组蛋白去乙酰化酶抑制剂促大鼠胚胎干细胞向神经元分化  

Histone deacetylase inhibitor promotes the neuronal differentiation of ratembryonic stem cells

作　　者：张西[1] 宣爱国[1] 徐丽萍[1] 龙大宏[1] 洪乐鹏[1] 

机构地区：[1]广州医科大学人体解剖教研室,510182

出　　处：《中华生物医学工程杂志》2014年第6期423-427,共5页Chinese Journal of Biomedical Engineering

基　　金：国家自然基金（81371217,30900728）;广东省自然基金（S2013010015464）;广东省优秀青年教师培养计划项目（Yq2013137）;广州市教育局项目（10A156）;广州医科大学博士基金项目（2012C01）

摘　　要：目的探讨组蛋白去乙酰化酶（HDAC）抑制剂丁酸钠（NaB）对大鼠胚胎干细胞（ESC）向神经元分化的影响。方法利用含bFGF、EGF的DMEM／F12培养基培养原代ESC；应用DAPI染色，荧光显微镜观察不同药物浓度NaB（0．2、1、2μmol／L）作用48h后对细胞凋亡的影响；免疫荧光检测NaB（1μmol／L）作用72h后和对照组（PBS）ESC双肾上皮质激素（DCX）和DAPI，计算DCX／DAPI的比值；免疫印迹检测不同浓度NaB（0．2、1、2μmol／L）作用48h后和对照组（PBS）ESC组蛋白H3、H4乙酰化水平。结果在1、2／μmol／LNaB组ESC出现明显凋亡，细胞形状不规则，核固缩，核内可见致密的颗粒荧光，视野下细胞碎片较多，且凋亡细胞百分比明显高于0．2μmol／LNaB组和对照组[（7．85±0．73）％、（18．42±2．04）％比（3．48±0．35）％、（2．16±0．32）％，均P〈0．05]。1μmol／LNaB组ESCDCX／DAPI比值高于对照组（38．51±4．33比14．81±1．77，P〈0．05）。随NaB药物浓度的增加，ESC蛋白H3、H4乙酰化程度较对照组增强，0．2±mol／L组处理后乙酰化组蛋白H3和H4表达变化不明显，1、2μmol／L时组蛋白H3和H4乙酰化程度明显增高（均P〈0．05）。结论HDAC抑制剂NaB可明显促使ESC向神经元分化。Objective To investigate the effect of sodium butyrate （NAB）, a histone deacetylase inhibitor （HDACi）, on the neuronal differentiation of rat embryonic stem cells （ESCs）. Methods Primary ESCs were cultured in the Dulbecco＇ s modified Eagle＇s medium （DMEM）/F12 supplemented with bFGF and EGF. By using DAPI staining, the apoptosis of ESCs was observed at 48 h after treatment with different concentrations （0.2, 1, 2 μmol/L） of NaB. Immunofluorescence labeling was used to measure the levels of doubleeortin （DCX） and DAPI in 1μmol/L NaB-treated ESCs for 72 h and the controls, and thereby the ratio of DCX/DAPI in either group was calculated. Western blotting was used to detect the levels of acetyl- H3 and acetyl-H4 in ESCs treated with 0.2, 1 and 2 μmol/L NaB and in the control group treated with PBS at 48 h. Results There was significant apoptosis in ESCs treated with 1 μmol/L and 2 μmol/L NaB, which presented as irregular shape of cells, karyopyknosis, intra-nuclear dense fluorescent granules, and massive cell debris under microscopy. The rate of apoptosis was higher in ESCs treated with 1 p.mol/L and 2 μmol/L NaB [ （7.85±0.73）% and （18.42±2.04） %, respectively ] than those in ESCs treated with 0.2 μmol/ L NaB [ （3.48±0.35）% ] or in controls [ （2.16±0.32）% ] （all P〈0.05）. Immunofluorescence showed that the ratio of DCX/DAPI was significantly higher in ESCs treated with 1 μmol/L NaB than that in controls （38.51±4.33 vs 14.81±1.77, P〈0.05）. The level of acetyl-H3 and acetyl-H4 did not show obvious change in

关 键 词：组蛋白去乙酰化酶抑制剂 丁酸钠 胚胎干细胞 神经元 

分 类 号：R733.7[医药卫生—肿瘤]