摘要
目的探讨人羊膜间充质干细胞(human amniotic mesenchymal stem cells,h AMSCs)是否具有MSCs特性,以及经TGF-β_1和VEGF联合诱导后是否具有向韧带成纤维细胞分化的能力。方法取自愿捐赠的足月产妇胎盘,采用胰蛋白酶胶原酶联合消化法分离培养h AMSCs,流式细胞术检测h AMSCs表型分子,免疫荧光染色检测h AMSCs其角蛋白-19(cytokeratin-19,CK-19)和波形蛋白表达情况。取第3代h AMSCs,分别使用含TGF-β_1和VEGF的L-DMEM/F12成韧带或纤维细胞诱导培养基(实验组)和普通L-DMEM/F12培养基(对照组)培养,细胞增殖-毒性检测试剂盒(cell counting kit-8,CCK-8)检测两组细胞增殖能力;培养5、10、15 d分别采用免疫荧光染色及实时荧光定量PCR检测韧带及血管生成相关特异性蛋白和基因表达。结果倒置相差显微镜观察示h AMSCs呈单层贴壁生长;流式细胞术结果示h AMSCs表达MSCs表型分子;免疫荧光染色示h AMSCs高表达波形蛋白、低表达CK-19;h AMSCs具有向成骨、成软骨及成脂细胞分化的能力。CCK-8法检测示,7 d时两组细胞均达增殖高峰,实验组细胞增殖能力于7 d后显著高于对照组(P<0.05)。免疫荧光染色示,培养5、10、15 d时实验组Ⅰ型胶原、Ⅲ型胶原、纤维连接蛋白(Fibronectin)、细胞连接素(Tenascin-C)表达染色均较对照组增强。实时荧光定量PCR结果示,随时间延长实验组Ⅰ型胶原、Ⅲ型胶原、Fibronectin、α-肌动蛋白(α-smooth muscle actin,α-SMA)、VEGF m RNA相对表达量均逐渐上调(P<0.05)。除培养5 d两组Ⅰ型胶原、Ⅲ型胶原、VEGF m RNA相对表达量比较差异无统计学意义(P>0.05)外,其余各时间点实验组Ⅰ型胶原、Ⅲ型胶原、Fibronectin、α-SMA和VEGF m RNA相对表达量均显著高于对照组(P<0.05)。结论 h AMSCs具有MSCs特征,且体外增殖能力良好,可作为组织工程种子细胞来源;体外诱导后韧带成纤维细胞及血管生成相关特异性基因表达上调,韧带成纤维细�
Objective To investigate whether human amniotic mesenchymal stem cells （hAMSCs） have the characteristics of mesenchymal stem cells （MSCs） and the differentiation capacity into ligament fibroblastsin vitro. Methods The hAMSCs were separated through trypsin and collagenase digestion from placenta, the phenotypic characteristics of hAMSCs were detected by flow cytometry, the cytokeratin-19 （CK-19） and vimentin expression of hAMSCs were tested through immunofluorescence staining. The hAMSCs at the 3rd passage were cultured with L-DMEM/F12 medium containing transforming growth factor β1 （TGF-β1） and vascular endothelial growth factor （VEGF） as the experimental group and with single L-DMEM/F12 medium as the control group. The morphology of hAMSCs was observed by inverted phase contrast microscope; the cellular activities and ability of proliferation were examined by cell counting kit-8 （CCK-8） method; the ligament fibroblasts related protein expressions including collagen type I, collagen type III, Fibronectin, and Tenascin-C were detected by immunofluorescence staining; specific mRNA expressions of ligament fibroblasts and angiogenesis including collagen type I, collagen type III, Fibronectin, α-smooth muscle actin （α-SMA）, and VEGF were measured by real-time fluorescence quantitative PCR. Results The hAMSCs presented monolayer and adherent growth under inverted phase contrast microscope; the flow cytometry results demonstrated that hAMSCs expressed the MSCs phenotypes; the immunofluorescence staining results indicated the hAMSCs had high expression of the vimentin and low expression of CK-19; the hAMSCs possessed the differentiation ability into the osteoblasts, chondroblasts, and lipoblasts. The CCK-8 results displayed that cells reached the peak of growth curve at 7 days in each group, and the proliferation ability in the experimental group was significantly higher than that in the control group at 7 days （P〈0.05）. The immunofluorescence staining results showed that the express
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2017年第5期582-593,共12页
Chinese Journal of Reparative and Reconstructive Surgery
基金
贵州省科学技术基金资助项目(黔科合SY字[2010]3091
黔科合LH字[2016]7477)~~
关键词
人羊膜间充质干细胞
韧带成纤维细胞
TGF—β1
VEGF
组织工程韧带
Human amniotic mesenchymal stem cells
ligament fibroblasts
transforming growth factor β1
vascular endothelial growth factor
tissue engineered ligament
