【作者】 钱海燕；

【导师】 李庚山；

【作者基本信息】 武汉大学 ， 内科学， 2004， 硕士

【摘要】 目的 分别使用布法罗大鼠肝细胞（Buffalo rats liver，BRL）条件培养基、鼠胚成纤维细胞（Mouse embryonic fibroblast，MEF）饲养层体外培养胚胎干细胞（embryonic stem cells，ESCs），并使用不同分化诱导物对ESCs分化为心肌细胞施加影响，以筛选理想的高效定向培养及分化方案。 方法 分别用BRL条件培养基和MEF饲养层两种培养方法培养ESCs，以抑制ESCs分化并保持其增殖。当ESCs增殖到足够数量时撤除MEF饲养层和BRL条件培养基，以维甲酸（retinoic acid，RA，2nmol／L）、二甲基亚砜（dimethyl sulfoxide，DMSO，0.6％）、转化生长因子β₁（transforming growth factor-β₁，TGF-β₁，2ng／ml）、激活素-A（activin-A，20ng／ml）为分化诱导剂加入分化培养基中，组合成六种分化培养基，分别为无诱导物组、TGF β₁组、activin-A组、RA+DMSO组、TGFβ₁+DMSO组、TGF β₁+activin-A组，采用悬滴、悬浮、贴壁三步法诱导ESCs分化为心肌细胞。经光镜观察、透射电镜分析及免疫细胞化学染色，以出现自发性收缩活动且心肌细胞特异结构蛋白免疫染色阳性的悬滴计为分化成功。计数各组分化成功的悬滴数，计量资料以x±s表示，组间比较用方差分析及t检验，以P＜0.05为差异有显著性，比较各组分化比率。 结果 （1） BRL条件培养基及MEF饲养层两种培养方法均能促进ESCs增殖并抑制其分化，ESCs呈巢状集落生长。（2） 六组分化诱导条件均能使ESCs分化为心肌细胞，以MEF饲养层法培养和BRL条件培养法培养的ESCs在六组分化诱导物作用下分化比率分别为无诱导物组（23.1±2.58％ vs 30.5±1.29％）、TGF-β₁组（61.2±3.21％ vs 68.5±2.65％）、activin-A组（58.5±2.26％ vs 65.3±1.71％）、RA+DMSO组（62.8±3.97％ vs 70.8±2.71％）、TGF-β₁+DMSO组（71.2±3.96％ vs 76.0±2.94％）、TGFβ₁+activin-A组（81.8±4.06％ vs 92.8±2.22％）。（3） 以BRL条件培养法培养的ESCs在六组分化比率均显著高于MEF饲养层法（P＜0.01）；在六种诱导条件之间比较，以TGF-β₁+activin-A组的分化率最高（P＜0.001）。 结论 以BRL条件培养基培养，TGF-β₁+activin-A组合是一种理想的ESCs体外分化体系。更多还原

【Abstract】 Objective To evaluate the effects of different conditions on inducing mouse embryonic stem cells (ESCs) in vitro to differentiate into cardiomyocytes.Methods BRL (Buffalo rats liver) conditioned medium and MEF (Mouse embryonic fibroblast) feeders were used to promote the growth of ESCs and maintain them in an undifferentiated state respectively. During the inducing process, retinoic acid (2nmol/L), dimethyl sulfoxide (0.6%), transforming growth factor- β1 (2ng/ml) and activin-A (20ng/ml) were used as inducing reagents, and made up six kinds of differentiating medium which are group no -reagents, group TGF β1, group activin-A, group RA+DMSO, group TGF β1+DMSO,and group TGF β1+activin-A. Then a three-step method inducing ESCs cultured in hanging drops, in suspension and in plating was used to induce the differentiation of ESCs. Light microscope, electrical microscope and immunocellularchemistry staining are used to identify the differentiation of ESCs. All data are given as mean values SD. Students t-test was applied as appropriate and a value of P<0.05 was considered significant.Results ESCs which were cultured respectively by BRL conditioned medium and MEF feeders could be induced in vitro to differentiate into cardiomyocytes. The differentiation rate of ESCs cultured in BRL conditioned medium were significantly more than that in MEF feeders (P<0.01). Of all groups, the highest diferentiation rate was obtained in that group TGF- β1 + activin-A in which 92.8% 2.2% of ESCs could be induced to differentiate into cardiomyocytes (P<0.001).Conclusion The system made up of BRL conditioned medium and the inducing reagents including TGF- β1 and activin-A was very valuable in inducing ESCs differentiate into cardiomyocytes.更多还原