细胞间接触诱导大鼠骨髓间质干细胞向平滑肌细胞的分化(英文)  

Differentiation of rat mesenchymal stem cells into smooth muscle cells induced by cell-to-cell contact

作　　者：徐正云 马爱群[2] 王亭忠[2] 蒋文慧[2] 胡志[2] 

机构地区：[1]长治市人民医院心内科,山西省长治市104600 [2]西安交通大学第一医院心内科,陕西省西安市710061

出　　处：《中国组织工程研究与临床康复》2007年第15期2980-2984,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:干细胞能够分化为血管平滑肌细胞,参与动脉粥样硬化发生、发展,其机制尚不清楚。“环境诱导分化学说”认为细胞-细胞接触、细胞因子可能在干细胞分化过程中起关键作用。骨髓间质干细胞具有向平滑肌细胞分化的潜能。目的:体外诱导骨髓间质干细胞分化为平滑肌细胞,观察已分化成熟平滑肌细胞及细胞因子对骨髓间质干细胞分化的影响。设计:以细胞为观察对象的重复观察测量,对照性体外实验。单位:西安交通大学第一医院心内实验室。材料:实验于2003-05/2004-05在西安交通大学第一医院心内实验室完成。SD大鼠60～80g、90～110g,雌雄不限,由本校动物中心提供。PE标记小鼠抗大鼠CD34(Santa Cruz),PE标记小鼠抗大鼠CD71(Oxford Biotechnology),FITC标记小鼠抗大鼠CD90(Oxford Biotechnology),小鼠抗人单克隆抗体SM-α-actin(NeoMarkers),小鼠抗人单克隆抗体Calponin(NeoMarkers),TRITC标记山羊抗小鼠IgG(SBA),pEGFP-N3(本实验室提供),Lipofectamine2000(Invitrogen)。方法:采用密度梯度离心和细胞贴壁法与组织贴块法分离培养骨髓间质干细胞与血管平滑肌细胞。通过流式细胞仪检测,平滑肌细胞抗体免疫荧光染色,鉴定骨髓间质干细胞与血管平滑肌细胞。将骨髓间质干细胞转染绿色荧光蛋白,以未转染的骨髓间质干细胞为对照。骨髓间质干细胞与血管平滑肌细胞分条件培养:①取3代骨髓间质干细胞传至12孔培养板,培养液为低糖DMEM加骨髓间质干细胞条件培养液各半,胎牛血清浓度为0%、5%、7.5%。细胞固定后,应用平滑肌细胞抗体免疫荧光染色检测结果。②将半透膜小室置于6孔培养板每一孔内,使每孔隔成内外两室。将骨髓间质干细胞在外室培养,平滑肌细胞在内室培养。以单独培养的骨髓间质干细胞为对照,胎牛血清浓度为3%、7.5%。用平滑肌细胞抗体免疫荧光染色检测结果。③骨髓间质干细胞BACKGROUND： It is conceivable that bone marrow stem cells can differentiate into smooth muscle cells （SMCs） and contribute to neointimal formation in atherogenesis. However, the mechanism remains unknown. The ＂milieu-induced-differentiation＂ hypothesis focuses on the key role of cell-to-cell contact and cytokine on the differentiation of stem cells. Bone marrow mesenchymal stem calls （MSCs） have the potential to differentiate into SMCs. OBJECTIVE： To induce MSCs into SMCs in vitro, and investigate the influence of the differentiated SMCs or call factors on MSCs differentiation. DESIGN ： Controlled experiment in vitro with repeated observation and measurement based on calls SETTING： Department of Cardiology, First Hospital of Xran Jiaotong University MATERIALS： The experiment was accomplished in the Laboratory of Cardiology, First Hospital of Xran Jiaotong University between May 2003 and May 2004. SD rats of either gender ware provided by the Animal Center of Xi＇an Jiao Tong University, 60-80 g, 90-110 g. The following antibodies were used： Mouse anti human SM-α-actin （NeoMarkers）, Mouse anti human Calponin （NeoMarkers）, TRITC-coupled goat anti mouse IgG antibody （SBA）. Mouse anti rat CD34 conjugated FITC （Santa Cruz）, Mouse anti rat CD71 conjugated FITC （Oxford Biotechnology）, Mouse anti rat anti-CD90 conjugated PE （Oxford Biotechnology）. Lipofectamine 2000 （Invitrogen）. PEGFP-N3 （the laboratory）. METHODS ： Bone marrow mesenchymal stem calls ware obtained from rat bone marrow by using percoll density gradient centrifugation. SMCs were isolated by using tissue explantation method. Flow cytometer was used to detect the immunofluorescence stain. Then MSCs and SMCs ware identified. MSCs were transfected with pEGFP-N3 by Lipofectamine 2000, while untransfected MSCs were taken as controls. Conditioned culture of MSCs and SMCs： ①MSCs at passage 3 were seeded on chamber slides in a 12-wall culture plate. The medium was DMEM containing 0%, 5%, 7.5%

关 键 词：骨髓间质干细胞 血管平滑肌细胞 细胞培养 细胞分化 

分 类 号：R394.2[医药卫生—医学遗传学]