siRNA干扰CAT基因表达对猪皮下前体脂肪细胞分化的影响  

Influence of Interfering CAT Gene by siRNA on Differentiation of Porcine(Sus scrofa) Subcutaneous Preadipocytes

作　　者：单保森 刘鑫[1] 罗武 邵勇钢[3] 韦伟[1] 陈杰[1] 张立凡[1,2,1] SHAN Bao-Sen;LIU Xin;LUO Wu;SHAO Yong-Gang;WEI Wei;CHEN Jie;ZHANG Li-Fan(College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China;National Experimental Teaching Demonstration Center of Animal Science, Nanjing Agricultural University, Nanjing 210095, China;College of Animal Science, XinjiangAgricultural University, Urumqi 830052, China)

机构地区：[1]南京农业大学动物科技学院,南京210095 [2]南京农业大学动物科学类国家级教学示范中心,南京210095 [3]新疆农业大学动物科学学院,乌鲁木齐830052

出　　处：《农业生物技术学报》2019年第4期666-676,共11页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育重大专项课题(No.2018ZX0800928B);江苏省自然科学基金(No.BK20181319);国家科技支撑计划(No.2015BAD03B01)

摘　　要：皮下脂肪沉积作为影响猪(Sus scrofa)生长和肉质的主要因素之一,在养猪生产中一直都是被关注的焦点问题。过氧化氢酶(catalase, CAT)是一种广泛存在于机体氧化代谢过程中的关键抗氧化酶,在动物的脂肪沉积中发挥着重要的作用。为了探究CAT基因在猪皮下前体脂肪细胞分化中的作用,本研究首先通过qRT-PCR检测CAT基因在二花脸猪皮下前体脂肪细胞分化过程中的表达变化,以及在二花脸猪不同组织的表达谱变化;通过小干扰RNA (small interfering RNA, siRNA)抑制内源CAT基因表达,运用油红O染色检测皮下前体脂肪细胞的分化能力;同时qRT-PCR检测沉默CAT基因后,前体脂肪细胞中过氧化物酶体增殖物激活受体γ基因(peroxisome proliferator activated receptorγ, PPARγ)、CCAAT增强子结合蛋白α基因(CCAAT enhancer binding proteinα, C/EBPα)、脂肪酸结合蛋白4基因(fatty acid binding protein4, FABP4)、载脂蛋白E基因(apolipoprotein E, ApoE)、脂联素基因(adiponectin, C1Q and collagen domain containing, ADIPOQ)、围脂滴蛋白1基因(perilipin 1, PLIN1)和激素敏感脂肪酶基因(hormone-sensitive lipase, HSL)等脂肪分化相关基因的表达变化;并进一步检测超氧化物歧化酶2基因(superoxide dismutase2, SOD2)、谷胱甘肽合成酶基因(glutathione synthetase, GSS)、过氧化物酶3基因(peroxiredoxin 3, PRDX3)等抗氧化酶相关基因的表达变化。结果显示,CAT基因主要在猪皮下脂肪、肾脏和肝脏组织中表达,且在猪皮下脂肪组织的表达极显著高于肌内脂肪组织(P<0.01)。在二花脸猪皮下前体脂肪细胞分化过程中,CAT基因的表达均显著升高(P<0.05或P<0.01),其中第4 d表达量最高。降低CAT基因的表达可显著增加细胞中的过氧化氢(H_2O_2)含量(P<0.01)和抑制猪皮下前体脂肪细胞的甘油三酯水平(P<0.05),尽管其对PPARγ、C/EBPα、FABP4、ApoE等基因的表达没有显著影响(P>0.05),但可以显著降低ADIPOQ (P<0.05As one of the major factors for affecting the growth and meat quality of pigs(Sus scrofa),subcutaneous fat deposition has always been a hot issue in pig production. Catalase(CAT) is a key antioxidant enzyme in the oxidative metabolism process and plays vital roles in the fat deposition of animals. To investigate the role of CAT gene in the porcine subcutaneous preadipocytes differentiation, the expression patterns of CAT during the preadipocyte differentiation process and CAT mRNA levels in various tissues of Erhualian pigs were detected using qRT-PCR. Moreover, CAT gene was knockdowned by small interfering RNA(siRNA) fragments, and then, the ability of subcutaneous preadipocytes differentiation was assessed via oil red O staining. The expression levels of important candidates in siCAT treated adipocytes, including the key adipocyte differentiation-related genes such as peroxisome proliferator activated receptor γ gene(PPARγ),CCAAT enhancer binding protein α gene(C/EBPα), fatty acid binding protein 4 gene(FABP4), apolipoprotein E gene(ApoE), adiponectin, C1 Q and collagen domain containing gene(ADIPOQ), perilipin 1 gene(PLIN1),and hormone-sensitive lipase gene(HSL), and the antioxidant-related genes such as superoxide dismutase 2 gene(SOD2), glutathione synthetase gene(GSS), and peroxidase 3 gene(PRDX3) were detected by q RT-PCR.The results indicated that CAT gene was mainly expressed in subcutaneous adipose, kidney, and liver tissues of pigs and its expression levels in subcutaneous adipose tissues were significantly higher than that in intramuscular adipose tissues(P<0.01). During the differentiation process, the expression levels of CAT gene increased significantly at different differentiation stages, with the highest expression levels on the 4 th day of the preadipocytes differentiation. Silencing of CAT increased the contents of hydrogen peroxide(H2 O2)(P<0.01) and also decreased the triglyceride levels of subcutaneous preadipocytes(P<0.05). Furthermore,interfering CAT expression reduced the expression l

关 键 词：过氧化氢酶基因(CAT) 小干扰RNA(siRNA) 二花脸猪 前体脂肪细胞分化 

分 类 号：S828[农业科学—畜牧学]