成人外周血平滑肌祖细胞的体外培养  

In vitro culture of smooth muscle progenitor cells from adult human peripheral blood

作　　者：周学凯[1] 倪旭东[1] 李飞[1] 张荣庆[1] 郭文怡[1] 

机构地区：[1]解放军第四军医大学西京医院心血管内科,陕西省西安市710032

出　　处：《中国组织工程研究与临床康复》2011年第14期2587-2591,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：陕西省科学技术研究发展计划项目(2010K01-171)"冠脉介入治疗术后再狭窄防治的基础与临床研究"~~

摘　　要：背景:外周血平滑肌祖细胞具有向平滑肌细胞分化的能力。目的:探索体外分离培养成人外周血平滑肌祖细胞的方法及其生长分化特性。方法:采用密度梯度离心法分离获得成人外周血单个核细胞,培养12d后,应用流式细胞仪鉴定分析平滑肌祖细胞并分选纯化,继续培养诱导分化。采用倒置显微镜观察平滑肌祖细胞的形态变化,免疫荧光染色法观察其α-肌动蛋白的表达,同时应用Westernblot法检测调宁蛋白、平滑肌肌球蛋白重链的表达情况。此外,观察平滑肌祖细胞的生长特性,绘制生长曲线。结果与结论:成人外周血单个核细胞诱导培养4d时开始出现细胞集落,12d时细胞呈明显梭形。流式细胞仪分析显示,CD14和CD105双阳性的平滑肌祖细胞占贴壁细胞的(71.8±7.2)%。分选纯化的平滑肌祖细胞培养到28d呈旋涡状生长。间接免疫荧光染色显示,α-肌动蛋白表达呈阳性。Westernblot检测显示,调宁蛋白和平滑肌肌球蛋白重链分别于14,21d开始表达,并逐渐增加。生长曲线表明,细胞生长至第6天进入对数生长期,第12天后进入平台期。提示通过对外周血单个核细胞的诱导培养,可以获得大量的平滑肌祖细胞。它能稳定增殖,并可进一步分化为平滑肌细胞。BACKGROUND：Smooth muscle progenitor cells（SPCs） from human peripheral blood have the capacity to differentiate into smooth muscle cells.OBJECTIVE：To investigate the method of in vitro purifying and culturing SPCs,and to observe differentiation and growth of SPCs.METHODS：Mononuclear cells were isolated from adult human peripheral blood by density gradient centrifugation.After 12 days of culture in vitro,SPCs were identified and sorted by flow cytometry.SPCs were then induced to differentiate and amplify.Inverted microscope was used to observe morphological changes.Smooth muscle cells specific markers（α-SMA,Calponin,SM-MHC） were then checked with immunofluorescent staining and Western blotting.In addition,Cell growth curve of culture was drawn.RESULTS AND CONCLUSION：Four days later,cell colonies were presented.Twelve days later,most adherent cells were showing spindle-shape.CD14/CD105 double positive cells were SPCs,which accounted for（71.8±7.2）% of adherent cells.Twenty-eight days later,cells arranged in order,showing whirlpool-shape.On day 28,immunostaining of adherent cells was positive for α-SMA.Expression of calponin and SM-MHC was found at 14 d and 21 d with Western blotting respectively,and they gradually enhanced.Growth curve of SPCs showed that increased logarithmic phase started on the 6th day,and platform phase started on the 12th days.Results indicated that SPCs could be harvested through culturing mononuclear cells from adult human peripheral blood.They could maintain the ability of proliferation,and differentiate into smooth muscle cells in vitro.So,SPCs could be selected as the seed cells.

关 键 词：平滑肌祖细胞 CD14 CD105 细胞培养 诱导分化 组织工程 种子细胞 

分 类 号：R394.2[医药卫生—医学遗传学]