人真皮间充质干细胞对增生性瘢痕成纤维细胞α-SMA和DCN表达的影响  

Influences of human dermis derived mesenchymal stem cells on α-SMA and DCN expressions of hypertrophic scars fibroblasts

作　　者：张文夺[1] 邓呈亮[1] 郭常敏[1] 聂开瑜[1] 唐修俊[1] 魏在荣[1] 王达利[1] 

机构地区：[1]遵义医学院附属医院烧伤整形外科,563003

出　　处：《中华整形外科杂志》2016年第4期285-292,共8页Chinese Journal of Plastic Surgery

基　　金：国家自然科学基金（81060157）;贵州省科技创新人才团队项目[黔科合人才团队2014（4016）]

摘　　要：目的 初步探讨人真皮间充质干细胞（human dermis derived mesenchymal stem cells, hDMSCs）对不同时期增生性瘢痕成纤维细胞（HSFB） α-平滑肌肌动蛋白（α smooth muscle actin,α-SMA）和核心蛋白多糖（decorin,DCN）表达的影响,以探讨间充质干细胞用于增生性瘢痕防治的可行性.方法 机械法联合酶消化法分离、培养hDMSCs,取第3代生长良好的细胞,以流式细胞仪检测hDMSCs的CD分子,免疫细胞化学检测角蛋白19和波形蛋白,向成脂、成软骨和成骨细胞诱导分化.根据临床上增生性瘢痕形成的病程将瘢痕标本分为6个月、1年、2年3组,每组3例.将3组HSFB分别与第3代生长良好的贴壁hDMSCs通过非接触式Transwell共培养体系培养21 d,并分别与相对应组HSFB普通6孔板培养21d进行对照,采用RT-PCR和Western Blot技术检测3组共培养后HSFB α-SMA和DCN的mRNA和蛋白的表达情况.结果 hDMSCs高表达CD73、CD105、CD44、CD90等间充质干细胞表面标志,不表达CD14、CD34、CD45等造血干细胞表面标志,波形蛋白阳性表达,不表达角蛋白19.经诱导可以向成脂、成软骨和成骨细胞分化,符合间充质干细胞的最低鉴定标准.普通6孔板培养的6个月、1年、2年3组HSFB的α-SMA mRNA及蛋白的表达量分别为198.20±15.46、0.29±0.070,175.24±17.04、0.38 ±0.110,125.73 ±6.99、0.33±0.085：DCNmRNA及蛋白的表达量分别为61.30 ±9.79、0.015 ±0.003,70.89±11.29、0.020±0.007,77.31±4.80、0.023±0.003.与5＆#215;104 hDMSCs共培养处理后6个月、1年、2年3组HSFB0α-SMA mRNA及蛋白的表达量分别48.40±6.42、0.100±0.020,192.16±11.37、0.110±0.014,73.33 ±6.29、0.ll0±0.016;DCN mRNA及蛋白的表达量分别为156.92±14.91、0.049±0.015,154.42±18.17、0.033±0.008,140.82±7.32、0.030±0.004.与普通6孔板培养相比较,共培养各组HSFB的α-SMA mRNA及蛋白表达下调,DCN mRNA及蛋白表达上调,并以增生性瘢痕形成早期即6个月组变化较明显.结论 hDMSCs�Objective To preliminarily explore the effects of human dermis derived mesenchymal stem cells （hDMSCs） on expressions of α-smooth muscle actin （α-SMA） and decorin （DCN） in hypertrophic scars fibroblasts （HSFB） at different periods,and to explore the feasibility of MSCs in prevention and treatment of HSFBs.Methods hDMSCs were cultured with mechanical method combined with enzyme digestion.The cells of the third generation which were well grown were taken,and flow cytometry （FCM） was used to detect CD molecules in hDMSCs.Immunocytochemistry was used to detect cytokeratin 19 （CK19） and vimentin and identify the separated cells.The cells were differentiated into lipoblasts,chondroblasts and osteoblasts.According to the formation course of hypertrophic scar,the scar specimens were divided into 6-month,l-year,and 2-year group with three cases in each group.HSFBs from different groups were co-cultured with well-adherent hDMSCs of the third generation in non-contact transwell co-culture system for 21 days.And HSFBs from the corresponding groups were cultured in normal six-well plate as the controls.Real-time fluorescent-polymerase chain reaction （RT-PCR） and Western Blot were used to detect the expressions of mRNA and proteins of α-SMA and DCN in HSFBs from different groups.Results hDMSCs highly expressed the surface markers including CD73,CD105,CD44 and CD90,etc.,but did not express hematopoietic stem cell surface markers including CD 14,CD34 and CD45.They positively expressed vimentin but not CK19.The cells can be differentiated into lipoblasts,chondroblasts and osteoblasts,which was in line with the minimum identification standards of mesenchymal stem cells.For HSFB cultured in normal six-well plates,α-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 198.20 ± 15.46/0.29 ± 0.070,175.24 ± 17.04/0.38 ± 0.110,and 125.73 ± 6.99/0.33 ±0.085,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 61.30 ± 9.79/0.015

关 键 词：真皮 间质干细胞 瘢痕 成纤维细胞 共同培养技术 

分 类 号：R622[医药卫生—整形外科]