CAJ | 학술논문

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48 h后对细胞凋亡的影响;免疫荧光检测NaB (1μmol/L)作用72 h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48 h后和对照组(PBS)ESC组蛋白H3、H4乙酰化水平.结果在1、2μmol/L NaB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/L NaB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/L NaB组ESC DCX/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,0.2μmol/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.
목적 탐토조단백거을선화매(HDAC)억제제정산납(NaB)대대서배태간세포(ESC)향신경원분화적영향.방법 이용함bFGF、EGF적DMEM/F12배양기배양원대ESC;응용DAPI염색,형광현미경관찰불동약물농도NaB(0.2、1、2μmol/L)작용48 h후대세포조망적영향;면역형광검측NaB (1μmol/L)작용72 h후화대조조(PBS)ESC쌍신상피질격소(DCX)화DAPI,계산DCX/DAPI적비치;면역인적검측불동농도NaB(0.2、1、2μmol/L)작용48 h후화대조조(PBS)ESC조단백H3、H4을선화수평.결과 재1、2μmol/L NaB조ESC출현명현조망,세포형상불규칙,핵고축,핵내가견치밀적과립형광,시야하세포쇄편교다,차조망세포백분비명현고우0.2μmol/L NaB조화대조조[(7.85 ± 0.73)%、(18.42±2.04)%비(3.48±0.35)%、(2.16±0.32)%,균P<0.05].1μmol/L NaB조ESC DCX/DAPI비치고우대조조(38.51±4.33비14.81±1.77,P<0.05).수NaB약물농도적증가,ESC단백H3、H4을선화정도교대조조증강,0.2μmol/L조처리후을선화조단백H3화H4표체변화불명현,1、2μmol/L시조단백H3화H4을선화정도명현증고(균P<0.05).결론 HDAC억제제NaB가명현촉사ESC향신경원분화.
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(0.2,1,2μmol/L)of NaB.Immunofluorescence labeling was used to measure the levels of doublecortin(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 2μmol/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 1μmol/L and 2μmol/L NaB[(7.85±0.73)%and(18.42±2.04)%,respectively]than those in ESCs treated with 0.2μmol/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 ESCs treated with 0.2 μmol/L,but was significantly elevated in those treated with 1 μmol/L and 2 μmol/L NaB.With the increasing NaB concentration,the levels of acetyl-H3 and acetyl-H4 in NaB-treated groups were higher than that in the control group.Conclusions HDACi obviously promotes the neuronal differentiation of ESCs.