鸡THRSP基因对LMH细胞脂质沉积的调控作用  

Regulatory Effect of Chicken(Gallus gallus)THRSP Gene on Lipid Deposition in LMH Cells

作　　者：刘世昊 魏志恒 姜新城 闫耀华 徐璐 郁建锋[1] 顾志良[1] LIU Shi-Hao;WEI Zhi-Heng;JIANG Xin-Cheng;YANYao-Hua;XU Lu;YU Jian-Feng;GU Zhi-Liang(School of Biology and Food Engineering,Changshu Institute of Technology,Changshu 215500,China)

机构地区：[1]常熟理工学院生物与食品工程学院,常熟215500

出　　处：《农业生物技术学报》2024年第7期1596-1605,共10页Journal of Agricultural Biotechnology

基　　金：江苏省自然科学基金(BK20191476);常熟理工学院2023大学生创业创新计划资助。

摘　　要：甲状腺激素应答spot 14(thyroid hormone responsive spot 14,THRSP)是一种转录调控因子,参与组织的脂类代谢调控。为探讨THRSP基因在鸡(Gallus gallus)脂质代谢中的作用及调控机制,本研究检测了THRSP基因在鸡12种组织中的表达情况;以鸡脂肪组织总RNA的反转录产物为模板,扩增获得THRSP基因全长CDS序列,并构建其真核表达载体;在鸡肝癌细胞系LMH(Leghorn male hepatoma)中过表达和敲低THRSP基因的表达,通过油红O染色检测脂滴沉积,利用比色法测定总胆固醇(total cholesterol,TC)和总甘油三酯(total triglyceride,TG)的变化,通过qRT-PCR方法检测脂质代谢相关基因的表达变化。结果显示,THRSP基因在鸡的肝脏和脂肪组织中高表达;Western blot检测显示,构建的过表达载体转染LMH细胞,能成功表达融合蛋白THRSP-Myc;THRSP基因过表达促进LMH细胞脂质沉积(P<0.01),TC(P<0.05)和TG(P<0.01)显著升高,过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ,PPARγ)(P<0.01)和二酰甘油酰基转移酶2(diacylglycerol acyltransferase2,DGAT2)(P<0.05)基因表达显著上调,丙酰辅酶A羧化酶亚基α(propionyl-CoA carboxylase subunitα,PCCA)(P<0.05)和磷酸烯醇丙酮酸羧激酶1(phosphoenolpyruvate carboxykinase 1,PCK1)(P<0.001)基因表达显著下调。敲低THRSP基因抑制LMH细胞脂质沉积(P<0.05),TC和TG含量有下降趋势(P=0.06),DGAT2基因表达显著下调(P<0.05),PCCA(P<0.01)和PCK1(P<0.001)基因表达显著上调。上述结果表明,THRSP基因可能通过调节PPARγ、DGAT2、PCCA和PCK1基因表达而发挥对细胞脂质沉积的促进作用。本研究为深入探讨鸡THRSP基因功能提供新的参考。Thyroid hormone responsive spot 14(THRSP)is a transcriptional regulator involved in the regulation of lipid metabolism in tissues.In order to understand the role of THRSP gene in lipid metabolism and its molecular mechanism,this study determined the expression of THRSP gene in 12 kinds of chicken(Gallus gallus)tissues,amplified the full-length CDS sequence of THRSP gene using the reverse transcription product of total RNA from chicken adipose tissue as a template,and constructed the eukaryotic expression vector of THRSP gene.After the THRSP gene was overexpressed and down-regulated in LMH(Leghorn male hepatoma)cells,the effect of lipid drop deposition was detected by oil red O staining,the content changes of total cholesterol(TC)and total triglyceride(TG)were determined using colorimetry method,and the expression changes of lipid metabolism-related genes were determined by qRT-PCR.The results showed that THRSP gene was highly expressed in adipose tissues and liver.Western blot analysis showed that THRSPMyc fusion protein was successfully expressed in LMH.It was found that overexpression of THRSP gene could promote lipid drop deposition in LMH cells(P<0.001),TC(P<0.05)and TG(P<0.01)content significantly increased,the gene expression of peroxisome proliferator activated receptorγ(PPARγ)(P<0.01)and diacylglycerol acyltransferase 2(DGAT2)(P<0.05)were up-regulated,and the gene expression of propionyl-CoA carboxylase subunitα(PCCA)(P<0.05)and phosphoenolpyruvate carboxykinase 1(PCK1)(P<0.001)were down-regulated.When THRSP gene was knocked down in LMH cells,lipid drop deposition were inhibited(P<0.05),TC and TG contents were decreased(P=0.06),DGAT2 gene expression was downregulated(P<0.05),while PCCA(P<0.01)and PCK1(P<0.001)gene expression were up-regulated.The above results indicate that THRSP gene might play a role in promoting cellular lipid deposition by regulating the gene expression of PPARγ,DGAT2,PCCA,and PCK1.This study provides a new reference for further exploring the function of chicken THRSP gene.

关 键 词：鸡 甲状腺激素应答spot 14(THRSP) 脂质代谢 鸡肝癌细胞系LMH(Leghorn male hepatoma) 

分 类 号：S831.2[农业科学—畜牧学]