谭秀文;游伟;刘晓牧;靳青;魏晨;万发春;张相伦;

• 1:山东省农业科学院畜牧兽医研究所山东省畜禽疫病防治与繁育重点实验室山东省肉牛生产性能测定中心山东省畜禽健康养殖工程技术中心

摘要(Abstract)：

本研究采用组织块法分离培养获得肉牛肌肉细胞并进行鉴定,利用过氧化氢(H_2O_2)构建氧化应激模型,为深入研究氧化应激对肉牛肌肉生长发育和肉品质的影响机制提供技术支撑。选用体重为252 kg的利鲁(利木赞×鲁西黄牛)杂交公牛背最长肌,利用组织块培养法分离培养肌肉细胞。通过形态学观察、免疫荧光法及流式细胞仪等手段对细胞进行鉴定。采用不同浓度(0、50、100、150、200、250、300、350、400、450μmol/L)的H_2O_2处理细胞24 h,通过观察细胞形态、检测细胞存活率及氧化还原状态,筛选适宜作用浓度。然后利用该浓度H_2O_2处理细胞不同时间(0、0.75、1.5、3、6、12、24 h),通过分析确定适宜作用时间。结果表明:1)分离获得的细胞长势良好,形状细长,呈纺锤体形和梭形。免疫荧光法和流式细胞仪鉴定肌肉细胞的特异性蛋白(生肌决定因子1和配对盒基因7)均得到较高表达。2)视野内细胞数量随H_2O_2浓度升高逐渐减少,至450μmol/L时,细胞发生明显死亡现象。细胞存活率随H_2O_2浓度的升高显著降低,与0浓度H202处理相比,300μmol/L时降至65.4%(P<0.05),450μmol/L时仅为6.7%(P<0.05)。细胞活性氧(ROS)水平先略微降低后逐渐升高(P<0.05),超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性先升高后逐渐降低(P<0.05),丙二醛(MDA)和蛋白质羰基(PC)含量逐渐升高(P<0.05),8-羟基脱氧鸟苷(8-OHdG)含量先升高后降低(P<0.05)。3)细胞存活率随300μmol/L H_2O_2处理时间的延长逐渐降低,与0 h相比,6 h降至61.2%(P<0.05),12 h降至47.4%(P<0.05),但在24 h时略有升高(P<0.05)。随着处理时间的延长,细胞ROS水平先升高后逐渐降低(P<0.05),SOD和CAT活性先升高后降低(P<0.05),MDA、PC和8-OHdG含量均逐渐升高(P<0.05)。综上所述,本研究通过组织块培养法分离培养获得较高纯度的肉牛肌肉细胞,并成功构建氧化应激模型,H_2O_2的适宜作用浓度和时间分别为300μmol/L和6 h。

关键词(KeyWords)： 肉牛;肌肉细胞;细胞培养;细胞鉴定;过氧化氢;氧化应激模型

Abstract:

Keywords:

基金项目(Foundation): 山东省自然科学基金项目（ZR2017BC043）;; 现代农业（肉牛牦牛）产业技术体系建设专项资金（CARS-37）;; 山东省农业科学院农业科技创新工程（CXGC2017B02）;山东省农业科学院畜牧兽医研究所资助项目

作者(Author): 谭秀文;游伟;刘晓牧;靳青;魏晨;万发春;张相伦;

Email:

DOI:

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