【作者】 赵雅丽；

【导师】 芒来； 张心壮；

【作者基本信息】 内蒙古农业大学 ， 畜牧（专业学位）， 2020， 硕士

【摘要】 本试验通过体外分离培养马骨骼肌卫星细胞、鉴定以及氧化应激调控机理的研究,建立了马骨骼肌卫星细胞的培养体系以及氧化应激模型,阐明氧化应激的调控机理,为运动马的骨骼肌氧化应激调控提供科学依据和研究基础。本试验主要分为以下三个部分,试验一体外分离培养蒙古马骨骼肌卫星细胞并对获得的细胞进行鉴定。试验二选取可正常增值,生长状况良好的骨骼肌卫星细胞作为材料,利用H2O2来诱导骨骼肌卫星细胞产生氧化损伤,研究不同浓度的H2O2对骨骼肌卫星细胞的损伤情况,建立起骨骼肌卫星细胞的氧化损伤模型,并筛选出H2O2最佳诱导剂量和作用时间。试验三在建立骨骼肌卫星细胞的正常对照模型和氧化损伤模型的基础上,设计与Nrf2-ARE信号通路相关的基因引物,通过基因的相对表达量以及下游调控的抗氧化基因的表达情况,进而确定Nrf2-ARE信号通路调控蒙古马骨骼肌卫星细胞氧化损伤的机理。本研究得出以下结果:1.试验一:马骨骼肌卫星细胞培养及鉴定选取健康的2岁蒙古马,通过利用胶原酶和含0.25%EDTA的胰酶,两步酶消化法获得骨骼肌卫星细胞,将分离得到的细胞经细胞筛过滤去除直径偏大的杂细胞和细胞碎片,并利用差速贴壁法纯化肌卫星细胞,通过免疫荧光,Q-PCR.等方法对肌卫星细胞进行鉴定分析。本试验成功从肌肉组织中分离培养了蒙古马骨骼肌卫星细胞,经过分化培养液诱导可使细胞分化形成肌管,差速贴壁法的结果表明骨骼肌卫星细胞的贴壁时间长于马的成纤维细胞,骨骼肌卫星细胞的特异性表达基因与成纤维细胞相比表达差异显著。免疫荧光结果显示:肌卫星细胞的特异性抗体呈阳性表达,其中Pax7、Myod在细胞核内表达,desmin分布在细胞质中。2.试验二:马骨骼肌卫星细胞氧化应激模型的建立选取可正常增值且生长良好的肌卫星细胞作为材料,采用H2O2诱导肌卫星细胞产生氧化损伤,研究不同浓度的H2O2对肌卫星细胞的影响,并筛选出H2O2最佳诱导剂量和作用时间,以此建立起肌卫星细胞的氧化损伤模型,结果表明:H2O2对于细胞的活力损伤具有时间和剂量依赖性,结合H2O2对肌卫星细胞凋亡率、ROS含量和抗氧化指标的影响,可得出当H2O2作用浓度为600 umol/L,处理时间为6h时,骨骼肌卫星细胞产生了明显的氧化损伤,可作为构建肌卫星细胞氧化损伤模型的最佳条件。3.试验三:Nrf2-ARE通过介导马骨骼肌卫星细胞的氧化应激调控机理在建立骨骼肌卫星细胞的正常对照模型和氧化应激模型的基础上,设计与Nrf2-ARE通路相关的基因引物,通过基因的相对表达量以及下游调控抗氧化酶活,进而确定Nrf2-ARE信号通路调控蒙古马骨骼肌卫星细胞氧化损伤的机理。结果表明:H2O2损伤组的骨骼肌卫星细胞的Nrf2表达量与对照组细胞相比显著降低。Nrf2-ARE信号通路调控的抗氧化酶基因SOD1、SOD2、GPX1、GPX3、TrxR1的表达结果同样也是H2O2处理组低于对照组细胞。这些结果说明H2O2抑制了 Nrf2-ARE信号通路导致细胞产生氧化应激。更多还原

【Abstract】 In this experiment,by isolating and culturing satellite skeletal muscle satellite cells in vitro,identification and oxidative stress regulation mechanism research,a skeletal muscle satellite cell culture system and oxidative stress model were established to elucidate the regulation mechanism of oxidative stress,which is used for sports horses.The regulation of skeletal muscle oxidative stress provides scientific basis and research basis.This test is mainly divided into the following three parts.Test one isolates and cultures Mongolian horse skeletal muscle satellite cells in vitro and identifies the obtained cells.In the second experiment,skeletal muscle satellite cells with normal value-added and good growth conditions were selected as materials.H2O2 was used to induce oxidative damage to skeletal muscle satellite cells.The damage of different concentrations of H2O2 on skeletal muscle satellite cells was studied to establish skeletal muscle satellite cells.Oxidative damage model,and screen out the optimal induction dose and duration of H2O2.Experiment 3 On the basis of establishing a normal control model of skeletal muscle satellite cells and an oxidative damage model,design gene primers related to the Nrf2-ARE signaling pathway,through the relative expression of genes and the expression of downstream regulated antioxidant genes,and then To determine the mechanism of Nrf2-ARE signaling pathway regulating oxidative damage of Mongolian skeletal muscle satellite cells.This study produced the following results:1.Experiment 1 Culture and identification of horse skeletal muscle satellite cellsA healthy 2-year-old Mongolian horse was selected,and skeletal muscle satellite cells were obtained by a two-step enzyme digestion method using collagenase and pancreatin containing 0.25%EDTA,and the separated cells were filtered through a cell sieve to remove large diameter miscellaneous cells and cells The fragments were purified by using the differential adhesion method,and the muscle satellite cells were identified and analyzed by immunofluorescence,Q-PCR and other methods.In this experiment,Mongolian horse skeletal muscle satellite cells were isolated and cultured from muscle tissue.After differentiation culture medium induction,the cells could differentiate into myotubes.The results of differential adhesion method showed that the adhesion time of skeletal muscle satellite cells was longer than that of horses.Fibroblasts and skeletal muscle satellite cells express significantly different genes compared with fibroblasts.Immunofluorescence results showed that the specific antibodies of muscle satellite cells were positively expressed,of which Pax7 and Myod were expressed in the nucleus,and desmin was distributed in the cytoplasm.2.Experiment 2 Establishment of oxidative stress model of horse skeletal muscle satellite cellsChoose muscle satellite cells that can normally increase in value and grow well as materials,use H2O2 to induce muscle satellite cells to produce oxidative damage,study the effect of different concentrations of H2O2 on muscle satellite cells,and screen out the optimal induction dose and action time of H2O2.An oxidative damage model of muscle satellite cells was established.The results showed that H2O2 had a time and dose-dependent effect on cell viability damage.Combined with the effect of H2O2 on muscle satellite cell apoptosis rate,ROS content and antioxidant indicators,it can be concluded that H2O2 When the concentration of action is 600 umol/L and the treatment time is 6h,the skeletal muscle satellite cells have obvious oxidative damage,which can be used as the best condition for the construction of muscle satellite cell oxidative damage model.3.Experiment 3 Nrf2-ARE regulates the mechanism of oxidative stress by mediating skeletal muscle satellite cells of horsesBased on the establishment of a normal control model of skeletal muscle satellite cells and an oxidative stress model,design gene primers related to the Nrf2-ARE pathway,and determine the Nrf2-ARE signal through the relative expression of genes and downstream regulation of antioxidant enzyme activities The pathway regulates the mechanism of oxidative damage of Mongolian skeletal muscle satellite cells.The results showed that the expression of Nrf2 in the skeletal muscle satellite cells of the H2O2 injured group was significantly lower than that of the control group.The expression results of the antioxidant enzyme genes SOD1,SOD2,GPX1,GPX3,and TrxR1 regulated by the Nrf2-ARE signaling pathway were also lower in the H2O2 treatment group than in the control group.These results indicate that H2O2 inhibits the Nrf2-ARE signaling pathway and causes oxidative stress in cells.更多还原