过表达生长激素的脂肪干细胞促进成纤维细胞增殖与迁移  

Adipose-derived stem cells overexpressing growth hormone promote proliferation and migration of fibroblasts

作　　者：高仪轩 王凌峰[1] 巴特[1] 李芳 曹胜军[1] 李俊亮[1] 周彪[1] 陈强 Gao Yixuan;Wang Lingfeng;Ba Te;Li Fang;Cao Shengjun;Li Junliang;Zhou Biao;Chen Qiang(Department of Burn Surgery,Third Affiliated Hospital of Inner Mongolia Medical University,Baotou 014010,Inner Mongolia Autonomous Region,China;Experimental Center,Third Affiliated Hospital of Inner Mongolia Medical University,Baotou 014010,Inner Mongolia Autonomous Region,China)

机构地区：[1]内蒙古医科大学第三附属医院烧伤外科,内蒙古自治区包头市014010 [2]内蒙古医科大学第三附属医院实验中心,内蒙古自治区包头市014010

出　　处：《中国组织工程研究》2023年第1期8-14,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81660318),项目负责人:王凌峰;包头市科技计划项目(2019P3038),项目负责人:王凌峰;内蒙古自治区2020年硕士研究生科研创新资助项目(SZ2020105),项目负责人:高仪轩。

摘　　要：背景:生长激素具有免疫调节、促进细胞增殖及蛋白合成等生理作用,已被证实可以促进急慢性创面愈合。目的:构建过表达生长激素的脂肪干细胞系(生长激素-脂肪干细胞),并探究其对成纤维细胞增殖迁移能力的影响及其分子机制。方法:①体外分离并鉴定脂肪干细胞;②构建生长激素过表达慢病毒,将脂肪干细胞分为生长激素组、空载组、对照组,以上3组分别转染生长激素过表达慢病毒、空载慢病毒或不进行传染;③RT-qPCR、Western blot、ELISA检测各组细胞内及上清中生长激素的mRNA和蛋白表达、ERK1/2通路相关基因的表达情况;④划痕实验和Transwell实验、MTS实验检测生长激素组和空载组细胞对成纤维细胞的迁移和增殖能力的影响、RT-qPCR检测生长激素-脂肪干细胞与成纤维细胞共培养后成纤维细胞内相关基因的表达变化;⑤ERK抑制剂抑制生长激素组细胞后,观察其与成纤维细胞共培养后成纤维细胞内相关基因的表达变化。结果与结论:①分离并鉴定脂肪干细胞,成功构建生长激素-脂肪干细胞系;②生长激素组细胞内生长激素的mRNA和蛋白表达较空载组和对照组显著升高(P<0.01),且上清中生长激素浓度更高;生长激素组细胞内ERK1/2及p-ERK1/2蛋白表达上调;③生长激素组较空载组促成纤维细胞迁移和增殖能力增强(P<0.05);④生长激素-脂肪干细胞与成纤维细胞共培养后成纤维细胞内基质金属蛋白酶2基因表达下调,Ⅰ型胶原蛋白基因表达上调,Ⅲ型胶原蛋白基因表达无显著变化;⑤抑制生长激素-脂肪干细胞内ERK蛋白后,成纤维细胞内上述基因mRNA表达差异消失;⑥结果表明,慢病毒转染法构建过表达生长激素的脂肪干细胞效率较高,且该细胞可持续分泌生长激素并促进成纤维细胞增殖迁移,该生理作用可能与上调细胞内ERK1/2信号通路有关。BACKGROUND:Growth hormone plays an important physiological role in immune regulation,cell proliferation,and protein synthesis.It has been proven that growth hormone can promote the healing of acute and chronic wounds.OBJECTIVE:To construct adipose-derived stem cell lines overexpressing growth hormone,and to investigate the effect of adipose-derived stem cell lines overexpressing growth hormone on proliferation and migration of fibroblasts and its molecular mechanism.METHODS:(1) Adipose-derived stem cells were isolated and identified in vitro.(2) Growth hormone-overexpressing lentivirus was constructed.Adiposederived stem cells were divided into growth hormone group,empty group,and control group.The above three groups were transfected with growth hormoneoverexpressing lentivirus,empty lentivirus,and no infection.(3) The mRNA and protein expression levels of growth hormone and related genes of ERK1/2 pathway in cells and supernatants of each group were detected by RT-qPCR,western blot assay,and ELISA.(4) The effects of growth hormone group and empty group on the migration and proliferation of fibroblasts were detected by the scratch test,Transwell test,and MTS test.The expression of related genes in fibroblasts after co-culture with adipose-derived stem cell lines overexpressing growth hormone was detected by RT-qPCR.(5) After inhibiting adipose-derived stem cell lines overexpressing growth hormone by ERK inhibitor,the expression of above-mentioned genes in fibroblasts was observed after co-culture with ERK inhibitor.RESULTS AND CONCLUSION:(1) Adipose-derived stem cells were isolated and identified and the adipose-derived stem cell lines overexpressing growth hormone were successfully constructed.(2) The mRNA and protein expression levels of growth hormone in the growth hormone group were significantly higher than those in the empty group and control group(P < 0.01),and growth hormone in the supernatant of the growth hormone group was higher.The expression of ERK1/2 and p-ERK1/2 protein was up-regulated in the grow

关 键 词：创面愈合 组织工程 脂肪干细胞 生长激素 ERK通路 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]