白绒山羊PPARγ基因RNA干扰慢病毒载体的构建及对肌内脂肪细胞增殖分化的影响  

Construction of Lentiviral RNAi Vector of PPARγ Gene in Cashmere Goat and Its Effect on Proliferation and Differentiation of Intramuscular Adipocytes

作　　者：杜琛[1] 李金泉[2] 陈秀娟[1] 

机构地区：[1]内蒙古医科大学附属医院,妇产科生殖医学中心,呼和浩特010050 [2]内蒙古农业大学动物科学学院,动物遗传育种与繁殖自治区重点实验室,呼和浩特010018

出　　处：《畜牧兽医学报》2016年第4期671-678,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30960246);内蒙古自然科学基金(2010Zd11);国家科技支撑项目(2011BAD28B05);国家绒毛用羊现代农业产业技术体系(CARS-40-05)

摘　　要：本研究以绒山羊肌内脂肪细胞为试验材料,通过靶向阻断过氧化物酶体增殖物激活受体PPARγ基因,建立稳定干扰的肌内脂肪细胞株,探讨绒山羊PPARγ基因在肌内脂肪细胞增殖和分化过程中的功能。采用慢病毒质粒包装系统构建特异靶向白绒山羊PPARγ基因的RNA干扰(RNA interference,RNAi)慢病毒载体,用以建立PPARγ基因稳定沉默的肌内脂肪细胞株。利用实时定量和Western blot方法检测PPARγ基因在干扰组和对照组不同时间点的表达情况,并通过MTT和油红O染色法研究绒山羊PPARγ基因对肌内脂肪细胞增殖和分化的影响。经测序证实合成的含PPARγ-shRNA慢病毒载体寡核苷酸链插入正确,对肌内脂肪细胞的感染效率为80%以上,荧光定量和Western blot检测慢病毒介导的shRNA可以有效降低PPARγ的表达,其mRNA和蛋白水平作用的时间相隔24h。沉默PPARγ基因后,脂肪细胞内甘油三脂的浓度明显低于对照组,相反MTT检测干扰组中细胞增殖能力高于对照组。本研究构建了绒山羊shRNA-PPARγ慢病毒干扰载体,成功转染至肌内脂肪细胞后可明显抑制脂肪细胞的分化,而促进肌内脂肪细胞的增殖,为进一步研究PPARγ基因在绒山羊脂肪细胞代谢通路中的作用及脂肪沉积机制奠定基础。The cashmere-goat intramuscular adipocytes were used as experimental materials,and aimed to establish the stable intramuscular adipocyte line for the interference through target interrupting the peroxisome proliferation-activated receptor（PPARγ ）gene,so as to explore the function of cashmere-goat PPARγ gene during the proliferation and differentiation processes of the intramuscular adipocytes.Lentivirural plasmids pack-aging system was adopted to establish a lentiviral vector for RNA interference of specific targeting White Cashmere-goat PPARγ gene,which was used to establish the intramuscular adipocyte line to stabilize and silence the PPARγ gene.Real-time quantitative and Western blot methods were used to detect the expression of PPARγ gene at different points in time in the interference group and control group,as well as the MTT and Oil Red O Staining methods were used to research the influence of cashmere-goat PPARγ gene on the proliferation and differentiation of intramuscular adipocytes.It was confirmed by sequencing that oligonucleotide chains containing PPARγ-shRNA lentivirus vector were inserted properly,its infection efficiency to intramuscular adipocytes was above 80%,lentivirus-mediated shRNA detected by fluorescence quantitative and Western blot could effectively reduce the expression of PPARγ gene,the reaction time interval between mRNA and protein level was 24 h.After silencing of PPARγ gene,the concentration of triglyceride within adipocytes was significantly lower than that in the control group;Contrarily,the cell proliferation ability in MTT test interference group was higher than that in control group.This study established cashmere-goat shRNA-PPARγ lentivirus interference vector,after successfully transfecting into the intramuscular adipocytes,it obviously inhibited the differentiation of adipocytes,and promoted the proliferation of intramuscular adipocytes,which lay foundation for further research the role of PPARγ gene in cashmere-goat metabolic pathways and in the fat deposit

关 键 词：肌内脂肪细胞 RNA干扰 PPARΓ 

分 类 号：S827[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]