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1.
Decorin, a small leucine‐rich proteoglycan, plays an important role in cellular activities through modification of growth factors. It also acts as a signaling molecule to non‐muscle cells through epidermal growth factor receptor or insulin‐like growth factor I receptor (IGF‐IR). However, it is unclear if decorin acts as a signaling molecule to myogenic cells. In this study, we investigated the effect of decorin on the differentiation of myoblasts and the signaling via IGF‐IR to myogenic cells. C2C12 myoblasts cultured in media containing decorin for 72 h showed more extensive formation of multinucleated myotubes than control cells cultured in the same media without decorin. The protein expressions of myogenin and myosin heavy chian were higher in decorn‐treated cells than in control cells. These results suggest that decorin enhances the differentiation of myoblasts. Western blot analysis and immunocytochemistry showed that IGF‐IR was expressed in myoblasts and myotubes. Furthermore, Akt, which is downstream of IGF‐IR, was more phosphorylated in myoblasts cultured in media containing decorin than those in media without decorin. These results suggest that decorin activates Akt downstream of IGF‐IR and enhances the differentiation of myogenic cells.  相似文献   

2.
【目的】试验旨在探究circRNA-Zfp609调节C2C12成肌细胞增殖和分化的潜在分子机制。【方法】利用RT-PCR和测序分析小鼠骨骼肌组织和C2C12成肌细胞中circRNA-Zfp609的表达,实时荧光定量PCR检测小鼠心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、骨骼肌组织及增殖12、24、36、48 h和分化0、1、3、5 d的C2C12成肌细胞中circRNA-Zfp609的相对表达量;实时荧光定量PCR检测分化0、1、3、5 d的C2C12成肌细胞中肌细胞生成素（MyoG）和肌球蛋白重链（MyHC）的相对表达量。用circRNA-Zfp609的干扰表达载体（siRNA）干扰细胞,通过CCK-8测定siRNA对C2C12成肌细胞增殖率的影响;用实时荧光定量PCR检测siRNA干扰对circRNA-Zfp609、MyoG和MyHC相对表达量的影响。通过TargetScan 7.0和miRDB软件预测circRNA-Zfp609上与肌肉分化相关的miRNA位点,将筛选的miRNAs的过表达载体转染HEK293T细胞,利用双荧光素酶报告试验验证circRNA-Zfp609与miRNA的互作关系。根据miRNAs对circRNA-Zfp609的互作,构建circRNA-Zfp609的野生型和突变型载体并转染HEK293T细胞,利用双荧光素酶报告试验验证circRNA-Zfp609对miRNA的靶向关系。【结果】PCR和测序结果表明,小鼠骨骼肌中可表达circRNA-Zfp609;circRNA-Zfp609在小鼠骨骼肌中表达水平最高,在其他组织中的表达量由高到低依次是肾脏、肺脏、心脏、肝脏、胃、脾脏和小肠。与12 h相比,在C2C12成肌细胞增殖的36和48 h circRNA-Zfp609的相对表达量显著增加（P<0.05）;与分化第0天相比,在C2C12成肌细胞分化的第1、3和5天circRNA-Zfp609的相对表达量均显著增加（P<0.05）,MyoG、MyHC的相对表达量均极显著增加（P<0.01）。与NC组相比,siRNA组C2C12成肌细胞的增殖率和circRNA-Zfp609相对表达量均极显著降低（P<0.01）,MyoG和MyHC的相对表达量显著降低（P<0.05）。circRNA-Zfp609上有miR-150-5p、miR-327、miR-344g-3p和miR-615-5p 4种与肌肉分化相关的miRNAs。circRNA-Zfp609与miR-615-5p的吸附能力最强,具有靶向结合作用。circRNA-Zfp609可以作为分子海绵与调控肌肉分化相关的miR-615-5p相互作用。【结论】circRNA-Zfp609在小鼠的组织中广泛表达,在骨骼肌中表达水平最高;circRNA-Zfp609在C2C12成肌细胞增殖和分化的不同时期差异表达,circRNA-Zfp609上有4个与肌肉分化相关的miRNAs,其中miR-615-5p与circRNA-Zfp609具有靶向关系。本研究结果可为与家畜骨骼肌生长发育相关的研究提供参考。  相似文献   

3.

Background

Fatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells.

Methods

To investigate the role of DGAT2 in regulating lipid accumulation, FA composition and the expression of adipogenic genes, we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2. Cells were then cultured in differentiation medium (DM) without FA, with a mixture of FAs (FA-DM), or containing a 13C stable isotope-labeled FA mixture (IFA-DM). The FA composition of adipocytes was analyzed by gas chromatography–mass spectrometry and gas chromatography-isotope ratio mass spectrometry. Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d.

Results

The triacylglyceride (TAG) content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells. When cultured in DM or FA-DM for 12 d, cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs (C16:1 and C18:1). However, when cells overexpressing DGAT2 were cultured with FA-DM for 30 min, the FA composition was almost identical to that of controls. Further, the proportion of stable isotope-labeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d. These results collectively indicate that the higher proportion of mono-unsaturated FAs, C16:1 and C18:1, may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium. This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA synthesis (ACACA, FASN, SCD1, and A-FABP) were significantly higher in cells overexpressing DGAT2 than in control cells.

Conclusions

In conclusion, our study revealed that TAG accumulation, the proportion of MUFAs, and the expression of adipogenic genes were higher in 3T3-L1 cells overexpressing DGAT2 than in control cells.  相似文献   

4.
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5.
Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin‐like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF‐1 mRNA was similarly expressed in M. longissimus thoracis of double‐muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF‐2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF‐2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM‐myoblasts) as compared with differentiating NM derived myoblasts (NM‐myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF‐2 mRNA expression and decreased myotube formation, but did not effect IGF‐1 mRNA expression. An activin‐like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM‐myoblasts, and restored the attenuated IGF‐2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF‐2 expression via ALK‐Smad signaling.  相似文献   

6.
本研究分析了共轭亚油酸(CLA)对C2C12肌细胞生脂转分化和生肌分化的影响。分别培养并诱导C2C12鼠源肌细胞生脂转分化和正常的生肌分化,同时分别使用终浓度为50μmol/L的c9,t11-CLA和t10,c12-CLA处理细胞,并设对照组,取生脂转分化第10天和生肌分化第8天的细胞用于实时定量PCR检测,观察c9,t11-CLA和t10,c12-CLA对C2C12肌细胞不同分化的影响。结果表明:1)与对照组相比,c9,t11-CLA促进了C2C12肌细胞的生脂转分化,显著增加了细胞内甘油三酯(TG)含量(P0.05),显著上调了细胞内脂肪酸合成酶(FAS)、CCAAT增强子结合蛋白α(C/EBPα)、过氧化物酶体增殖剂激活受体γ(PPARγ)和脂肪酸结合蛋白4(FABP4)基因的表达水平(P0.05);与对照组相比,t10,c12-CLA则抑制了C2C12肌细胞的生脂转分化,显著减少了细胞内TG含量(P0.05),显著下调了细胞内C/EBPα、PPARγ和FA BP4基因的表达水平(P0.05)。免疫印迹杂交结果显示FAS和FABP4的蛋白质表达水平也发生了与基因表达相一致的变化。2)与对照组相比,t10,c12-CLA抑制了C2C12肌细胞的生肌分化,显著减少了细胞内肌管数/细胞数(P0.05),显著下调了细胞内肌细胞生成素(MYOG)和成肌分化抗原(MYOD)基因的表达水平(P0.05);与对照组相比,c9,t11-CLA则显著上调了细胞内MYOG基因的表达水平(P0.05),对C2C12肌细胞的生肌分化有一定程度的促进作用。免疫印迹杂交结果显示MYOG和MYOD的蛋白质表达水平也发生了与基因表达相一致的变化。以上结果表明,CLA对动物骨骼肌细胞的正常生肌分化和生脂转分化都具有重要的调节作用。  相似文献   

7.
In this study, we tested the hypothesis that in ovo feeding (IOF) of L‐arginine (L‐Arg) enhances nitric oxide (NO) production, stimulates the process of myogenesis, and regulates post‐hatching muscle growth. Different doses of L‐Arg were injected into the amnion of chicken embryos at embryonic day (ED) 16. After hatching, the body weight of individual male chickens was recorded weekly for 3 weeks. During in vitro experiments, myoblasts of the pectoralis major (PM) were extracted at ED16 and were incubated in medium containing 0.01 mm L‐Arg, 0.05 mm L‐Arg, and (or) 0.05 mm L‐nitro‐arginine‐methyl‐ester (L‐NAME), an inhibitor of nitric oxide synthase (NOS). When 25 mg/kg L‐Arg/initial egg weight was injected, no difference was observed in body weight at hatch, but a significant decrease was found during the following 3 weeks compared to that of the non‐injected and saline‐injected control, and this also affected the growth of muscle mass. L‐NAME inhibited gene expression of myogenic differentiation antigen (MyoD), myogenin, NOS, and follistatin, decreased the cell viability, and increased myostatin (MSTN) gene expression. 0.05 mm L‐Arg stimulated myogenin gene expression but also depressed muscle cell viability. L‐NAME blocked the effect of 0.05 mm L‐Arg on myogenin mRNA levels when co‐incubated with 0.05 mm L‐Arg. L‐Arg treatments had no significant influence on NOS mRNA gene expression, but had inhibiting effect on follistatin gene expression, while L‐NAME treatments had effects on both. These results suggested that L‐Arg stimulated myoblast differentiation, but the limited number of myoblasts would form less myotubes and then less myofibers, while the latter limited the growth of muscle mass.  相似文献   

8.
The present study describes the isolation, cloning and characterization of adipogenic progenitor cells from rat skeletal muscle. Among the obtained 10 clones, the most highly adipogenic progenitor, 2G11 cells, were further characterized. In addition to their adipogenicity, 2G11 cells retain myogenic potential as revealed by formation of multinucleated myotubes when co‐cultured with myoblasts. 2G11 cells were resistant to an inhibitory effect of basic fibroblast growth factor on adipogenesis, while adipogenesis of widely used preadipogenic cell line, 3T3‐L1 cells, was suppressed almost completely by the same treatment. In vivo transplantation experiments revealed that 2G11 cells are able to possess both adipogenicity and myogenicity in vivo. These results indicate the presence of bipotent progenitor cells in rat skeletal muscle, and suggest that such cells may contribute to ectopic fat formation in skeletal muscle.  相似文献   

9.
A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow‐twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3 days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow‐twitch muscle fibers and mitochondrial function‐related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho‐AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow‐twitch muscle fibers and mitochondrial function‐related gene expression. Finally, we showed that Sirt1 was required for leucine‐induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow‐twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.  相似文献   

10.
In this study, we examined the effects of several egg white proteins (ovalbumin, ovomucoid, ovotransferrin and lysozyme) on proliferation and myotube growth in C2C12 murine myoblast cells. Cell proliferation was measured using a water‐soluble tetrazolium salt (WST‐8)‐based assay and then validated using Giemsa staining. Significant proliferative activities of C2C12 cells were observed in response to the addition of 10?5–10?4 mol/L ovalbumin or ovomucoid. Ovotransferrin decreased C2C12 cell proliferation and lysozyme showed no significant effects on the proliferation of C2C12 cells. In contrast, the proliferative effects of ovalbumin and ovomucoid were not observed in 3T3‐L1 murine preadipocyte cells. We also measured the effects of ovalbumin and ovomucoid on C2C12 myotube diameters by using histological analysis. In comparison to control cells, myotube diameters were significantly increased in cells cultured in 10?6–10?4 mol/L ovalbumin or ovomucoid, suggesting that ovalbumin and ovomucoid stimulate the growth of myotubes. Thus, our results clearly demonstrated that ovalbumin or ovomucoid stimulated the proliferation of myoblasts and growth of myotubes.  相似文献   

11.
旨在研究RNF20及其介导的组蛋白H2B第120位赖氨酸的单泛素化（H2Bub）对小鼠棕色脂肪细胞成脂分化的影响。采集1日龄和2月龄雄性C57BL/6小鼠的棕色脂肪组织（n=3），用Western blot方法检测RNF20的表达及其介导的H2Bub水平。利用胶原酶消化法分离获得1日龄小鼠的棕色前体脂肪细胞。分别诱导棕色前体脂肪细胞和C3H10T1/2细胞系成脂分化，通过油红O染色检测其分化效果，进一步通过Western blot检测细胞分化前后（0和8 d）RNF20的表达及其介导的H2Bub水平。通过siRNA干扰Rnf20基因在C3H10T1/2细胞系中的表达，油红O染色方法观察Rnf20基因对成脂分化的影响，利用qPCR和Western blot技术检测Rnf20基因的干扰效率及其介导的H2Bub水平。结果显示，2月龄小鼠棕色脂肪组织中RNF20表达量及其介导的H2Bub水平均显著高于1日龄小鼠。脂肪细胞分化标记蛋白PPARγ和CEBPα的表达水平，RNF20表达量及其介导的H2Bub水平在棕色前体脂肪细胞及C3H10T1/2细胞成脂分化后均显著增加。此外，在C3H10T1/2细胞中敲降Rnf20基因后，与阴性对照组相比，RNF20及其介导的H2Bub水平显著降低，成脂分化后脂滴明显减少。综上表明，RNF20对小鼠棕色脂肪细胞的分化是必需的，敲降Rnf20基因导致组蛋白H2Bub水平显著降低，且降低了C3H10T1/2细胞的成脂分化效率。本研究丰富了小鼠棕色脂肪细胞分化过程中的表观遗传调控研究，为深入理解动物脂肪细胞分化提供了新的基因素材。  相似文献   

12.
试验旨在揭示ACTC1基因在秦川牛中的时空表达规律，为进一步研究ACTC1基因在肉牛肌肉发育和脂肪沉积等方面的功能奠定基础。利用实时荧光定量PCR技术检测了ACTC1基因在24月龄成年秦川牛13种组织和4日龄新生秦川牛12种组织中的表达规律，同时研究分析了该基因在秦川牛成肌细胞和前体脂肪细胞不同分化阶段（0、2、4、6、8 d）的表达特性。结果显示，ACTC1基因在秦川牛心脏中的表达量最高，骨骼肌中次之；除瘤胃和肾脏外，24月龄成年牛各组织中的表达量显著或极显著高于4日龄新生牛（P<0.05；P<0.01）；ACTC1基因在成肌细胞中的表达随着分化程度的增加呈现先升高后降低的趋势，在成肌细胞分化第2、4、6和8天ACTC1基因的表达量与第0天相比差异极显著（P<0.01），分别是第0天的2.6、4.7、5.6和4.2倍，这与成肌细胞的分化速率一致；在脂肪细胞中ACTC1基因的表达趋势为先降后升，第2、4天的表达量与第0天相比差异极显著（P<0.01），从分化第2天开始表达量随脂肪细胞分化程度增加而增加。ACTC1基因在不同年龄牛肌肉组织（心肌和骨骼肌）中的表达量最高，且其表达特性与肌细胞分化整体趋势一致；另外，ACTC1基因在脂肪细胞中的表达也有一定的规律。综上所述，推测ACTC1基因可能会影响牛肌肉组织的生长发育和脂肪沉积。  相似文献   

13.
The aim of the present study was to define the effect of TGF-beta1 on C2C12 myoblasts myogenesis. TGF-beta1 together with its receptor is a negative auto-paracrine regulator of myogenesis, which influences the proliferation, differentiation, and functions of muscle cells. TGF-beta1 exerts highly significant inhibitory effect on differentiation of C2C12 mouse myoblasts manifested by the impairment of cell fusion and very low expression of myosin heavy chain. The study of differentiating C2C12 mouse myoblasts treated with TGF-beta1 revealed 502 genes (436 down-regulated and 66 up-regulated) with statistically different expression. TGF-beta1-regulated genes were identified to be involved in 29 biological processes, 29 molecular functions groups and 59 pathways. The strongest inhibiting effect of TGF-beta1 was observed in the cadherin and Wnt pathways. The key-genes that could play the role of TGF-beta1 targets during myoblasts differentiation was identified such as: Max, Creb1, Ccna2, Bax, MdfL, Tef, Tubg1, Cxcl5, Rho, Calca and Lgals4.  相似文献   

14.
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15.
脂联素（Adp）是主要由脂肪组织分泌的细胞因子,有重要的生理作用。本试验旨在研究重组脂联素（rAdp）对皖南花猪脂肪细胞脂联素及其受体2,AMP激活蛋白激酶（AMPK）、过氧化物增殖剂活化受体α（PPARα）mR-NA表达的影响。选择10d皖南花猪皮下脂肪组织分离前体脂肪细胞,增殖培养至80%融合后换分化培养基培养,细胞分化后用0、2和10mg/L rAdp分别处理12和48h。油红O染色法鉴定脂肪细胞,MTT方法检测细胞活力;酶法测定培养液中甘油释放量,荧光定量RT-PCR方法检测脂肪细胞脂联素（Adp）、脂联素受体1（AdpR1）、脂联素受体2（AdpR2）、PPARα和AMPK mRNA表达。结果显示,rAdp处理后,脂肪细胞活力总体有降低趋势,10mg/L处理48h达到显著水平（P〈0.05）;rAdp处理对甘油释放的抑制作用未达到差异水平。rAdp处理12h后,脂肪细胞AdpR1和AdpR2mRNA表达显著升高（P〈0.01）,但无剂量依赖性;rAdp处理48h后,脂肪细胞AdpmRNA表达显著下降（P〈0.05）。rAdp处理12h后,脂肪细胞PPARαmRNA表达显著升高（P〈0.01）,且有剂量效应性;而AMP AMPK mRNA表达均无显著性变化。结果提示,重组脂联素处理猪原代脂肪细胞有降低细胞活力和抑制脂肪细胞甘油释放量的趋势,能显著上调AdpR1、AdpR2和PPARα基因的表达,从而刺激脂肪酸氧化和甘油三酯的水解作用。  相似文献   

16.
Marbling, or i.m. fat, is an important factor determining beef quality. Both adipogenesis and hypertrophy of existing adipocytes contribute to enhanced marbling. We hypothesized that the fetal stage is important for the formation of i.m. adipocytes and that AMP-activated protein kinase (AMPK) has a key role in adipogenesis during this stage. The objective of this study was to assess the role of AMPK in adipogenesis in fetal sheep muscle and 3T3-L1 cells. Nonpregnant ewes were randomly assigned to a control (Con, 100% of NRC recommendations, n = 7) or overfed (OF, 150% of NRC, n = 7) diet from 60 d before to 75 d after conception, when the ewes were killed. The fetal LM was collected at necropsy for biochemical analyses. The activity of AMPK was less in the fetal muscle of OF sheep. The expression of peroxisome proliferator-activated receptor (PPAR)gamma, a marker of adipogenesis, was greater in OF fetal muscle compared with Con fetal muscle. To further show the role of AMPK in adipogenesis, we used 3T3-L1 cells. The 3T3-L1 cells were incubated in a standard adipogenic medium for 24 h and 10 d. Activation of AMPK by 5-aminoimidazole-4-car-boxamide-1-beta-d-ribonucleoside dramatically inhibited the expression of PPARgamma and reduced the presence of adipocytes after 10 d of differentiation. Inhibition of AMPK by compound C enhanced the expression of PPARgamma. In conclusion, these data show that AMPK activity is inversely related to adipogenesis in fetal sheep muscle and 3T3-L1 cells.  相似文献   

17.
Weaning is characterized by intestinal inflammation, which is a big challenge in pig industry. Control of intestinal inflammation is important for improvement of growth performance and health. Therefore, the study was focused on the anti‐inflammatory activity of low‐molecular‐weight chitosan oligosaccharide (LCOS) in a porcine small intestinal epithelial cell line (IPEC‐J2). The results showed that TNF‐α, as inflammation inducer, significantly upregulated the mRNA expression of IL‐8 and MCP‐1. Afterwards, LCOS significantly attenuated mRNA expression of IL‐8 and MCP‐1 induced by TNF‐α in the cells. Mannose (MAN), as ligand of mannose receptor, had no effect on the anti‐inflammatory activity of LCOS, which suggested that mannose receptor may not involve in the anti‐inflammatory activity of LCOS in IPEC‐J2 cells. Interestingly, N‐[2‐(p‐bromocinnamylamino)ethyl]‐5‐isoquinolinesulfonamide 2HCl hydrate (H89), as PKA (protein kinase A)‐specific inhibitor, reversed the mRNA expression of IL‐8 when co‐cultured with LCOS. Furthermore, LCOS concentration dependent downregulated the mRNA expression of claudin‐1 compared with TNF‐α treatment. However, the trans‐epithelial electric resistance (TEER) was not affected by LCOS when co‐cultured with TNF‐α in 3 hr. In conclusion, LCOS have a potent anti‐inflammatory activity, and as a feed additives, may be useful for the inhibition of inflammatory process in weaning period of pigs with intestinal inflammation occurring.  相似文献   

18.
Increased susceptibility to intracellular pathogens in HIV-infected individuals and FIV-infected cats is attributed to a defective T-helper 1 (Th1) immune response. However, little is known about specific cytokine responses to secondary pathogens. To address this question, control and FIV-infected cats were challenged with Toxoplasma gondii, and lymph node cells analyzed for cytokine mRNA expression. Twenty-four weeks post-FIV infection, prior to T. gondii challenge, IL2 and IL12 mRNAs were depressed, whereas IL10 and IFNgamma mRNAs were increased in CD4+ and CD8+ subsets. Following T. gondii challenge, control cats showed increased expression of IL2, IFNgamma, IL10, IL12, and IL6 mRNAs. In contrast, IL2, IL6, IFNgamma, and IL12 mRNAs were suppressed in FIV-T. gondii co-infected cats, whereas IL10 remained at the high prechallenge levels. IFNgamma and IL10 mRNAs were produced by both CD4+ and CD8+ cells in FIV-T. gondii cats. Elevated IL10 may suppress a Th1 cytokine response to T. gondii challenge.  相似文献   

19.
Ide基因通过AKT调控成肌细胞增殖和分化的研究   总被引：1，自引：1，他引：0  
旨在了解胰岛素降解酶（insulin-degrading enzyme,Ide）在猪不同组织中的表达情况,及其在C2C12成肌细胞增殖和分化中的作用。本研究利用RT-PCR和Western blotting技术检测了Ide在8月龄雄性小型猪不同组织中的表达;利用siRNA技术干扰Ide表达,检测了Ide在C2C12成肌细胞增殖、凋亡和分化中的作用,试验分为Ide-siRNA处理组和NC-siRNA（negative control）对照组,每组3个重复。结果显示,Ide在猪的不同组织（包括大脑、股四头肌、股二头肌、背最长肌、心、肝、脾、肺、肾和睾丸）中广泛表达,其中,在股四头肌、肾和睾丸中表达量相对更高。Ide-siRNA转染成肌细胞48 h后,Ide表达量极显著下降（P<0.001）。CCK-8检测显示,干扰Ide表达促进了成肌细胞的增殖,细胞周期相关基因表达也显著升高,同时发现干扰Ide表达未引起成肌细胞凋亡。利用2%马血清诱导成肌细胞分化的同时转染Ide-siRNA以干扰其表达,在分化的第2和5天发现,与对照组相比,分化相关基因Myog、Myhc等在干扰组中的表达均显著下降,提示成肌细胞的分化受到了抑制。进一步的研究还发现,在分化的第5天,Akt2和磷酸化的AKT（P-AKT）表达量下降。综上,Ide在猪的不同组织中广泛表达,以肌肉、肾和睾丸中表达量更高。干扰Ide表达促进C2C12成肌细胞的增殖,分化时干扰Ide表达则通过Akt2/P-Akt/Myog途径抑制C2C12成肌细胞的分化。  相似文献   

20.
Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C(2)C(12) myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K/Akt activator, in ASB15+ was able to partially override the previously observed phenotype of delayed differentiation, whereas administration of the PI3K/ Akt inhibitor, LY294002, decreased phosphorylation of Akt and differentiation of all cell lines similar to the untreated ASB15+ myoblasts. These results provide initial evidence that ASB15 has a role in early myoblast differentiation and that its effects may be mediated in part by the PI3K/Akt signal transduction pathway.  相似文献   

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