卵巢肿瘤去泛素化酶7A基因与鹅肥肝形成关系的研究

doi:10.16431/j.cnki.1671-7236.2022.04.011

摘要/Abstract

摘要： 【目的】研究卵巢肿瘤去泛素化酶7A(OTUD7A)基因与鹅肥肝形成的关系。【方法】选取健康、体重一致的70日龄朗德鹅公鹅40只,随机分为2组,对照组自由采食,试验组进行填饲试验,其中填饲第1~5天每日采食量为500 g,第6~12天每日采食量为800 g,第13~19天每日采食量为1 200 g。在填饲第7、14和19天时,每组随机选取6只鹅屠宰,取肝脏,采用实时荧光定量PCR测定不同填饲阶段肝脏中OTUD7A基因的表达水平。采用Ⅳ型胶原酶消化法分离23胚龄的鹅原代肝细胞,分别用浓度为0(空白组)、125、250 mmol/L的葡萄糖,0(空白组)、50、100、200 nmol/L的胰岛素,0(空白组)、0.125、0.250 mmol/L的油酸、亚油酸及0(空白组)、0.25、0.50 mmol/L棕榈酸处理鹅原代肝细胞,并用实时荧光定量PCR检测这些脂肪肝形成相关因子对OTUD7A基因表达水平的影响。构建过表达OTUD7A基因的载体pcDNA3.1-OTUD7A,并将构建好的过表达重组质粒转染鹅原代肝细胞,通过转录组学测序(RNA-Seq)进行差异表达基因的筛选,并对获得的差异表达基因进行GO功能富集分析。【结果】在填饲第7、14天时,鹅肝脏中的OTUD7A基因表达显著高于对照组(P<0.05)。与空白组相比,250 mmol/L葡萄糖处理以及0.125 mmol/L和0.250 mmol/L棕榈酸处理均显著提高鹅原代肝细胞中OTUD7A基因的表达水平(P<0.05)。转录组学测序结果表明,OTUD7A基因过表达后共筛选到34个差异表达基因,其中有19个基因表达上调、15个基因表达下调。GO功能富集分析发现,差异表达基因注释到对微生物的防御反应、对外界生物刺激的反应、T细胞分化、细胞外外泌体等条目,其中CLMP、PROCR、SH3BP1、ARHGAP28、ACE、OTUD7A、LOC106045877、LOC106048002基因的表达上调,TNFSF8、DDX60、PLAC8、RSAD2、MX1、RSAD2、GBP1基因的表达下调。【结论】鹅肥肝形成过程中OTUD7A基因的表达水平升高,OTUD7A基因可能通过调控TNFSF8、RSAD2、MX1、GBP1、CLMP和PROCR等基因的表达参与鹅肥肝的形成。

关键词: 鹅; 脂肪肝; OTUD7A基因; 葡萄糖; 胰岛素; 脂肪酸

Abstract: 【Objective】 The experiment was aimed to study the relationship between the ovaries tumor deubiquitinase 7A (OTUD7A) gene and the formation of goose fatty liver.【Method】 40 70-day-old male Landes geese with similar body weight were selected and randomly divided into two groups.The geese of control group were free to feed,and the geese of experimental group were overfeeding.The daily intake was 500 g for the 1st to 5th day of filling,800 g for the 6th to 12th day,and 1 200 g for the 13th to 19th day.After overfeeding for 7,14 and 19 days,six geese in each group were randomly selected and slaughtered for liver samples.The expression levels of OTUD7A gene in livers of different overfeeding stages were determined by Real-time quantitative PCR.The goose primary hepatocytes were isolated from Landes goose embryos after 23 days of incubation using type Ⅳ collagenase digestion method.Goose primary hepatocytes were treated with 0 (blank control group),125 and 250 mmol/L glucose,0 (blank control group),50,100 and 200 nmol/L insulin,0 (blank control group),0.125 and 0.250 mmol/L oleic acid,linoleic acid,and 0 (blank control group),0.25 and 0.50 mmol/L palmitic acid,respectively,and the effects of these factors that related to fatty liver formation on the expression level of OTUD7A gene were detected by Real-time quantitative PCR.In addition,the overexpression vector pcDNA3.1-OTUD7A was constructed,and the constructed overexpression recombinant plasmid was transfected into goose primary hepatocytes,and the differentially expressed genes were screened by transcriptomic sequencing (RNA-Seq),and the obtained differentially expressed genes were analyzed by GO function enrichment.【Result】 OTUD7A gene expression in fatty liver was significantly higher than that in control group at days 7 and 14 of overfeeding (P<0.05).Compared to blank control group,OTUD7A gene expression in goose primary hepatocytes was up-regulated by 250 mmol/L of glucose,and 0.125 or 0.250 mmol/L of palmitate (P<0.05).Transcriptomic sequencing results revealed that a total of 34 differentially expressed genes were screened after OTUD7A gene overexpression,of which 19 genes were up-regulated and 15 genes were down-regulated.The GO function enrichment analysis of RNA-Seq showed that the entries were annotated as defense response to other organism,response to external biotic stimulus,T cell differentiation,extracellular exosome and so on.The expressions of CLMP,PROCR,SH3BP1,ARHGAP28,ACE,OTUD7A,LOC106045877 and LOC106048002 genes were up-regulated,and the expressions of TNFSF8,DDX60,PLAC8,RSAD2,MX1,RSAD2 and GBP1 genes were down-regulated.【Conclusion】 The expression level of OTUD7A gene was increased during the formation of goose fatty liver,OTUD7A might participate in the development of goose fatty liver through regulating the expressions of TNFSF8,RSAD2,MX1,GBP1,CLMP and PROCR.

Key words: goose; fatty liver; OTUD7A gene; glucose; insulin; fatty acids

中图分类号:

S835

孙青云, 范翔, 邢娅, 赵敏孟, 刘龙, 耿拓宇, 龚道清. 卵巢肿瘤去泛素化酶7A基因与鹅肥肝形成关系的研究[J]. 中国畜牧兽医, 2022, 49(4): 1295-1303.

SUN Qingyun, FAN Xiang, XING Ya, ZHAO Minmeng, LIU Long, GENG Tuoyu, GONG Daoqing. Study on the Relationship Between OTUD7A Gene and Goose Fatty Liver Formation[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1295-1303.

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