黄芪多糖活化AMPK减轻游离脂肪酸对C2C12成肌细胞的细胞毒性

目的: 探讨黄芪多糖(APS)减轻游离脂肪酸(free fatty acids, FFAs)对骨骼肌细胞的毒性作用及其机制。方法: 培养C2C12成肌细胞分5组:对照组、APS组、5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核糖苷(5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside, AICAR)组、FFAs组和FFAs+APS组。MTT法检测C2C12细胞存活率;透射电镜观察细胞超微结构;Western blotting检测细胞内腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)和磷酸化乙酰辅酶A羧化酶(p-ACC)表达水平;高效液相色谱法测定细胞胞浆AMP/ATP比值。结果: (1)与FFAs组比较,APS显著提高细胞存活率(P<0.05);(2)透射电镜观察发现,APS作用24 h后可以减轻FFAs导致的线粒体肿胀;(3)Western blotting结果显示:与FFAs组比较,APS作用24 h后显著增加p-AMPK表达(P<0.01)及 p-ACC表达(P<0.05),而不影响总AMPK表达 (P>0.05);(4)AMP/ATP比值检测结果:与FFAs组比较,APS作用24 ｈ后细胞内AMP/ATP比值增加 (P<0.01)。结论: APS可以减轻FFAs对骨骼肌细胞的毒性,其机制可能与保护线粒体、激活AMPK途径有关。     

黄芪多糖通过活化AMPK和促进骨骼肌FAT/CD36转位改善成肌细胞FFAs代谢

 目的：探讨黄芪多糖(Astragalus polysaccharides, APS)对骨骼肌游离脂肪酸（free fatty acids, FFAs）代谢的影响及其机制。方法：培养小鼠C2C12成肌细胞;MTT法检测不同浓度FFAs作用不同时间对细胞活性的影响。根据MTT结果选取FFAs最适浓度和时间处理细胞并用APS干预,采用乙酰辅酶A合成酶-乙酰辅酶A氧化酶法检测APS干预前后培养液FFAs浓度;Western blotting测APS干预前后细胞膜脂肪酸转位酶(FAT/CD36)、总FAT/CD36、磷酸化腺苷酸活化蛋白激酶（phosphorylated AMP-activated protein kinase, p-AMPK）和总AMPK蛋白表达。结果：FFAs对细胞的毒性呈浓度和时间依赖性。与FFAs组比较,FFAs+APS组细胞膜FAT/CD36及p-AMPK蛋白表达增加(P<0.05),而总FAT/CD36及总AMPK蛋白表达无明显差异(P＞0.05),同时培养液FFAs浓度降低,细胞活性增加(P<0.05)。结论：APS可以增加骨骼肌细胞对FFAs的摄取利用,其机制可能与活化AMPK和促进FAT/CD36转位有关。     

小檗碱对LPS诱导神经元损伤的作用机制研究

为探究小檗碱(berberine, Ber)对LPS诱导的神经元损伤及腺苷一磷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase, AMPK)-mTOR-p70核糖体蛋白S6激酶(ribosomaiprotein S6 kinase, S6K)信号通路的影响,MTT法筛选最佳的Ber处理HT22神经元浓度。将HT22细胞分为NC组、LPS组、Ber+LPS组和AMPK抑制剂Compound C(Com.C)+Ber+LPS组。FACS检测HT22细胞凋亡率;qRT-PCR法检测HT22细胞IL-1β、IL-6和TNF-α的mRNA相对表达量;DCFH-DA荧光探针检测HT22细胞中活性氧类(reactive oxygen species, ROS)含量;TBA法、比色法和WST-1法分别检测HT22细胞中丙二醛(malondialdehyde, MDA)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)和总超氧化物歧化酶(superoxide dismutase, SOD)的含量;Weste...     

SH2A 基因对细胞信号转导的影响及其亚细胞定位

目的 研究Src同源域2(src homology 2，SH2)A基因在细胞信号转导中的作用并进行亚细胞定位。方法 通过RT—PCR方法扩增SH2A cDNA编码序列，构建真核重组表达载体pcDNA3．1-SH2A，利用脂质体转染肝癌Bel7402细胞、COS7细胞，检测蛋白酶C(protein kinaseC，PKC)、酪氨酸蛋白激酶(tyrosine protein kinase，TPK)、丝裂原激活蛋白激酶(mitogen activated protein kinase，MAPK)活性的改变；另构建pEGEP—SH2A，转染同前，荧光显微镜观察荧光定位。结果 测序结果显示真核重组表达载体pcDNA3．1-SH2A及pEGFP—SH2A中均含有SH2AcDNA编码序列；肝癌Bel7402细胞、COS7细胞转染pcDNA3．1-SH2A后，胞浆PKC的活性下降了40％左右，MAPK和TPK活性未见明显改变。荧光显微镜观察发现SH2A基因在细胞质中表达。结论 SH2A基因编码蛋白在PKC信号转导通路中起抑制作用；SH2A基因编码蛋白定位于细胞质。     

负荷渐增式训练对老年小鼠骨骼肌卫星细胞AMPK磷酸化的影响

目的 探讨负荷渐增式训练对老年小鼠骨骼肌卫星细胞腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）磷酸化的影响。方法 实验小鼠分为 3 组：青年对照组（YC组，n=12）、老年对照组（OC组，n=12）与老年运动组（OT组，n=12）。OT组进行负荷渐增式训练，流式细胞分选技术分离CD45-/CD31-/Sca1-/VCAM(CD106)+细胞群体，分选细胞通过desmin、Myod肌原性染色以及成肌分化诱导培养进行肌卫星细胞鉴定，免疫组化结合Western blotting方法检测肌卫星细胞p-AMPK水平。结果 YC组骨骼肌卫星细胞AMPK及p-AMPK表达水平显著高于OC组（P<0.05）；OT组与OC组AMPK表达无明显变化（Ｐ>0.05），而OT组p-AMPK表达水平显著高于OC组（P<0.05）。结论 负荷渐增式训练可促进老年小鼠骨骼肌卫星细胞AMPK磷酸化，改善老年小鼠骨骼肌能量代谢。     

AMPK调节骨骼肌细胞GLUT4基因表达的机制研究

腺苷酸活化蛋白激酶(AMPK)能调节运动/肌肉收缩所引起的骨骼肌细胞葡萄糖转运蛋白4(GLUT4)基因的表达,但至今它的调节机制不清.研究显示在非运动刺激引起的细胞信号事件中由组蛋白去乙酰化酶(HDACs)以及组蛋白乙酰化酶(HATs)控制的组蛋白乙酰化状态是调节基因表达的重要机制,所以我们假设AMPK信号途径是通过征用HDACs中的HDAC5(在骨骼肌细胞内高表达)来实现对运动/肌肉收缩引起的GLUT4基因表达控制.细胞分为正常浓度葡萄糖对照组(NGLU组)、正常浓度AICAR组(NGLU AICAR组)、高浓度对照组(HGLU组)、高浓度AICAR组(HGLU AICAR组).用5 mmol/L和20 mmol/L葡萄糖浓度培养骨骼肌细胞后,NGLU AICAR组和HGLU AICAR组与肌肉收缩模拟信号刺激5-氨基-4-甲酰胺咪唑核糖核苷酸(AICAR)孵育.AICAR能激活NGLU组骨骼肌细胞AMPKα2、减少骨骼肌细胞核HDAC5蛋白、促使HDAC5与骨骼肌细胞加强因子(MEF2)蛋白分离和上调GLUT4基因的表达;相反,高浓度葡萄糖延迟由AICAR引起的AMPKα2磷酸化、AMPKα2向细胞核转入、HDAC5向细胞核转出和GLUT4基因的表达.实验结果说明在不同葡萄糖浓度下的骨骼肌细胞GLUT4基因表达变化都对应着上游AMPK蛋白和下游HDAC5蛋白的变化,AMPK可能是征用转录抑制子HDAC5来调节MEF2的活性而达到控制肌肉收缩所引起的GLUT4基因表达.     

二甲双胍对自然衰老小鼠神经元损伤的保护作用机制

目的 探讨二甲双胍对自然衰老小鼠神经元损伤的保护作用及其作用机制.方法 采用4月龄小鼠作为年轻对照组,18月龄小鼠作为老龄鼠,15月龄给予250mg/kg/d二甲双胍灌胃给药3个月作为治疗组,采用苏木精-伊红染色检测脑组织形态学,利用Morris Water Maze方法进行水迷宫实验;利用人神经母细胞瘤细胞SH-SY5Y构建神经元氧化应激损伤模型,细胞实验分4组:对照组、H2O2组、H2O2+二甲双胍组与H2O2+二甲双胍+Compound C组;采用蛋白质免疫印迹法检测自噬及凋亡相关蛋白的表达.结果 18月龄小鼠学习记忆能力与年轻组相比显著下降,自噬水平降低,凋亡增加,经二甲双治疗后有所缓解;神经元细胞在受到刺激损伤后,自噬相关蛋白Beclin1、LC3Ⅱ/Ⅰ减少,凋亡增加;给予二甲双胍治疗后,腺苷单磷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)被激活,自噬水平升高,凋亡相关蛋白Bax/Bcl-2比值降低;给予AMPK抑制剂Compound C后自噬水平没有明显变化.结论 二甲双胍通过激活AMPK引起自噬水平升高,进而保护H2O2诱导的神经元细胞免于凋亡和衰老相关神经退行性病变.     

利拉鲁肽通过microRNA-33对2型糖尿病小鼠肝损伤的治疗作用

目的:观察利拉鲁肽对2型糖尿病小鼠肝组织微小RNA-33(microRNA-33,miR-33)、腺苷酸活化蛋白激酶(AMPK)及肝细胞凋亡通路相关蛋白的影响,探讨其可能作用机制,为临床应用提供药效学依据。方法:采用高脂饮食联合链脲佐菌素(STZ)腹腔注射复制C57BL/6小鼠2型糖尿病模型。小鼠随机分为4组,每组15只:对照组为正常小鼠皮下注射等量的生理盐水;模型组为T2DM小鼠皮下注射等量的生理盐水;利拉鲁肽低剂量组为了2DM小鼠皮下注射100μg·kg~(-1)·d~(-1)利拉鲁肽;利拉鲁肽高剂量组为T2DM小鼠皮下注射200μg·kg~(-1)·d~(-1)利拉鲁肽。给药4周后进行口服葡萄糖耐量试验(OGTT),检测各组小鼠空腹血糖(FBG)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、丙氨酸氨基转移酶(ALT)及天冬氨酸氨基转移酶(AST)的含量;HE染色检测肝组织病理学变化;免疫荧光检测肝组织的cleaved caspase-3;Western blot法检测p-AMPK/AMPK及凋亡相关蛋白Bcl-2、caspase-3的水平;实时定量PCR检测肝组织miR-33的水平。结果:与模型组相比,利拉鲁肽高、低剂量组小鼠FBG、TG、TC、LDL-C、ALT及AST含量明显降低,HDL-C含量明显升高(P0.05);肝组织病理结构的紊乱得到明显改善;免疫荧光结果可见,利拉鲁肽高、低剂量给药组小鼠的cleaved caspase-3明显降低;Western blot实验结果可见,利拉鲁肽高、低剂量给药组小鼠肝组织的Bcl-2表达明显增高,caspase-3表达显著下降(P0.05)肝组织AMPK磷酸化水平显著增加(P0.01);且肝组织miR-33水平显著降低(P0.01),且呈一定的剂量依赖性。结论:利拉鲁肽可以缓解2型糖尿病小鼠肝损伤,其机制可能与降低肝组织miR-33表达,从而增加AMPK磷酸化水平,进而抑制肝细胞凋亡有关。     

氯沙坦通过激活AMPK抑制LPS诱导的小鼠海马GFAP表达

目的:探讨氯沙坦对脂多糖(LPS)诱导的星形胶质细胞活化标志物胶质纤维酸性蛋白(GFAP)表达的影响,以及其机制是否与激活腺苷酸活化蛋白激酶(AMPK)有关。方法:将成年雄性昆明小鼠分为正常对照组、LPS模型组、氯沙坦给药组及氯沙坦与compound C合用组。侧脑室注射相同剂量LPS(24μg/d,每天1次,共2次),以建立中枢神经炎症损伤模型;氯沙坦(0.5、1或5 mg·kg~(-1)·d~(-1))于LPS注射前14 d开始连续每日腹腔注射给药;AMPK抑制剂Compound C(10 mg·kg~(-1)·d~(-1))于LPS注射前2 d开始连续每日腹腔注射给药。在LPS末次注射后的第3天取各组小鼠的海马脑区组织,利用Western blot法检测GFAP、AMPK、p-AMPK、哺乳动物雷帕霉素靶蛋白(mTOR)和p-mTOR的蛋白水平。结果:2次LPS注射后可显著诱导GFAP在海马脑区的表达(P0.01),而氯沙坦可浓度依赖地抑制LPS诱导的GFAP表达,当氯沙坦剂量为5 mg·kg~(-1)·d~(-1)时可显著抑制GFAP表达(P0.05),同时,在该剂量下,氯沙坦显著提高了AMPK的磷酸化水平(P0.01),但对mTOR的磷酸化水平无明显调节作用;而AMPK抑制剂Compound C可显著逆转氯沙坦对GFAP表达及AMPK磷酸化的调节作用(P0.05)。结论:氯沙坦可抑制LPS诱导的海马GFAP表达,该作用可能与其激活AMPK有关,但并不依赖于mTOR信号通路。     

3,4-二羟基苯乙酮通过AMPK途径降低肝细胞及肝脏组织的甘油三酯含量

目的:探讨3,4-二羟基苯乙酮(3,4-dihydroxyacetophenone,3,4-DHAP)调节肝细胞脂质代谢的机制。方法:传代培养人正常肝细胞L02,分为正常对照组、模型组、3,4-DHAP组和辛伐他汀组;药物处理8 h后收集细胞;应用试剂盒检测细胞内甘油三酯(triglyceride,TG)含量; RT-qPCR检测细胞中AMP活化蛋白激酶(AMPactivated protein kinase,AMPK)的mRNA表达水平;采用Western blot法检测细胞AMPK、磷酸化AMPK(phosphorylated AMPK,p-AMPK)、磷酸化固醇调节元件结合蛋白1c(phosphorylated sterol regulatory element binding protein 1c,p-SREBPs-1c)和磷酸化乙酰辅酶A羧化酶(phosphorylated acetyl-CoA carboxylase,p-ACC)的蛋白水平。高脂饲养新西兰大白兔,随机分为正常对照组、模型组、3,4-DHAP组和辛伐他汀组,给药处理12周后,收集肝脏标本,油红O染色;应用试剂盒分析各组TG含量;采用Western blot法检测肝脏组织中AMPK、p-AMPK、p-SREBP-1c和p-ACC的蛋白水平。结果:在细胞水平,3,4-DHAP处理后,与模型组比较,TG含量明显降低,AMPK在mRNA及蛋白水平的表达皆显著增加,AMPK磷酸化明显增加,即活性明显增强; SREBP-1c及ACC磷酸化亦显著增多。在动物实验中,经3,4-DHAP处理后,肝脏组织中的TG含量明显减少,AMPK蛋白表达显著增多、p-AMPK、p-SREBP-1c和pACC蛋白水平明显升高。结论:3,4-DHAP可能通过AMPK途径降低肝细胞及肝脏组织中的TG。     

The changing AMPK expression profile in differentiating mouse skeletal muscle myoblast cells helps confer increasing resistance to apoptosis

AMP-activated protein kinase (AMPK) functions as a alpha/beta/gamma heterotrimer to preserve ATP levels and so cell viability during stressful conditions. However, its role in aiding survival of adult skeletal muscle precursor cells is unclear. Using the differentiating mouse C2C12 postnatal skeletal muscle myoblast cell line, we have determined that proteins for the AMPK subunit isoforms alpha2 and gamma2 are constitutively expressed, while those for alpha1, beta1 and beta2 are undetectable in undifferentiated myoblasts but increasingly expressed with differentiation to myotubes. Although the gamma3 subunit is expressed at a low level in myoblasts, it too is expressed increasingly with differentiation to myotubes. The p50 but not the p72 isoform of the embryonic alpha subunit homologue MELK is expressed only in proliferating myoblasts, while the ARK5 alpha subunit homologue is increasingly expressed with differentiation. Myotubes displayed higher basal and stimulated alpha1/alpha2 AMPK activation than myoblasts. Furthermore, serum starvation resulted in less apoptosis of differentiated myotubes than of undifferentiated myoblasts. This reflects, in part, the increased expression of functional AMPK in the myotubes, since specific inhibition of AMPK activity with 6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-alpha] pyrimidine (Compound C) exacerbated the apoptosis resulting from serum withdrawal. If these in vitro events can also occur in vivo, they could have implications for pathologies such as muscle wasting, in which undifferentiated satellite stem cells may be easier apoptotic targets than their differentiated counterparts. Furthermore, these results suggest that when interpreting results from in vitro or in vivo experiments on AMPK, the subunit expression profile should be taken into account.     

Silibinin improves palmitate-induced insulin resistance in C2C12 myotubes by attenuating IRS-1/PI3K/Akt pathway inhibition

The present study investigated the effect of silibinin, the principal potential anti-inflammatory flavonoid contained in silymarin, a mixture of flavonolignans extracted from Silybum marianum seeds, on palmitate-induced insulin resistance in C2C12 myotubes and its potential molecular mechanisms. Silibinin prevented the decrease of insulin-stimulated 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) uptake and the downregulation of glutamate transporter type 4 (GLUT4) translocation in C2C12 myotubes induced by palmitate. Meanwhile, silibinin suppressed the palmitate-induced decrease of insulin-stimulated Akt Ser473 phosphorylation, which was reversed by wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K). We also found that palmitate downregulated insulin-stimulated Tyr632 phosphorylation of insulin receptor substrate 1 (IRS-1) and up-regulated IRS-1 Ser307 phosphorylation. These effects were rebalanced by silibinin. Considering several serine/threonine kinases reported to phosphorylate IRS-1 at Ser307, treatment with silibinin downregulated the phosphorylation of both c-Jun N-terminal kinase (JNK) and nuclear factor-κB kinase β (IKKβ), which was increased by palmitate in C2C12 myotubes mediating inflammatory status, whereas the phosphorylation of PKC-θ was not significantly modulated by silibinin. Collectively, the results indicated that silibinin prevented inhibition of the IRS-1/PI3K/Akt pathway, thus ameliorating palmitate-induced insulin resistance in C2C12 myotubes.     

Vascular endothelial growth factor B inhibits lipid accumulation in C2C12 myotubes incubated with fatty acids

To investigate (1) the effect of vascular endothelial growth factor B (VEGFB) on lipid accumulation and the alteration of fatty acids and fatty acid-related enzymes in C2C12 myotubes incubated with fatty acids and (2) the regulatory effect of VEGFB on skeletal muscle lipid metabolism. Mouse C2C12 myotubes were incubated with oleic acid (OA) and palmitic acid (PA), and differentiated mature C2C12 myotubes were treated with VEGFB. Oil-red O staining, BODIPY staining and cell triglycerides (TG) content were examined. Total RNA was isolated, and real-time PCR analysis was performed. Treatment with 100?μM OA and 50?μM PA induced lipid droplet accumulation and increased TG content (p?<?.01), and 100?ng/mL VEGFB reduced lipid droplet accumulation and decreased TG content (p?<?.01). Treatment with 100?ng/mL VEGFB significantly induced the mRNA expression of fatty acid transport protein 1 (FATP1) (p?<?.01) and FATP4 (p?<?.01). Treatment with 100?ng/mL VEGFB significantly induced the mRNA expression of adipose TG lipase and hormone-sensitive lipase (p?<?.01) as well as carnitine palmitoyltransferase I (p?<?.01), peroxisome proliferator-activated receptor-γ coactivator-1α (p?<?.01), acyl-coa dehydrogenase very long chain (p?<?.05), acyl-coa synthetase long-chain family member 1 (p?<?.01), peroxisomal acyl-coenzyme A oxidase 1 (p?<?.05), and mitochondrial uncoupling protein 3 (p?<?.01). VEGFB enhanced FATP1and FATP4 expression, promoted C2C12 myotube fatty acid oxidation and TG decomposition, and inhibited C2C12 myotube fatty acid re-esterification, thus inhibiting lipid accumulation in C2C12 myotubes incubated with fatty acids.     

由BMP-4基因转入C2C12细胞引起的成肌细胞到成骨细胞的表型转变

目的:研究BMP-4基因对C2C12细胞分化的影响。方法:BMP-4基因在pCI-neo质粒中构建后转入C2C12细胞。转入pCI-neo-BMP-4重组质粒的细胞、转入pCI-neo质粒的细胞及没有转染的细胞在相同的条件下培养。结果:转入pCI-neo质粒的细胞及没有转染的细胞在增殖期和分化期均不表达BMP-4,在未分化期表现为典型的星形形态,并相互融合成细长而多核的肌小管。而转入pCI-neo-BMP-4重组质粒的细胞在增殖期和分化期均大量表达BMP-4,形态上不形成肌小管,而是形成类似成骨细胞的形态,并高水平表达碱性磷酸酶、在培养基中分泌骨钙素。结论:表明将BMP-4基因转入C2C12细胞可改变其肌源的分化途径,而分化为成骨细胞系,且这种分化转向是基因水平上的。     

Runx2通过抑制细胞巨自噬以诱导C2C12细胞向成骨细胞分化

 目的：探讨细胞巨自噬与Runx2诱导C2C12细胞成骨分化的关系。方法： 在强力霉素(doxycycline, Dox)诱导Runx2表达的细胞系C2C12/Runx2Dox中进行研究。Dox (10 mg/L) 处理0 d、1 d、3 d及6 d后,real-time qPCR检测LC3b、Beclin-1、p62和LAMP-2表达情况,Western blotting分析LC3-I/LC3-II比值。设置不同的3-甲基腺嘌呤(3-methyladenine, 3-MA)或雷帕霉素(rapamycin, Rap)浓度,Dox处理14 d后分析碱性磷酸酶(alkaline phosphatase,ALP)活性。用3-MA (5 mmol/L)或Rap (10 μmol/L)与Dox共同处理1 d、3 d及6 d后检测ALP及骨钙素 (osteocalcin,OC)表达情况。结果： (1) C2C12细胞向成骨分化时,LC3b 与Beclin-1显著下调,p62与LAMP-2无明显变化;(2) LC3-I向LC3-II转换的过程被抑制;(3) 3-MA (5 mmol/L)可增强ALP 活性,而Rap(10 μmol/L)则抑制其活性;(4) 3-MA可上调ALP及OC表达,Rap则下调二者表达。结论： Runx2通过下调LC3和Beclin-1、抑制LC3-I向LC3-II转换的方式阻碍自噬体形成,以诱导C2C12细胞分化为成骨细胞。     

Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells

C2C12 cells offer a useful model to study the differentiation of non-muscle cells to skeletal muscle cells. Myosin phosphorylation and changes in related enzymes, with an emphasis on myosin phosphatase (MP) were analyzed over the first 6 days of C2C12 differentiation. There was a transition from myosin phosphatase target subunit 1 (MYPT1), predominant in the non-muscle cells to increased expression of MYPT2. Levels of MYPT1/2 were estimated, and both isoforms were higher in non- or partially differentiated cells compared to the concentrations in the differentiated isolated myotubes from day 6. A similar profile of expression was estimated for the type 1 protein phosphatase catalytic subunit, delta isoform (PP1c delta). Phosphatase activities, using phosphorylated smooth and skeletal muscle myosins, were estimated for total cell lysates and isolated myotubes. In general, smooth muscle myosin was the preferred substrate. Although the expression of MYPT1/2 and PP1c delta was considerably reduced in isolated myotubes the phosphatase activities were not reduced to corresponding levels. Most of the MP activity was due to PP1c, as indicated by okadaic acid. In spite of relatively high expression of MYPT1/2 and PP1c delta, marked phosphorylation of non-muscle myosin (over 50% of total myosin) was observed at day 2 (onset of expression of muscle-specific proteins) and both mono- and diphosphorylated light chains were observed. Partial inhibition of MLCK by 1-(5-chloronaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) or by a construct designed from the autoinhibitory domain of MLCK, resulted in an increase in small myotubes (3-5 nuclei) after 3 days of differentiation and a decrease in larger myotubes (compared to control). The effect of ML-9 was not due to a reduction in intracellular Ca2+ levels. These results suggest that phosphorylation of non-muscle myosin is important in growth of myotubes, either in the fusion process to form larger myotubes or indirectly, by its role in sarcomere organization.     

敲减钙离子/钙调蛋白依赖性激酶Ⅳ抑制干扰素-γ诱导C2C12细胞免疫分子的表达

目的 探究敲减钙离子/钙调蛋白依赖性激酶Ⅳ（CaMKⅣ）基因对炎性培养条件下C2C12细胞免疫分子表达的影响。 方法 采用慢病毒基因干扰技术敲减C2C12细胞的CaMKⅣ基因,以2%马血清将C2C12细胞分化成肌管,以干扰素-γ（IFN-γ）刺激C2C12细胞,采用Real-time PCR和Western blotting检测CaMKⅣ的表达,采用Western blotting和免疫荧光检测免疫分子主要组织相容性复合体（MHC） class Ⅰ(H-2K^b)、MHC class Ⅱ(H2-Ea)、Toll样受体3（TLR3）的表达,采用Real-time检测肌细胞分泌型促炎性细胞因子白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α、巨噬细胞炎性蛋白（MIP）-1α和单核细胞趋化因子（MCP）-1的基因表达。 结果 IFN-γ可诱导成肌干细胞以及分化肌管表达或上调表达免疫分子MHC-Ⅰ、MHC-Ⅱ、TLR3,同时上调C2C12细胞促炎性细胞因子IL-1β、IL-6、TNF-α、MIP-1α和MCP-1的基因表达量;敲减CaMKⅣ基因导致上述上调趋势被抑制。 结论 敲减CaMKⅣ基因可有效抑制IFN-γ诱导的免疫分子的表达,可能提示CaMKⅣ参与调节肌细胞的免疫学特性。     

香烟提取物通过减少HDAC2表达诱导小鼠C2C12成肌细胞衰老

目的:探讨香烟提取物(CSE)能否引起小鼠C2C12成肌细胞衰老,并研究成肌细胞衰老与组蛋白去乙酰化酶2(HDAC2)的关系。方法:培养C2C12细胞株,分化为成熟成肌细胞,观察CSE干预对成肌细胞衰老和HDAC2表达的影响,采用real-time PCR和Western blot方法分别检测HDAC2 mRNA和蛋白的表达;β-半乳糖苷酶染色检测衰老细胞的百分率。结果:MTT法测定最佳CSE浓度与干预时间分别为60 m L/L和24 h。CSE干预后β-半乳糖苷酶染色阳性细胞数增加,同时伴有HDAC2 mRNA和蛋白表达的减少。四溴苯三唑(TBB)在促进HDAC2 mRNA和蛋白表达的同时,β-半乳糖苷酶染色阳性细胞数减少;用HDAC2的特异性阻滞剂丙戊酸抑制HDAC2 mRNA和蛋白的表达时,β-半乳糖苷酶染色阳性细胞数增加。结论:香烟提取物可通过减少小鼠C2C12成肌细胞HDAC2的表达促进细胞老化。