OSBPL2基因缺陷型巴马小型猪肥胖相关特征的分析

Obesity-related phenotypic analysis of OSBPL2 gene knockout Bama minature pigs

目的:分析氧化固醇结合样蛋白2(oxysterol binding protein-like 2,OSBPL2)基因缺陷型巴马小型猪肥胖相关的表型特征和可能机制。方法:将3月龄野生型(wild type,WT)和OSBPL2缺陷型(mutant type,MT)巴马小型猪饲喂高脂饲料(high fat diet,HFD)。喂食9个月后检测体重、皮下脂肪厚度,HE染色观察肝脏脂肪变性,电镜观察肝脏组织亚细胞结构,检测血清甘油三酯、总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、丙氨酸氨基转移酶、天门冬氨酸氨基转移酶等生化指标,实时荧光定量PCR(qRT-PCR)法检测肝脏脂肪合成分化相关基因的变化,Western blot及免疫组织化学法检测脂肪酸结合蛋白4(adipocyte fatty acid binding protein 4,FABP4)及酰基辅酶A氧化酶1(acylcoenzyme A oxidase 1,ACOX1)蛋白表达水平。结果:MT-HFD组体重、皮下脂肪厚度明显高于WT-HFD组(P<0.05);HE染色结果显示,MT-HFD组脂肪细胞显著增大(P<0.001);MT-HFD组肝脏脂肪空泡变性明显多于WT-HFD组。qRT-PCR、Western blot及免疫组织化学染色结果发现,MT-HFD组肝组织中FABP4蛋白含量明显增加,ACOX1蛋白明显减少;电镜结果显示MT-HFD组肝脏线粒体相关内质网膜长度变短。结论:OSBPL2缺陷引起巴马小型猪肥胖相关表型特征,可能与抑制脂肪酸β-氧化、影响能量代谢平衡及促进脂肪分化有关。

Objective:This study aims to analyze the obesity-related phenotypic characteristics and possible mechanism of oxysterol binding protein-like 2(OSBPL2)gene knockout in Bama miniature pigs.Methods:Three month-old wild-type(WT)Bama miniature pigs and OSBPL2 mutant type(MT)Bama miniature pigs were fed with high-fat diet(HFD).Body weight and subcutaneous fat thickness were measured after feeding 9 months;liver steatosis was observed by HE staining;liver subcellular structure was observed by electron microscopy;serum was collected to detect total cholesterol,total triglycerid,high density lipoprotein cholesterol,low density lipoprotein cholesterol,alanine aminotransferase and aspartate aminotransferase;the mRNA expression of genes involved fat synthesis and adipose differentiation in liver were measured by real-time quantitative PCR;the protein expression levels and distributions of adipocyte fatty acid binding protein(FABP4)and peroxisomal acylcoenzyme A oxidase 1(ACOX1)were detected by Western blot and immunohistochemistry.Results:The body weight and subcutaneous fat thickness significantly increased in MT-HFD group(P<0.05).HE staining results showed the area of subcutaneous fat cells increased significantly in MT-HFD group(P<0.001),and a large number of fat vacuoles were observed in the liver tissue of MT-HFD group.the qRT-PCR,Western blot and IHC results showed that FABP4 mRNA and protein expression increased,while ACOX1 expression decreased in pigs of MT-HFD group.Electron microscopy analysis results showed the length of mitochondria-associated endoplasmic reticulum membrane decreased in MT-HFD group.Conclusion:OSBPL2 deficiency may cause obesity-related phenotypic characteristics in Bama miniature pigs,which may be associated with the inhibition of fatty acidβ-oxidation,the influence of energy metabolism balance and the promotion of adipose differentiation.