粒细胞巨噬细胞刺激因子对大鼠创面哺乳动物西罗莫司靶蛋白信号通路的作用  

Effect of granulocyte-macrophage colony-stimulating factor on the mammalian target of sirolimus signaling pathway in the wound of rat

作　　者：崔文慧[1] 黄宏[3] 徐祥[3] 郭敏[3] 代卉[2] 简宇[3] 郭韡[3] 邢伟[3] 蒋建新[3] 杨戎[1] 

机构地区：[1]重庆医科大学基础医学院生理教研室,400016 [2]重庆医科大学基础医学院神经科学研究中心,400016 [3]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

出　　处：《中华烧伤杂志》2012年第1期36-41,共6页Chinese Journal of Burns

基　　金：基金项目：国家重点基础研究发展计划（2012CB518105）;创伤、烧伤与复合伤国家重点实验室自主研究课题（SKLZZ200907）;重庆市自然科学基金（CSTC,2010BB5036）

摘　　要：目的 了解应用粒细胞巨噬细胞刺激因子（GM-CSF）对大鼠创面愈合的影响及对哺乳动物西罗莫司（雷帕霉素）靶蛋白（mTOR）信号通路的作用. 方法 将50只SD大鼠按照随机数字表法分为治疗组和对照组,每组25只,于其背部制作约2 cm×2 cm全层皮肤缺损创面.治疗组大鼠创面外涂重组人粒细胞巨噬细胞刺激因子（ rhGM-CSF）凝胶,10 μg/cm2,rhGM-CSF实际作用量为1×10-4 μg/cm2；对照组创面涂抹不含任何药物的凝胶基质,10 μg/cm2.2组每日给药1次直至创面愈合.于致伤后1、3、5、7、14 d,每组每时相点处死5只大鼠：（1）观察并计算创面愈合率.（2）于前4个时相点取创面组织标本,行HE染色观察组织病理学改变,ELISA法检测GM-CSF含量,蛋白质印迹法检测GM-CSF、CD31及mTOR信号通路相关分子P70S6K、磷酸化（p-）P70S6K、4E-BP1、p-4E-BP1、mTOR、p-mTOR表达量.对数据行t检验. 结果 （1）伤后1d,2组大鼠创面愈合率接近（t=0.307,P＞0.05）；伤后3～ 14 d,治疗组创面愈合率明显高于对照组（t值为2.704～ 4.030,P ＜0.05或P ＜0.01）.（2）HE染色可见,与对照组同时相点相比,治疗组创面肉芽组织及微血管数量明显增多,创缘角化上皮细胞增殖明显.（3）ELISA法和蛋白质印迹检测结果显示,2组大鼠创面GM-CSF含量及蛋白表达量均在伤后3d达到峰值,对照组分别为（720.9±0.9）pg/mL、2.45±0.10,治疗组分别为（910.5±1.3）pg/mL、2 80±0.48.治疗组各时相点GM-CSF含量均明显高于对照组（t值为105.743～298.971,P值均为0.000）,治疗组伤后1、5、7 d GM-CSF蛋白表达量明显高于对照组（t值为4.070～5.275,P值均小于0.01）.（4）治疗组伤后1、3、7d创面组织中CD31表达量均明显高于对照组（t值为7.237～26.401,P值均小于0.01）.（5）治疗组伤后各时相点创面组织中mTOR和p-mTOR表达量均高于对照组（t值为2.921 ～23.143,P ＜0.05或P＜0.01）.治疗组P70S6K表达量�Objective To investigate the effects of granulocyte-macrophage colony-stimulating factor （GM-CSF）on wound healing and mammalian target of sirolimus（rapamycin）signaling pathway in rats.Methods Fifty SD rats were divided into control group（ n =25 ）and treatment group（ n =25）according to the random number table.All rats were inflicted with 2 cm × 2 cm full-thickness skin wound on the back.Recombinant human GM-CSF gel（10 μg/cm2）was applied onto the wounds in treatment group,and the actual quantity was 1 × 10-4 μg/cm2.Gel vehicle（ 10 μg/cm2 ）without any medicine was applied onto the wounds in control group.The treatment was conducted once a day up to the day of wound healing.Five rats from two groups were sacrificed on post injury day（PID）1,3,5,7,14 respectively to observe and determine the wound healing rate.Wound tissue samples were collected at the former 4 time points to observe the histopathological changes with HE staining,and to detect the content of GM-CSF with enzyme-linked immunosorbent assay,and the expression levels of GM-CSF,CD31,and the mTOR signal pathway associated molecules P70S6K,phosphorylated（p-）P70S6K,4E-BP1,p-4E-BP1,mTOR,p-mTOR with Western blotting.Data were processed with t test. Results （ 1 ）Wound healing rates in control group and treatment group were close on PID 1（ t =0.307,P 〉 0.05 ）.Wound healing rate in treatment group was obviously higher than that in control group on PID 3,5,7,and 14（ with t values from 2.704 to 4.030,P 〈0.05 or P〈 0.01 ）.（2）Compared with those in control group,more abundant granulation tissue was observed in treatment group,in which an increase in the number of microveseels and obvious proliferation of keratinized epithelial cells in wound margin were observed at each time point.（3）The content and the protein expression level of GM-CSF peaked on PID 3 in two groups,and they were（720.9 ± 0.9）pg/mL,2.45 ±0.10 in control group and（910.5 ± 1.3）pg/mL,2.80 ± 0.48 in treatment group.The content of GM-

关 键 词：粒细胞巨噬细胞集落刺激因子 信号传导 哺乳动物西罗莫司(雷帕霉素)靶蛋白 创面愈合 

分 类 号：R641[医药卫生—外科学] R-332[医药卫生—临床医学]