摘要
目的探讨脂肪干细胞(ADSCs)过表达miR-34a后释放的外泌体对人增生性瘢痕成纤维细胞(HSF)增殖及凋亡的影响。方法从人脂肪组织中分离原代ADSCs,流式细胞术鉴定其表面标记分子,茜素红和碱性磷酸酶染色观察其成骨分化能力;采用miR-34a过表达慢病毒感染ADSCs,获得miR-34a过表达的ADSCs(ADSCs/miR-34a)和阴性对照的ADSCs(ADSCs/miR-NC),然后提取各组细胞上清液中的外泌体,获得ADSCs外泌体(ExoADSCs)、miR-34a过表达的ADSCs外泌体(ExoADSCs/miR-34a)和阴性对照的ADSCs外泌体(ExoADSCs/miR-NC),利用透射电镜观察外泌体形态,Western blot检测外泌体表面标志蛋白CD81和CD63;再将得到的外泌体分别与HSF共培养,分为对照组(HSF+PBS)、ExoADSCs处理组(HSF+ExoADSCs)、ExoADSCs/miR-NC处理组(HSF+ExoADSCs/miR-NC)和ExoADSCs/miR-34a处理组(HSF+ExoADSCs/miR-34a)。采用qRT-PCR法检测各组ADSCs及外泌体中miR-34a的表达水平;MTT法检测各组HSF细胞的增殖能力;流式细胞术检测各组HSF细胞的凋亡水平。Western blot法检测各组HSF细胞凋亡相关蛋白(cleaved-Caspase-3、Bax和Bcl-2)、纤维化相关蛋白(COLⅠ、COLⅢ)及TGF-β/Smad信号通路相关蛋白(TGF-β1、p-Smad2和p-Smad3)的表达水平。结果成功分离得到ADSCs,并具有成骨分化能力;透射电镜可见直径在50~100 nm呈双层膜结构的圆形或椭圆形囊泡状小体,Western blot显示外泌体标志性蛋白CD81和CD63阳性表达;miR-34a过表达可增加ADSCs及其外泌体中miR-34a表达水平;与ExoADSCs处理组和ExoADSCs/miR-NC处理组比较,ExoADSCs/miR-34a干预可抑制HSF增殖(P <0. 05),提高其凋亡率(P <0. 05),并上调cleavedCaspase-3和Bax蛋白表达水平(P <0. 05),并下调Bcl-2、COLⅠ、COLⅢ、p-Smad2、p-Smad3和TGF-β1等蛋白表达水平(P <0. 05)。结论过表达miR-34a的ADSCs外泌体可能通过下调TGF-β/Smad信号通路抑制HSF细胞增殖,促进其凋亡。
Objective To investigate the effect of exosomes(Exo)released from adipose derived stem cells(ADSCs)with overexpressed miR-34 a on proliferation and apoptosis of human hypertrophic scar fibroblasts(HSF).Methods Primary ADSCs were isolated from human adipose tissue and their surface markers were identified by flow cytometry.The osteogenic differentiation of ADSCs was observed by using alizarin red and alkaline phosphatase staining.ADSCs were infected by lentiviruses containing miR-34 a to obtain over-expressed miR-34 a ADSCs(ADSCs/miR-34 a)and negative control ADSCs(ADSCs/miR-NC).The exosome was extracted from cell supernatant of each group and ADSCs exosome(ExoADSCs),over-expressed miR-34 a ADSCs exosome(ExoADSCs/miR-34 a)and negative control ADSCs exosome(ExoADSCs/miR-NC)were obtained.The morphology of exosomes was observed by transmission electron microscope(TEM),and the surface marker proteins CD81 and CD63 were detected by Western blot.The exosomes were co-cultured with HSF and divided into control group(HSF+PBS),Exo ADSCs treatment group(HSF+ExoADSCs),ExoADSCs/miR-NC treatment group(HSF+ExoADSCs/miR-NC)and ExoADSCs/miR-34 a treatment group(HSF+ExoADSCs/miR-34 a).Quantificational real-time PCR(qRT-PCR)was used to detect the expression of miR-34 a in ADSCs and exosomes.The proliferation of HSF cells was detected by MTT assay.The apoptotic rate of HSF cells was detected by flow cytometry.The expression levels of apoptosis related proteins(cleaved-Caspase-3,Bax and Bcl-2),fibrosis-related proteins(COLⅠ,COLⅢ)and TGF-β/Smad signal pathway related proteins(TGF-β1,p-Smad 2 and p-Smad3)were detected by Western blot assay.Results ADSCs were successfully isolated and possessed osteogenic differentiation ability.TEM showed round or oval vesicle bodies with a diameter of 50~100 nm with bilayer membrane structure.Western blot showed positive expression of CD81 and CD63,which were the marker proteins of exosomes.Overexpression of miR-34 a could increase the expression of miR-34 a in ADSCs and exosomes.Compared with ExoADSCs group and ExoADSCs/miR-NC group,ExoADSCs/miR-34 a intervention could inhibit HSF cell proliferation(P<0.05),increase cell apoptosis rate(P<0.05),up-regulate the expression of cleaved-Caspase-3 and Bax protein(P<0.05),and down-regulate the expression of Bcl-2,COLⅠ,COLⅢ,p-Smad2,p-Smad3 and TGF-β1 protein(P<0.05).Conclusion The exosome of miR-34 a overexpressed ADSCs may inhibit the HSF cell proliferation and promote cell apoptosis by down-regulating TGF-β/Smad signaling pathway.
作者
肖向阳
郑德义
王宝云
李自力
Xiao Xiangyang;Zheng Deyi;Wang Baoyun(Dept of Burn and Plastic Surgery,Guizhou People’s Hospital,Guiyang 550002)
出处
《安徽医科大学学报》
CAS
北大核心
2020年第6期887-893,共7页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81760021)
贵州省人民医院青年基金(编号:GZSYQN[2015]12号)。
作者简介
肖向阳,男,硕士研究生;责任作者:郑德义,男,博士,主任医师,硕士生导师,Email:zhongliying1014@163.com。
