5-氮胞苷体外诱导大鼠骨髓基质干细胞向心肌样细胞的分化

背景:骨髓基质干细胞具有扩增迅速、分化潜能广泛等特点,因此建立骨髓基质干细胞的体外定向诱导模型有利于组织工程学研究.目的:探讨应用5-氮胞苷体外诱导大鼠骨髓基质干细胞向心肌样细胞分化的可能性.设计、时间及地点:细胞学体外观察,于2005-06/2008-06在辽宁医学院附属第一医院中心实验室完成.材料:SD大鼠20只,由辽宁医学院实验动物中心提供.5-氮胞苷为Sigma公司产品.方法:大鼠麻醉后,分离股骨和胫骨,全骨髓法+贴壁法分离培养骨髓基质干细胞,待细胞生长融合至90%后传代扩增.取P3代骨髓基质干细胞,以2×10~4/孔密度接种于24孔培养板,分别加入5,10,15,20 μmol/L 5-氮胞苷诱导培养,同时设立不加诱导剂的空白对照,诱导24 h后更换为正常培养液继续培养至3周.主要观察指标:细胞形态及生长曲线,诱导后细胞形态变化,诱导后细胞连接蛋白43和α-横纹肌肌动蛋白的表达.结果:①培养的骨髓基质干细胞大多呈梭形,有时可见细胞融合,P3代细胞接种后第1,2天为静止生长期,从第3天开始进入对数生长期,第9天达高峰,以后进入平台期,第12天细胞数量开始减少.②5 μmol/L 5-氮胞苷诱导后,骨髓基质干细胞形态无明显变化;10 μmol/L 5-氮胞苷诱导后骨髓基质干细胞变长,宽大,并朝一个方向延伸,具有肌管形成细胞的形态特点;15 μmol/L 5-氮胞苷诱导后,24孔板周边区有少量细胞存活;20 μmol/L 5-氮胞苷诱导后细胞死亡.③经10 μmol/L5-氮胞苷诱导3周后.骨髓基质干细胞胞浆内可见连接蛋白43和α-横纹肌肌动蛋白的表达;空白对照组均呈阴性表达.结论:骨髓基质干细胞自身无法向心肌细胞方向转化,体外经5-氮胞苷诱导后具有向心肌细胞分化的潜能.5-氮胞苷浓度过低不能起到诱导分化作用,浓度过高则会造成细胞大量死亡,10 μmol/L为较适宜的诱导浓度.

Differentiation from rat bone marrow stromal stem cells into cardiomyocytes induced by 5-azacitidine in vitro

BACKGROUND: Bone marrow stromal stem cells (BMSCs) are characterized by rapid amplification and wide differentiation.Thus, to establish in vitro BMSC induction models contributes to the study of tissue engineering.OBJECTIVE: To investigate the possibility of inducing the differentiation of rat BMSCs into cardiomyocytes by 5-azacitidine in vitro.OESING, TIME AND SETTING: The cytological in vitro study was performed at the Central Laboratory of First Affiliated Hospital of Liaoning Medical University from June 2005 to June 2008.MATERIALS: A total of 20 Sprague Dawley rats were supplied by the Experimental Animal Center, Liaoning Medical University.5-azacitidine (Sigma, USA) was used.MHEHODS: Following anesthesia, the rats were used to isolate the femur and tibia. BMSCs were isolated and cultured by the whole bone marrow method + adherent method. When 90% BMSCs were confluence, BMSCs were passaged. BMSCs at the third passage were incubated in a 24-well plate at 2×10~4/well, with 5,10, 15, 20 μmol/L 5-azacitidine. Simultaneously, a blank control group (without inductor) was set. Following 24 hours of induction, BMSCs were incubated in normal medium for 3 weeks.MAIN OUTCOME MEASURES: The following parameters were measured: cell appearance and growth curve, morphological changes following induction, and expression of connexin-43 and a-striated muscle actin.RESULTS: Cultured BMSCs were spindle-shape, with some cell confluence. P3 cells following incubation entered static phase at 1 and 2 days, and entered logarithmic phase at 3 days, reached a peak at 9 days, and then entered platform stage. Cell number became decreased at 12 days. Following induction of 5 μmol/L 5-azacitidine, no significant difference was found in BMSCs.Following induction of 10 μmol/L 5-azacitidine, BMSCs became long and big, extended towards a direction, with the property of myotube formation cells. Following induction of 15 umol/L 5-azacitidine, a few cells survived surrounding the 24-well plate.Following induction of 20 μmol/L 5-azacitidine, cells died. Following induction of 10 μmol/L 5-azacitidine for 3 weeks, expression of connexin-43 and a-striated muscle actin was determined in BMSCs. However, a negative expression was detected in the blank control group.CONCLUSION: BMSCs cannot differentiate into cardiomyocytes by itself. Following in vitro induction, BMSCs can differentiate into cardiomyocytes. Low-dose 5-azacitidine concentration cannot induce the differentiation, but high-dose 5-azacitidine concentration will induce death in a large number of cells. Thus, 10 μmol/L is an optimal concentration.