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

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骨髓间质干细胞与平滑肌细胞混合培养:于骨髓间质干细胞转染绿色荧光蛋白后24h,向每孔内加入等量的平滑肌细胞混合培养,观察细胞形态变化。以单独培养的骨髓间质干细胞为对照。主要观察指标:①骨髓间充质干细胞流式细胞仪检测结果。②不同条件培养下平滑肌细胞抗体免疫荧光染色结果。结果:①免疫荧光染色示,平滑肌细胞抗SM-α-actin阳性,抗calponin阳性。骨髓间质干细胞抗SM-α-actin阳性,而抗calponin阴性。②流式细胞仪检测,骨髓间质干细胞不表达CD34,弱表达CD71,高表达CD90。③骨髓间质干细胞转染绿色荧光蛋白持续表达约2周～3周。④加入平滑肌细胞培养液或不同浓度的胎牛血清,骨髓间充质干细胞生长良好。骨髓间充质干细胞与平滑肌细胞分层培养中,骨髓间充质干细胞生长对胎牛血清呈浓度依赖性。骨髓间质干细胞与平滑肌细胞混合培养7d后免疫荧光染色,可见绿色荧光蛋白和抗SM-α-actin、Calponin的双标细胞存在。而加平滑肌细胞条件培养液与分层培养组中骨髓间质干细胞均不表达Calponin。结论:①平滑肌细胞培养液及细胞因子只能促进骨髓间充质干细胞生长,细胞胞浆颗粒增多,并不能诱导骨髓间充质干细胞分化为平滑肌细胞。②细胞间直接接触对诱导骨髓间质干细胞定向分化为平滑肌细胞起决定作用。

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

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 cells (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 cell factors on MSCs differentiation.DESIGN: Controlled experiment in vitro with repeated observation and measurement based on cells.SETTING: Department of Cardiology, First Hospital of Xi'an Jiaotong University.MATERIALS: The experiment was accomplished in the Laboratory of Cardiology, First Hospital of Xi'an Jiaotong University between May 2003 and May 2004. SD rats of either gender were 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 cells were 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 were 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-well culture plate. The medium was DMEM containing 0%, 5%,7.5% fetal bovine serum (FBS) and SMCs conditioned medium containing 0%, 5%, 7.5% FBS, respectively. The cells were cultured for 10-14 days and immunofluorescence analysis was performed by using monoclonal antibodies against SM-α-actin, calponin.②Indirect co-culture of MSCs with SMCs were established using a semi-permeable membrane cell culture insert. The inserts were plated into culture well. SMCs were cultured on the inside of inserts while MSCs were added to the outside of inserts, respectively. MSCs were culture alone in medium containing 3%, 7.5% FBS and immunofluorescence analysis was performed by using monoclonal antibodies against SM-α-actin, calponin.③MSCs were transfected with pEGFP-N3. After 24 hours, the MSCs were cocultivated with SMCs at an equal density for 7-14 days.As a control, MSCs were cultured alone. MSCs co-cultured were stained with antibodies against calponin, SM-α-actin. MAIN OUTCOME MEASURES: ①Identification of MSCs by floe cytometer.②cytoplasmic antigen expression of SMCs. RESULTS: ①Immunofluorescence analysis showed that MSCs expressed SM-α-actin, but did not express calponin. As a control, SMCs expressed both SM-α-actin and calponin.②Flow cytometry showed that MSCs expressed CD71 of low level, CD90 of high level and no expression of CD34. ③The MSCs transfected with green fluorescence protein continued to express for 2-3 weeks. ④MSCs grew well in SMCs conditioned medium or different concentrations of FBS. Cell growth was FBS concentration dependent in indirect co-culture system of MSCs and SMCs. Several double-positive cells in direct co-culture system were detected enhanced green fluorescence protein and antibodies against calponin, SM-α-actin. CONCLUSION: ①SMCs conditioned medium and cell factor only promote MSCs growth and cytoplasmic granules increase. But these do not induce MSCs differentiate into SMCs. ②The cell-to-cell contact is essential for MSCs differentiation to SMCs.