体外诱导人羊膜间充质干细胞向韧带细胞分化的实验研究  

EXPERIMENTAL RESEARCH OF DIFFERENTIATION OF HUMAN AMNIOTIC MESENCHYMAL STEM CELLS INTO LIGAMENT CELLS IN VITRO

作　　者：金瑛[1] 李豫皖[1] 张承昊[2] 吴术红[1] 陈代雄[3] 刘毅[1] 

机构地区：[1]遵义医学院附属医院骨科,贵州遵义563000 [2]四川大学华西医院骨科 [3]遵义医学院附属医院贵州省细胞工程实验室,贵州遵义563000

出　　处：《中国修复重建外科杂志》2016年第2期237-244,共8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]2091)~~

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的特性及经诱导后在体外能否向韧带细胞分化。方法取产妇胎盘通过酶消化法获得h AMSCs,流式细胞术鉴定h AMSCs表型特征。取第3代h AMSCs分别加入含不同诱导液的L-DMEM/F12培养基:A组TGF-β1+b FGF,B组透明质酸(hyaluronic acid,HA),C组TGF-β1+b FGF+HA,D组为空白对照组。倒置相差显微镜观察诱导后21 d各组细胞形态学变化;磺酰罗丹明B(sulforhodamine B,SRB)比色法检测各组细胞生长活性;诱导后7、14、21 d分别采用免疫组织化学染色及实时荧光定量PCR检测韧带特异性蛋白和基因Ⅰ型胶原、Ⅲ型胶原、肌腱蛋白C(tenascin C,TNC)表达。结果流式细胞术鉴定结果表明h AMSCs表达MSCs表型。倒置相差显微镜观察示诱导21 d A、B、C组细胞均呈长梭形纤维细胞样生长,C组细胞形态更单一、有明显方向性且较A组排列更紧凑。SRB比色法检测示各组细胞于培养6 d左右达增殖高峰,6 d时A、B、C组细胞活性均显著高于D组。免疫组织化学结果示:诱导培养7 d,各组均未检测到TNC表达;7、14、21 d A、B、C组Ⅰ型胶原表达均显著高于D组,14、21 d A、B、C组Ⅲ型胶原表达显著高于D组,差异均有统计学意义(P<0.001)。B组Ⅰ型胶原表达以及A、C组Ⅲ型胶原、TNC表达随时间增加均呈逐渐增高趋势,各时间点间比较差异均有统计学意义(P<0.001)。实时荧光定量PCR结果示:诱导培养21 d,C组Ⅰ型胶原、TNC m RNA相对表达量显著高于A、B组,B组Ⅲ型胶原m RNA相对表达量显著高于A、C组,差异均有统计学意义(P<0.05)。A、C组Ⅰ型胶原、TNC以及C组Ⅲ型胶原m RNA相对表达量随时间增加呈逐渐上调趋势,各时间点间比较差异均有统计学意义(P<0.001)。结论 h AMSCs具有MSCs的特性,有良好增殖能力,经体外诱导后韧带特异性基因表达上调,韧带特异性蛋白合成增加,可作为韧带组织Objective To discuss whether human amniotic mesenchymal stem cells（hAMSCs） possesses the characteristic of mesenchymal stem cells,and could differentiate into ligament cells in vitro after induction.Methods The hAMSCs were separated through enzyme digestion,and the phenotypic characteristics of hAMSCs were tested through flow cytometry.The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor β_1（TGF-β_1）＋basic fibroblast growth factor（bFGF）（group A）,containing hyaluronic acid（HA）（group B）,containing TGF-β_1＋bFGF＋HA（group C）,and simple L-DMEM/F12 medium（group D） as control group.The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction;the cellular activities and proliferation were examined by sulforhodamine（SRB） colorimetric method;and specific mRNA and protein expressions of ligament including collagen type Ⅰ,collagen type Ⅲ,and tenascin C（TNC） were measured by real-time fluorescence quantitative PCR and immunohistochemical staining.Results The flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype.After 21 days of induction,the cells in groups A,B,and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope,and cells showed single shape,obvious directivity,and compact arrangement in group C.The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days;the cellular activities of groups A,B,and C were significantly higher than that of group D at 6 days after induction.Also,the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days;expressions of collagen type Ⅰ in groups A,B,and C were significantly higher than that in group D at 7,14,and 21 days（P〈0.001）;the expressions of collagen type Ⅲ in groups A,B,and C were significantly higher than that in group D at 14 and 21 days（P〈0.001）.There was an increa

关 键 词：人羊膜间充质干细胞 韧带组织工程 TGF-Β1 BFGF 透明质酸 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]