摘要
目的探讨组蛋白乙酰化/去乙酰化在C2C12肌细胞分化过程中的作用。方法选择C2C12肌原细胞为研究对象,按照培养基及干预条件的不同,将其分为对照组、马血清诱导分化组、1mmol/L丙戊酸(VPA)组、2mmol/L VPA组、4mmol/L VPA组及8mmol/L VPA组,每组样品数为6个,其培养基内分别含10%胎牛血清、2%马血清、2%马血清+1mmol/L VPA、2%马血清+2mmol/L VPA、2%马血清+4mmol/L VPA及2%马血清+8mmol/L VPA。比较不同浓度VPA组与马血清诱导分化组肌小管分化率差异。采用实时荧光定量聚合酶链反应(RT-PCR)与Western蛋白质印迹法(Western blotting)检测肌相关蛋白[包括肌球蛋白(Myosin)、肌钙蛋白(Troponin)I-SS、成肌细胞分化(Myod)1蛋白]和组蛋白去乙酰化酶(HDAC,包括HDAC1,2,3)在C2C12细胞分化过程中及使用不同浓度VPA干预C2C12细胞分化过程中的转录及蛋白表达水平,并比较其表达水平差异。结果本研究结果为:1马血清诱导分化组较对照组的肌相关蛋白mRNA和蛋白表达水平均显著升高,且差异有统计学意义(P<0.01);而HDAC mRNA和蛋白表达水平比较,差异均无统计学意义(P>0.05)。2各浓度VPA组肌小管分化率分别较马血清诱导分化组显著下降,且差异有统计学意义(P<0.05)。34mmol/L VPA组及8mmol/L VPA组肌相关蛋白及HDAC mRNA和蛋白表达水平分别较马血清诱导分化组显著下降,且差异有统计学意义(P<0.05);4给予VPA干预后,不同浓度VPA组C2C12细胞的Myosin、Troponin ISS、Myod1、HDAC1,2,3的mRNA及蛋白表达水平均呈浓度依赖性,随着VPA浓度增加,表达水平逐步下降(r=-0.92,-0.89,-0.93,-0.84,-0.93,-0.97;r=-0.99,-0.86,-0.92,-0.95,-0.91,-0.96;均为P<0.05)。结论组蛋白乙酰化/去乙酰化在C2C12肌原细胞成肌分化过程中具有重要意义,HDAC表达抑制可导致肌相关蛋白表达水平下降,进而抑制成肌分化。
Objective To explore the effects of histone acetylation and deacetylation on C2C12 myoblasts differentiation.Methods Based on the differentiation of C2C12 myoblasts into myotubes using high glucose dulbecco′s modified eagle medium（DMEM）containing 2%horse serumin vitro,valproic acid（VPA）was given to C2C12 myoblasts during differentiation with different concentrations,which contained1mmol/L VPA,2mmol/L VPA,4mmol/L VPA and 8mmol/L VPA in the final concentrations with 2% horse serum and high glucose DMEM.So that this experiment was scheduled into 6 groups as control group（contain 10% fetal bovine serum in growth medium）,horse serum induced differentiation group,1mmol/L VPA group,2mmol/L VPA group,4mmol/L VPA group and 8mmol/L VPA group according to different growth medium.There were 6 samples in each group.The myotube differentiation rate were compared in different concentration VPA groups and horse serum induced differentiation group.Besides,mRNA and protein expression levels of muscle-related proteins（including Myosin,Troponin I-SS,myogenic differentiation 1）and histone deacetylases（HDAC,including HDAC1,2,3）were also evaluated with real time polymerase chain reaction（RT-PCR）and Western blotting.The mRNA and protein expression levels of them were compared and analyzed.Results 1 The mRNA and protein expression levels of muscle-related proteins of horse serum induced differentiation group were significantly higher than those of control group,and the differences were statistically significant（P〈0.01）;but compared mRNA and protein expression levels of HDAC had no significant differences between two groups（P〉0.05）.2 The myotube differentiation rate in every concentration VPA group compared with horse serum induced differentiation group were significantly lower,and the differences were statistically significant（P〈0.05）.3 The mRNA and protein expression levels of muscle-related proteins and HDAC in 4mmol/L VPA group or 8mmol/L VPA group compared with horse serum induced different
出处
《中华妇幼临床医学杂志（电子版）》
CAS
2014年第5期36-41,共6页
Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition)
基金
国家自然科学基金(81070136
81270226)
四川省科技支撑计划项目(2011SZ0099
2014SZ0009)
国家教育部长江学者和创新团队发展计划(IRT0935)~~
