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

doi:10.11843/j.issn.0366-6964.2016.04.005

Abstract

Abstract:

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 deposition mechanism.

CLC Number:

S827
S813.3

DU Chen，LI Jin-quan，CHEN Xiu-juan. Construction of Lentiviral RNAi Vector of PPARγ Gene in Cashmere Goat and Its Effect on Proliferation and Differentiation of Intramuscular Adipocytes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(4): 671-678.

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References

［1］WOOD J D，ENSER M，FISHER A V，et al.Fat deposition，fatty acid composition and meat quality：A review［J］.Meat Sci，2008，78(4)：343-358.
［2］MUELLER E.Understanding the variegation of fat：Novel regulators of adipocyte differentiation and fat tissue biology［J］.Biochim Biophy Acta，2014，1842(3)：352-357.
［3］CRISAFULLI C，CUZZOCREA S.The role of endogenous and exogenous ligands for the peroxisome proliferator-activated receptor alpha (PPAR-alpha) in the regulation of inflammation in macrophages［J］.Shock，2009，32(1)：62-73.
［4］PYPER S R，VISWAKARMA N，YU S，et al.PPARalpha：energy combustion，hypolipidemia，inflammation and cancer［J］.Nucl Recept Signal，2010，8：e002.
［5］SHUII S，OLSON P，SEARS D D，et al.PPARγ activation in adipocytes is sufficient for systemic insulin sensitization［J］.Proc Natl Acad Sci U S A，2009，106(52)：22504-22509.
［6］SCHUPP M，LAZAR M A.Endogenous ligands for nuclear receptors：digging deeper［J］.J Biol Chem，2010，285(52)：40409-40415.
［7］MEI C L，HE P，CHENG B，et al.Chlamydia pneumoniae induces macrophage-derived foam cell formation via PPARα and PPARγ-dependent pathways［J］.Cell Biol Int，2009，33(3)：301-308.
［8］MITTAL V.Improving the efficiency of RNA interference in mammals［J］. Nat Rev Genet，2004，5(5)：355-365.
［9］LOIS C，HONG E J，PEASE S，et al.Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors［J］.Science，2002，295(5556)：868-872.
［10］杜琛，付绍印，韩志玲，等.绒山羊肌内前体脂肪细胞的基因表达分析［J］.畜牧兽医学报，2013，44(10)：1532-1538.
DU C，FU S Y，HAN Z L，et al.Gene expression analysis on intramuscular preadipocytes in the cashmere goats［J］.Acta Veterinaria et Zootechnica Sinica，2013，44(10)：1532-1538.(in Chinese)
［11］MUELLER S，GAUSSON V，VODOVAR N，et al.RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila［J］.Proc Natl Acad Sci U S A， 2010，107(45)：19390-19395.
［12］BRACKENY D E，SCOTT J C，SAGAWA F，et al.C6/36 aedes albopictus cells have a dysfunctional antiviral RNA interference response［J］.PLoS Negl Trop Dis，2010，4(10)：e856.
［13］LÉGER P，LARA E，JAGLA B，et al.Dicer-2-and Piwi-mediated RNA interference in Rift Valley fever virus-infected mosquito cells［J］.J Virol， 2013，87(3)：1631-1648.
［14］CHARLEY P A，WILUSZ J.Sponging of cellular proteins by viral RNAs［J］.Curr Opin Virol，2014，9：14-18.
［15］KLIEWER S A，MANGELSDORF D J.Fibroblast growth factor 21：from pharmacology to physiology［J］.Am J Clin Nutr，2010，91(1)：254S-257S.
［16］AHMADIAN M，DUCAN R E，SUL H S.The skinny on fat：lipolysis and fatty acid utilization in adipocytes［J］.Trends Endocrinol Metab，2009，20(9)：424-428.
［17］SUGII S，OLSON P，SEARS D D，et al.PPARgamma activation in adipocytes is sufficient for systemic insulin sensitization［J］.Proc Natl Acad Sci U S A，2009，106(52)：22504-22509.
［18］王义生，王小刚，李月白，等.靶向PPARγ基因RNA干扰对激素抑制家兔骨髓基质细胞成骨分化的影响［J］.郑州大学学报(医学版)，2009，44(2)：275-279.
WANG Y S，WANG X G，LI Y B，et al.Effects of RNAi for target on PPARγ to mainta in osteogenic differentiation of MSCs of rabbit suppressed by steroid［J］. Journal of Zhengzhou University (Medical Sciences)，2009，44(2)：275-279.(in Chinese)
［19］TONTONOZ P，HU E，GRACES R A，et al.mPPAR gamma 2：tissue-specific regulator of an adipocyte enhancer［J］.Genes Dev，1994，8(10)：1224-1234.
［20］HEGELE R A，POLLEX R L.Genetic and physiological insights into the metabolic syndrome［J］.Am J Physiol Regul Integr Comp Physiol，2005，289(3)：R663-R669.
［21］王丽，那威，王宇祥，等.鸡 PPARγ 基因的表达特性及其对脂肪细胞增殖分化的影响［J］.遗传，2012，34(5)：454-464.
WANG L，NA W，WANG Y X，et al.Characterization of chicken PPARγ expression and its impact on adipocyte proliferation and differentiation［J］.Hereditas(Beijing)，2012，34(5)：454-464.(in Chinese)

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

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