究  

Expressions of the key proteins of the protein kinase B/mammalian target of rapamycin signaling pathway in skin tissue and wound tissue of diabetic rats

作　　者：黄宏[1] 邱伟[1] 朱明[1] 张娅[1] 崔文慧[1] 邢伟[1] 李向云[1] 安天琛 陈民佳[1] 郭韡[1] 徐祥[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所,创伤、烧伤与复合伤国家重点实验室,重庆400042

出　　处：《中华烧伤杂志》2016年第10期588-593,共6页Chinese Journal of Burns

基　　金：国家重点基础研究发展计划（2012CB518105）;国家自然科学基金（81372059、81571912、81571913、81372060）

摘　　要：目的探讨糖尿病大鼠皮肤组织和创面组织中蛋白激酶B／哺乳动物雷帕霉素靶蛋白（Akt／mTOR）信号通路关键蛋白的表达变化，并探讨相关机制。方法选取7～8周龄SD大鼠78只，按照随机数字表法分为糖尿病组和非糖尿病组，每组39只。糖尿病组大鼠一次性快速腹腔注射20mg／mL链脲佐菌素液（65mg／kg，采用柠檬酸缓冲液配制），建立糖尿病模型；非糖尿病组大鼠同法注射等量的柠檬酸缓冲液。于注射后1～8周2组每周各取3只大鼠，于背部切取约1．0cm×1．0cm的全层皮肤，行HE染色观测表皮厚度。注射后1周，2组各取15只大鼠同前取皮造成全层皮肤缺损，于伤后即刻及1、3、5、7d每组各取3只大鼠处死，伤后即刻每只大鼠沿创缘切取1块皮肤组织、伤后1～7d每只大鼠切取创面组织，取一部分组织采用蛋白质印迹法检测皮肤组织及创面组织中的Akt、磷酸化Akt、mTOR、磷酸化mTOR的蛋白表达水平；取剩余组织行免疫荧光染色，观察皮肤组织及创面组织中的磷酸化Akt与波形蛋白表达。对数据行析因设计方差分析、多重t检验。结果（1）注射后1、2周，2组大鼠表皮厚度相似（t值分别为0．25、1．33，P值均大于0．05）；与非糖尿病组比较，注射后3周起糖尿病组大鼠表皮厚度明显变薄（t值为4．44～9．71，P〈0．05或P〈0．01）。（2）与伤后即刻皮肤组织比较，非糖尿病组大鼠伤后1～7d创面组织Akt、磷酸化Akt、roTOR（伤后3d除外）及磷酸化mTOR蛋白表达水平明显升高，t值为3．75～21．44，P〈0．05或P〈0．01。与伤后即刻皮肤组织比较，糖尿病组大鼠伤后1～7d创面组织中Akt、roTOR蛋白表达水平（伤后ldmTOR除外）无明显变化（t值为0．03～2．32，P值均大于0．05）；伤后1～7d创面组织中磷酸化Akt（伤后1d除外）、磷酸化roTOR蛋白表达水平均明显升高（t值为3．79～8．11，P〈0．Objective To explore the changes in the expressions of key proteins of the protein kinase B/mammalian target of rapamycin （Akt/mTOR） signaling pathway in skin tissue and wound tissue of diabetic rats, and to elucidate the associated mechanisms. Methods Seventy-eight SD rats aged from 7 to 8 weeks were divided into diabetes group and non-diabetes group according to the random number table, with 39 rats in each group. Rats in diabetes group were intraperitoneally injected with 20 mg/mL streptozotocin fluid in the dose of 65 mg/kg （ dissolved in citrate buffer solution） for once to establish the model of diabetes mellitus. Rats in non-diabetes group were injected with the equivalent volume of citrate buffer solution in the same way. Three rats of each group were respectively selected in each week from post injection week （PIW） 1 to 8 for collection of full-thickness skin samples on the back with area approximately of 1.0 cm × 1.0 cm to determine epidermal thickness with HE staining. Fifteen rats of each group were inflicted with full-thickness skin defect by resection of skin as above in PIW 1. Three rats of each group were respectively sacrificed im- mediately after injury and on post injury day （PID） 1, 3, 5 and 7. One piece of skin tissue around the wound edge in each rat was cut off immediately after injury, and wound tissue in each rat was cut off from PID 1 to 7. One part of the tissue was used for determination of protein expression levels of Akt, phosphorylated Akt, roTOR, and phosphorylated roTOR in skin tissue and wound tissue with Western blotting. Surplus tissue was used for observation of expressions of phosphorylated Akt and vimentin in skin tissue and wound tissue with immunofluorescent staining. Data were processed with analysis of variance of factorial design and multiple t test. Results （1） The epidermal thicknesses in rats between the two groups were similar in PIW 1 and 2 （ with t values respectively 0.25 and 1.33, P values above 0.05）. From PIW 3 on, the epidermal thickn

关 键 词：糖尿病 伤口愈合 皮肤 蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路 

分 类 号：R587.1[医药卫生—内分泌]