骨髓间充质干细胞体外诱导分化为平滑肌细胞的实验

Differentiation of bone marrow mesenchymal stem cells into the smooth muscle cells in vitro

目的:探讨新西兰兔骨髓间充质干细胞在体外不同条件下定向分化为阴茎海绵体平滑肌细胞的可行性。方法:实验于2004-10/2005-05在中山大学第二附属医院实验中心完成。采用全骨髓贴壁法分离骨髓间充质干细胞;分别通过添加血管内皮生长因子和碱性成纤维细胞生长因子进行体外直接诱导以及与阴茎海绵体平滑肌细胞不同比例(据骨髓间充质干细胞与阴茎海绵体平滑肌根细胞数量比,共培养诱导分为4∶1组、2∶1组以及1∶1组。每6孔作为1个诱导组,每孔兔骨髓间充质干细胞终浓度为1×10L-1,阴茎海绵体平8滑肌细胞相应终浓度分别为0.25×10L-1、0.5×108L-1及1×10L-1)进行88共培养两种方法诱导干细胞分化为阴茎海绵体平滑肌细胞。培养2d后的细胞爬片进行荧光免疫细胞化学染色测定抗α平滑肌肌动蛋白抗体表达,全程避光进行。完成染色后,立即进行激光共聚焦显微镜镜检。每孔爬片随机取5个视野,计算出每孔中Hoechst33342与FITC(Cy3)或双阳性细胞数及Hoechst33342单阳性的细胞数的比值以计算每孔的诱导率,应用SPSS软件进行完全随机设计的方差分析。结果:①阴茎海绵体平滑肌细胞的生长特性、形态及鉴定:组织块贴壁后细胞渐游出,呈梭形或长条形,原代细胞汇合90%约需16～19d,传代后生长加快,90%汇合时1∶2传代后约3d可再次传代。细胞融合时呈现典型的“峰-谷”样形态。传至10代之后细胞生长放缓,胞体渐变宽大,胞内颗粒增多。荧光免疫细胞化学检测特异性抗α平滑肌肌动蛋白抗体见绝大部分细胞呈阳性。②直接诱导以及共培养诱导的初步分析结果:骨髓间充质干细胞之细胞核标记良好,诱导后见胞浆表达多量抗α平滑肌肌动蛋白抗体,与阳性对照组相似;高倍镜下可清晰见平滑肌肌动蛋白肌丝,而阴性对照组只有核显色,无抗α平滑肌肌动蛋白抗体表达。③不同组别诱导率方差分析S-N-K检验得各组总体均数差异性检验的(F=44.831P<0.05),表明统计学上有显著性差异;共培养1∶1,组效率最高,依次是共培养2∶1组、共培养4∶1组,血管内皮生长因子组、血管内皮生长因子+碱性成纤维细胞生长因子组最低,后两者间诱导效果在统计学上无差别。结论:骨髓间充质干细胞可通过添加生长因子和共培养的方法诱导分化为阴茎海绵体平滑肌细胞,其中以共培养的方法效率较高。

AIM： To explore the feasibility of differentiation of bone marrow mesenchymal stem cells （MSCs） into corporal smooth muscle cells in vitro under different condition, METHODS：The experiment was carried out in the laboratory of the Second Affiliated Hospital of Sun Yat-sen University from October 2004 to May 2005, MSCs from New Zealand rabbits were isolated by using differential attachment method and characterized. The cells were induced to differentiate into corporal smooth muscle cells in two ways： direct induction by adding （VEGF）/（VEGF and b-FGF） and co-culture with rabbit corporal smooth muscle cells in vitro. Co-culture group was subdivided into 3 groups with the ratios of MSCs to corporal smooth muscle cells being 4：1, 2：1 and 1：1,respectively, and the constant concentration of MSCs in each subgroup was 1×10^8 L^-1~. After 2-day induction, immunocytochemistry was performed to or-smooth muscle actin antibody and induction rates of every group were determined. RESULTS： ①Spindle cells were seen to migrate from the explants of corporal tissue and reached 90% confluence at 16-19 days after tissue attachment. When confluenced, the cells displayed ＂hill-valley＂ morphology. ② After induction, α-smooth muscle actin antibody could be detected within the induced MSCs, while in control group noct-smooth muscle actin antibody was detected. ③Tests of One-way ANOVA showed that there was statistically significance of the differentiation rates in different groups （F=44,831, P〈 0.05）. The efficiency of induction in a decreasing order was coculture group of 1：1 〉 co-culture group of 2：1 〉 coculture group of 4：1 〉 direct induction group adding （VEGF） = direct induction group adding （VEGF＋b-FGF）. CONCLUSION： MSCs could be induced to differentiate into CSMC through adding growth factors and co-culturing with CSMC in vitro, among which the latter could achieve better differentiation rate.