Cre-LoxP技术构建肝脏特异性HIF-2α基因敲除小鼠模型
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摘要
目的探讨采用Cre-LoxP技术构建肝脏特异性HIF-2α基因敲除小鼠模型的可行性。方法由美国Jackson实验室引进loxP标记的HIF-2α基因小鼠,采用Cre/loxP重组酶系统和Alb-Cre转基因小鼠,通过多次繁殖杂交,建立肝脏特异性HIF-2α基因敲除小鼠模型。利用PCR技术鉴别小鼠基因型,并用RT-qPCR检测小鼠肝脏、脂肪及肌肉组织HIF-2αmRNA水平,同时检测野生型小鼠和Alb-Cre~+/HIF-2α~(fl/fl)的纯合子小鼠血糖、血脂以及肝功能水平。3组HIF-2αmRNA数据比较采用单因素方差分析和Turkey检验。结果 PCR法成功检测出子代小鼠基因型,包括Alb-Cre~+/HIF-2α~(fl/fl)和Alb-Cre~-/HIF-2α~(fl/fl)纯合子小鼠。肝脏HIF-2α基因敲除鼠肝脏组织HIF-2αmRNA表达量为0.10±0.02,明显低于野生型小鼠的1.00±0.10(HSD=-1.87,P<0.05)。结论采用Cre-LoxP技术可成功构建肝脏特异性HIF-2α基因敲除小鼠。
Objective To investigate the feasibility of establishing a mouse model with liverspecific HIF-2α gene knockout using Cre-LoxP technique. Methods The loxP-labeled mice with HIF-2α gene were introduced from the Jackson Laboratory of the United States. A liver-specific HIF-2αgene knockout mouse model was established by using Cre/loxP recombinase system and Alb-Cre transgenic mice through multiple generations of hybridization. The genotypes of mouse were identified by PCR. The expression levels of HIF-2α mRNA in the liver, fat and muscular tissues were detected by RT-qPCR. The blood glucose, blood lipid and liver function levels in the wild-type mice and Alb-Cre~+/HIF-2α~(fl/fl) homozygous mice were detected. The levels of HIF-2α mRNA among three groups were statistically compared by one-way ANOVA and Turkey test. Results The genotypes of the 2 nd generation mouse were successfully identified,including Alb-Cre~+/HIF-2α~(fl/fl) and Alb-Cre~-/HIF-2α~(fl/fl) homozygous mice. The expression level of HIF-2αmRNA in the liver tissues of HIF-2α gene knockout mice was 0.10±0.02, significantly lower than 1.00±0.10 of the wild-type mice(HSD=-1.87, P<0.05). Conclusion The mouse models with liver-specific HIF-2α gene knockout can be successfully established using Cre-LoxP technique.
Objective To investigate the feasibility of establishing a mouse model with liverspecific HIF-2α gene knockout using Cre-LoxP technique. Methods The loxP-labeled mice with HIF-2α gene were introduced from the Jackson Laboratory of the United States. A liver-specific HIF-2αgene knockout mouse model was established by using Cre/loxP recombinase system and Alb-Cre transgenic mice through multiple generations of hybridization. The genotypes of mouse were identified by PCR. The expression levels of HIF-2α mRNA in the liver, fat and muscular tissues were detected by RT-qPCR. The blood glucose, blood lipid and liver function levels in the wild-type mice and Alb-Cre~+/HIF-2α~(fl/fl) homozygous mice were detected. The levels of HIF-2α mRNA among three groups were statistically compared by one-way ANOVA and Turkey test. Results The genotypes of the 2 nd generation mouse were successfully identified,including Alb-Cre~+/HIF-2α~(fl/fl) and Alb-Cre~-/HIF-2α~(fl/fl) homozygous mice. The expression level of HIF-2αmRNA in the liver tissues of HIF-2α gene knockout mice was 0.10±0.02, significantly lower than 1.00±0.10 of the wild-type mice(HSD=-1.87, P<0.05). Conclusion The mouse models with liver-specific HIF-2α gene knockout can be successfully established using Cre-LoxP technique.
引文
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[18]Ramakrishnan SK,Shah YM.A central role for hypoxia-inducible factor(HIF)-2αin hepatic glucose homeostasis[J].Nutr Healthy Aging,2017,4(3):207-216.
[2]Nishiyama Y,Goda N,Kanai M,et al.HIF-1αinduction suppresses excessive lipid accumulation in alcoholic fatty liver in mice[J].Hepatol,2012,56(2):441-447.
[3]Loboda A,Jozkowicz A,Dulak J.HIF-1 and HIF-2 transcription factors--similar but not identical[J].Mol Cells,2010,29(5):435-442.
[4]De Minicis S,Day C,Svegliati-Baroni G.From NAFLD to NASHand HCC:pathogenetic mechanisms and therapeutic insights[J].Curr Pharmaceut Des,2013,19(29):5239-5249.
[5]Rosmorduc O,Fartoux L.HCC and NASH:how strong is the clinical demonstration[J].Clin Res Hepatol Gastroenterol,2012,36(3):202-208.
[6]Said A,Ghufran A.Epidemic of non-alcoholic fatty liver disease and hepatocellular carcinoma[J].World J Clin Oncol,2017,8(6):429-436.
[7]Alexander J,Torbenson M,Wu TT,et al.Non-alcoholic fatty liver disease contributes to hepatocarcinogenesis in non-alcoholic liver:a clinical and pathological study[J].J Gastroenterol Hepatol,2013,28(5):848-854.
[8]Sanyal A,Poklepovic A,Moyneur E,et al.Population-based risk factors and resource utilization for HCC:US perspective[J].Curr Med Res Opin,2010,26(9):2183-2191.
[9]Younossi ZM,Otgonsuren M,Henry L,et al.Association of nonalcoholic fatty liver disease(NAFLD)with hepatocellular carcinoma(HCC)in the United States from 2004 to 2009[J].Hepatology,2015,62(6):1723-1730.
[10]Singal AG,El-Serag HB.Hepatocellular carcinoma from epidemiology to prevention:translating knowledge into practice[J].Clin Gastroenterol Hepatol,2015,13(12):2140-2151.
[11]Younossi ZM,Koenig AB,Abdelatif D,et al.Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence,incidence,and outcomes[J].Hepatology,2016,64(1):73-84.
[12]Estes C,Razavi H,Loomba R,et al.Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease[J].Hepatology,2018,67(1):123-133.
[13]Turnbull CD.Intermittent hypoxia,cardiovascular disease and obstructive sleep apnoea[J].J Thorac Dis,2018,10(Suppl 1):S33-39.
[14]Liu Y,Ma Z,Zhao C,et al.HIF-1αand HIF-2αare critically involved in hypoxia-induced lipid accumulation in hepatocytes through reducing PGC-1α-mediated fatty acidβ-oxidation[J].Toxicol Lett,2014,226(2):117-123.
[15]Cao R,Zhao X,Li S,et al.Hypoxia induces dysregulation of lipid metabolism in HepG2 cells via activation of HIF-2α[J].Cell Physiol Biochem,2014,34(5):1427-1441.
[16]Baguet JP,Barone-Rochette G,Tamisier R,et al.Mechanisms of cardiac dysfunction in obstructive sleep apnea[J].Nat Rev Cardiol,2012,9(12):679-688.
[17]Yang SL,Wu C,Xiong ZF,et al.Progress on hypoxia-inducible factor-3:its structure,gene regulation and biological function(review)[J].Mol Med Rep,2015,12(2):2411-2416.
[18]Ramakrishnan SK,Shah YM.A central role for hypoxia-inducible factor(HIF)-2αin hepatic glucose homeostasis[J].Nutr Healthy Aging,2017,4(3):207-216.