摘要
目的探讨先心病婴幼儿骨髓间充质干细胞（bone marrow stromal cells，BMSCs）向心肌样细胞诱导分化的可行性，为心肌组织工程提供新的种子细胞的来源。方法无菌条件下抽取先天性心脏病婴幼儿胸骨骨髓3～5ml，经密度梯度离心（Percoll分离液，T为1．073），接种获得BM～SCs，常规培养。流式细胞仪检测细胞表面抗原。将已传2代的细胞应用5-氮杂胞苷（5-Azacytidine，5Aza）诱导24h，更换为完全培养基（LG-DMEM）后继续培养，2周后行免疫细胞化学及透射电镜行超微结构检查。结果先心病婴幼儿BMSCs基本上为梭型成纤维细胞样形态，表面标记为CD29、CD44、CD71、CD90表达阳性，CD3、CD14、CD34、CD45、HLA-DR不表达。BMSCs经5-Aza诱导2周后α-actin，Desmin，cTnI，Cx-43免疫细胞化学染色阳性，透射电镜示诱导后的细胞有明显的肌丝，单个细胞核呈椭圆形，位于细胞的中央。结论先心病婴幼儿骨髓阃充质干细胞可在体外经5-Aza诱导分化为心肌样细胞，有望成为应用自体细胞体外构建工程化心肌重要的种子细胞。
Objective To explore the feasibility of differentiation of bone marrow stromal ceils （BMSCs） from infants with congenital heart diseases into myocardial like cells. Methods The bone marrow was extracted from the sternum of infants with congenital heart diseases under sterile conditions. BMSCs were isolated by density gradient centrifugation （Percoll, T 1. 073） and expanded by cell culture. The cell surface antigens were detected by the flow cytometer. The third generation BM- SCs were induced by 5-Azacytidine （5-Aza） for 24 hours, and then were cultured in low-glucose Dulbecco Eagle＇s minimum essential medium （LG-DMEM） for 2 weeks. Cell phenotypes were identified by immunohistochemistry staining and transmission electron microscopy （SEM）. Results BMSCs from infants with congenital heart diseases showed fusiform fibroblast-like appearance. And they were positive expression of C1329, CD44, CD71 and CD90 while negative expression of CD3, CD14, CD34, CD45 and HLA-DR. The cellular irrtmunochemistry showed that the induced BMSCs were positive ex pression of α-actin, Desmin, cardiac troponin I （cTn I）, and connexin-43 （Cx-43）. SEM showed that the induced cell had transparent myofilaments and an oval-shape nucleus located in the center of the cell. Conclusions BMSCs from infants with congenital heart diseases could be induced into myocardial like ceils by 5-Aza in vitro. BMSCs might become the autologous seed cells for the engineered myocardial tissue.
出处
《中华小儿外科杂志》
CSCD
北大核心
2006年第2期65-68,共4页
Chinese Journal of Pediatric Surgery
关键词
心脏病
先天性
骨髓间充质千细胞
细胞分化
Heart diseases
congenltal
Bone marrow stromal cells
Cell differentiation
