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为探讨甘氨酰谷氨酰胺(Gly Gln)对猪离体骨骼肌卫星细胞增殖和分泌IGF 1的作用,取10日龄仔猪的半腱肌和背最长肌,将分离出的半腱肌卫星细胞和背最长肌卫星细胞分别按2×10 5mL-1密度接种于培养板上,采用浓度为0 6、1 2和2 4mmol/L的Gly Gln处理离体培养的猪半腱肌和背最长肌卫星细胞,观察Gly Gln对离体骨骼肌卫星细胞增殖和分泌IGF 1的影响.结果表明:用上述浓度的Gly Gln处理细胞,对离体培养的猪半腱肌卫星细胞的增殖有显著促进作用(P <0 0 5 ) ,而对离体培养的猪背最长肌卫星细胞的增殖有极显著的促进作用(P <0 0 1) ;同时发现:添加0 6mmol/LGly Gln对猪背最长肌卫星细胞分泌IGF 1有显著的促进作用,提示:不同浓度的Gly Gln对猪离体骨骼肌卫星细胞的增殖均有促进作用,但不同部位的骨骼肌卫星细胞对Gly Gln的反应性不同  相似文献
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【目的】卵泡抑素(Follistatin)能够调节骨骼肌肥大和脂肪沉积,可促进骨骼肌卫星细胞增殖。拟采用体外重组 Follistatin处理增殖期的鸭骨骼肌卫星细胞,阐明TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制。【方法】以孵化14 d的鸭胚为试验材料,采用差速贴壁的方法分离骨骼肌卫星细胞,待细胞长到70%—80%时,将培养基换成含有浓度分别为0、1、10、100 ng·mL-1的Follistatin培养基,继续培养36 h后,采用CCK-8检测骨骼肌卫星细胞增殖情况;使用抗pax7抗体染色,DAPI染核,鉴定骨骼肌卫星细胞;采用real-time qPCR方法检测Follistatin对骨骼肌卫星细胞增殖过程中的标记基因PCNA、生肌因子基因MyoD和TGF-β信号通路中TGF-β、Smad2和Smad3的表达的影响。【结果】在倒置显微镜下观察,传代培养12 h鸭骨骼肌细胞一部分未贴壁呈圆形,一部分贴壁呈梭形。24 h后细胞全部贴壁,细胞略有变长。2 d后细胞继续增多,且呈长梭形。3 d后细胞数目增加,个别细胞融合。4 d后细胞数目进一步增加,细胞变粗,个别细胞融合。5 d后有少量细胞开始分化,细胞进一步融合。Pax7免疫荧光染色分析显示,95%以上的细胞中Pax7呈阳性表达;CCK-8检测细胞增殖分析表明,不同浓度的Follistatin处理鸭骨骼肌卫星细胞后,各处理组细胞增殖均显著高于对照组(P<0.01),且10 ng·mL-1 Follistatin处理鸭骨骼肌卫星细胞增殖效果最明显,为最佳处理浓度;与对照组相比,10 ng·mL-1 Follistatin处理组的MyoD基因表达量显著下降(P<0.05),PCNA基因表达量显著升高(P<0.05),Myf5基因表达量显著升高,TGF-β和Smad2基因表达显著升高(P<0.05),且Smad3基因表达量极限著升高(P<0.01);Western blotting检测蛋白表达水平结果表明,与对照组相比,TGF-β、Smad2 和Smad3磷酸化水平也显著升高。【结论】10 ng·mL-1 Follistatin能显著促进鸭骨骼肌卫星细胞增殖,这一过程可通过TGF-β/Smad信号通路实现。使用最佳Follistatin处理浓度能够显著促进鸭骨骼肌卫星细胞增殖,该研究为鸭骨骼肌生长发育调控机理研究奠定分子基础。  相似文献
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 【目的】探讨体外培养条件下北京油鸡骨骼肌卫星细胞分离、培养及鉴定的方法,建立适于北京油鸡骨骼肌卫星细胞体外扩增的培养体系,为今后进一步研究北京油鸡骨骼肌卫星细胞提供技术平台。【方法】以15日龄的鸡胚胸肌为材料,采用联合酶消化法分离骨骼肌卫星细胞,差速贴壁法纯化细胞,使用Pax7、Desmin、Myod等骨骼肌卫星细胞的特异性标志对所得细胞进行免疫荧光鉴定,随后进行成肌诱导分化,并比较了3种培养体系对骨骼肌卫星细胞增殖的影响。【结果】细胞免疫荧光鉴定结果呈阳性,证实所培养的细胞为北京油鸡骨骼肌卫星细胞;成肌诱导后,细胞相互融合形成多核的肌管,成肌特异性标志MHC表达呈阳性;对不同扩增培养体系比较,结果表明,培养体系DMEM/F12+15%FBS+2.5ng?mL-1bFG最有利于北京油鸡骨骼肌卫星细胞的增殖。【结论】该试验成功地分离并鉴定了北京油鸡骨骼肌卫星细胞,建立了适于北京油鸡骨骼肌卫星细胞体外扩增的培养体系,同时成功地进行了成肌诱导分化,为今后研究北京油鸡骨骼肌生长和发育的机理提供了技术平台。  相似文献
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A synthetic isoflavone (ISO-S) or genistein was added in culture medium at different concentrations (0,10,20,30,40,and 80 pmol L-1) to investigate the effects of soybean isoflavones on antioxidative capacity of porcine skeletal muscle satellite cells.After 48 h incubation,the suspension was cryopreserved for the determination of superoxide dismutase (SOD),catalase (CAT),glutathione peroxidase (GSH-Px) activities,and malondialdehyde (MDA) content.The mRNA levels of SOD,CAT,and GSH-Px gene in cells were detected with Taqman fluorescent probe method.The results showed that the content of MDA and the activities and the mRNA levels of SOD of porcine skeletal muscle satellite cells were influenced by supplemented soybean isoflavone (P<0.05) when adding 10-80 gmol L-1 ISO-S or genistein in the medium.The MDA contents,SOD and CAT activities and their mRNA expression levels of porcine skeletal muscle cells responded quadratically P(<0.05) as the level of ISO-S or genistein increased.Pre-incubation of porcine skeletal muscle satellite cells with ISO-Sor genistein at 10-40 pmol L-t elevated the activities and the mRNA expression levels of SOD and CAT in cells concurrently and decreased the cellular content of MDA (P<0.05).The results indicated that pre-incubation of ISO-S or genistein at 10-40 pmol L-1 could improve the antioxidative capacity of porcine skeletal muscle satellite cells.  相似文献
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采用胶原酶和胰酶联用的酶消化法分离小鼠骨骼肌卫星细胞,应用差速贴壁法进行纯化,并利用RT-PCR和免疫荧光染色方法对分化前后细胞的标志基因进行鉴定。结果显示:分离出的肌卫星细胞生长状态良好,RT-PCR和免疫荧光染色显示肌卫星细胞Pax7和MyoD呈阳性表达,诱导分化形成肌管后,分化标志基因MyoG和MHC呈阳性表达。本研究成功从小鼠肌肉组织中分离出了肌卫星细胞,并具有很好的体外分化能力,可以为肌肉的发育分化和损伤修复研究提供良好的细胞模型。  相似文献
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【Objective】 The aim of this study was to investigate the effects of proteasome subunit beta type-5(PSMB5) on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells. 【Method】 In this study, the in vitro induced myogenic differentiation model of bovine skeletal muscle satellite cells was used to simulate the growth and development of bovine skeletal muscle. Firstly, the expression of PSMB5 in bovine skeletal muscle satellite cells before and after the differentiation was detected. The mRNA expression level of PSMB5 was detected by qRT-PCR, and the protein expression level of PSMB5 was detected by western blotting. Then, the small interfering RNA si-RNA-PSMB5 (si-PSMB5) was designed and synthesized, and PSMB5 overexpression plasmid pcDNA3.1-PSMB5 (pcDNA-PSMB5) was constructed, which were transfected into bovine skeletal muscle satellite cells by lipofectamine 3000, and the transfection effect was detected by qRT-PCR and Western blotting. Finally, EdU staining was used to detect the effects of PSMB5 interference and overexpression on the proliferation of bovine skeletal muscle satellite cells. Further, in vitro myogenic differentiation of bovine skeletal muscle satellite cells was induced, and the myotube formation of bovine skeletal muscle satellite cells was observed under light microscope. The expression of myosin heavy chain (MyHC) protein, a marker of differentiation, was detected by western blotting. The effects of PSMB5 on proliferation and differentiation of bovine skeletal muscle satellite cells were analyzed. 【Result】There was a significant difference in the expression level of PSMB5 before and after the differentiation of bovine skeletal muscle satellite cells. After the differentiation of bovine skeletal muscle satellite cells, the expression level of PSMB5 mRNA and protein was significantly higher than that in the proliferation period (P<0.05). When PSMB5 was interfered or overexpressed, there was no significant difference in EdU positive cell rate between the two groups (P>0.05). After interfering with the expression of PSMB5, the number of myotubes formed by cell differentiation was significantly less than that of the control group, and the protein expression level of MyHC was significantly lower than that of the control group (P<0.05); however, after overexpression of PSMB5, the number of myotubes formed by cell differentiation was significantly higher than that of the control group, and the protein expression level of MyHC was significantly higher than that of the control group (P<0.05). 【Conclusion】The results showed that the PSMB5 had no significant effect on the proliferation of bovine skeletal muscle satellite cells, but it had a significant regulatory effect on the myogenic differentiation process of bovine skeletal muscle satellite cells. Interference with PSMB5 expression could inhibit the myogenic differentiation process, while overexpression of PSMB5 could promote the myogenic differentiation process of bovine skeletal muscle satellite cells in vitro. In this study, the specific regulatory effect of PSMB5 on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells was explored, which laid a foundation for further study on the regulatory mechanism of PSMB5 in bovine muscle myogenic differentiation.  相似文献
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【目的】构建骨骼肌特异表达目的基因卵泡抑制素(follistatin,FST)的真核表达载体pCFCDs-red和 pSPFCDs-red,分别转染到不同细胞系中,检测FST及下游基因在RNA和蛋白水平的表达情况,同时测定骨骼肌特异启动子SP和α-actin的启动效率,以研究FST的特异表达对肌肉发育的影响。【方法】利用脂质体分别将真核表达载体pCFCDs-red和pSPFCDs-red转染到绵羊胎儿成纤维细胞(SFFCs)、骨骼肌卫星细胞(SMSCs)和SMSCs诱导肌管中,获得转基因细胞;对获得细胞的生长状况、细胞周期、细胞形态大小以及目的基因和下游基因的表达情况,分别用流式细胞仪、实时定量PCR和western blotting等方法进行检测分析。【结果】①转基因细胞系的生长趋势与非转基因细胞相似,转基因SMSCs细胞增殖的速度高于转基因SFFCs细胞;②流式细胞仪分析表明,转基因细胞系转染后70%以上细胞均处于G0/G1期,保持旺盛的分裂能力、具有单一峰值、细胞的整倍性好、细胞电子体积显著增大;③实时定量PCR及western blotting检测结果表明,转基因细胞系中FST的RNA及蛋白水平相比非转基因细胞系均上调;④转基因SMSCs和肌管相比转基因SFFCs细胞的FST表达量高,转pSPFCDs-red转基因细胞中FST的表达水平比转pCFCDs-red细胞系高。【结论】骨骼肌特异性启动子SP及α-actin能够在SMSCs及肌管中有效启动FST目的基因的表达,且在肌管中具有较高的表达量,SP启动子的启动效率较高于α-actin。在肌源性细胞中,FST的上调可以引起MSTN蛋白水平的下调,FST对骨骼肌细胞的生长发育具有一定促进作用。  相似文献
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【目的】探索胎牛骨骼肌卫星细胞分离、培养、鉴定及基因转染的方法。【方法】分别采用Ⅰ型胶原酶消化、Ⅰ型胶原酶与胰蛋白酶二步消化及链霉蛋白酶消化法对胎牛骨骼肌卫星细胞进行培养,比较了3种不同分离方法所得细胞数及其存活率的差异;利用差速贴壁法和Percoll密度梯度离心相结合的方法纯化骨骼肌卫星细胞,对纯化后的细胞进行myostatin基因RT-PCR 以及结蛋白(Desmin)免疫细胞化学染色鉴定,最后通过电转染法对纯化的骨骼肌卫星细胞进行EGFP基因转染研究。【结果】3种消化培养方法中,以链霉蛋白酶消化法分离得到的胎牛骨骼肌卫星细胞数显著高于其它2种方法(P<0.05),但细胞存活率较低(P<0.05);而采用Ⅰ型胶原酶与胰蛋白酶二步消化法可以得到相对较高的细胞数及存活率。利用差速贴壁和Percoll密度梯度离心相结合的方法可以得到纯化的骨骼肌卫星细胞;电转染法适用于骨骼肌卫星细胞的基因转染。【结论】建立了胎牛骨骼肌卫星细胞分离、培养、纯化、鉴定及基因转染的方法,为通过转基因方法改良秦川牛产肉性能研究奠定了基础。  相似文献
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