siRNA干扰<em>CAT</em>基因表达对猪皮下前体脂肪细胞分化的影响

doi:10.3969/j.issn.1674-7968.2019.04.010

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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 receptor γ, PPARγ)、CCAAT增强子结合蛋白α基因(CCAAT enhancer binding protein α, C/EBPα)、脂肪酸结合蛋白4基因(fatty acid binding protein 4, 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 dismutase 2, 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(H₂O₂)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<0.05)、PLIN1基因的表达(P<0.01)和升高HSL基因的表达(P<0.05),而对抗氧化酶相关基因SOD2、GSS、PRDX3的表达则没有显著影响(P>0.05)。上述结果表明,干扰CAT基因可以抑制二花脸猪皮下前体脂肪细胞的分化和脂质沉积,这可能与其能够降低ADIPOQ和PLIN1等成脂基因的表达,同时促进HSL基因表达有关。本研究为进一步揭示CAT基因对猪脂肪生成的作用机制,以及通过氧化调控降低猪的皮下脂肪沉积提供新的科学依据。

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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

Abstract：As 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, C1Q 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 qRT-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 4th day of the preadipocytes differentiation. Silencing of CAT increased the contents of hydrogen peroxide (H₂O₂) (P<0.01) and also decreased the triglyceride levels of subcutaneous preadipocytes (P<0.05). Furthermore, interfering CAT expression reduced the expression levels of ADIPOQ (P<0.05) and PLIN1 (P<0.01) and simultaneously increased the levels of HSL gene (P<0.05), with non-significant effect on the expression levels of PPARγ, C/EBPα, FABP4, ApoE, SOD2, GSS, and PRDX3 genes (P>0.05). Taken together, this study demonstrated CAT gene knockdown could inhibit the differentiation and lipid deposition of subcutaneous preadipocytes of Erhualian pigs, which might result from downregulating lipogenesis-related genes of ADIPOQ and PLIN1 and simultaneously upregulating the expressions of lipolysis-related HSL gene. These results could provide new evidence and ideas for further revealing the mechanism of CAT gene on adipogenesis and reducing the subcutaneous fat deposition of pigs.

Key words： Catalase gene (CAT) Small interfering RNA (siRNA) Erhualian pig Preadipocyte differentiation

收稿日期: 2018-10-30

ZTFLH:

S828

基金资助: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)和国家科技支撑计划(No. 2015BAD03B01)

通讯作者: lifanzhang@njau.edu.cn

引用本文:

单保森, 刘鑫, 罗武, 邵勇钢, 韦伟, 陈杰, 张立凡. siRNA干扰CAT基因表达对猪皮下前体脂肪细胞分化的影响[J]. 农业生物技术学报, 2019, 27(4): 666-676.
SHAN Bao-Sen, LIU Xin, LUO Wu, SHAO Yong-Gang, WEI Wei, CHEN Jie, ZHANG Li-Fan. Influence of Interfering CAT Gene by siRNA on Differentiation of Porcine (Sus scrofa) Subcutaneous Preadipocytes. 农业生物技术学报, 2019, 27(4): 666-676.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.04.010 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I4/666

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