Knockout Apolipoprotein E Gene Of Guangxi Mini Pig To Build Atherosclerosis Models & Hepatic Phenotype Analysis

In our country,especially north cities,cardiovascular diseases have high morbidity and mortality which related to environment and dietary structure.Most cardiovascular diseases such as coronary heart disease and cerebral infarction are caused by atherosclerosis.AS is always related to disorders of lipid metabolism and inflammation,especially to hypercholesterolemia.AS is chronic disease and has many influenced factors,and it can be fatal when it breaks out.In 1973,apolipoprotein E was found as component of very low-density lipoprotein which plays key role in metabolism including lipid transportation,regulation of enzyme activity and combination of cellular surface receptor.ApoE polymorphisms have different ability of binding to lipids and are related to neurodegenerative disease and cardiocascular disease.Besides,different apoE isforms have different susceptibility to these diseases.The relationship between apoE and diseases are simple in animals.ApoE gene knockout mouse and rabbit have elevated cholesterol level,and develope hyperlipidemia and spontaneously atherosclerosis.But these two transgenic animal models are not perfect to study on human AS.The size of mouse is too small to manipulation and surgery,and the major lipoprotein is HDL which protects mouse from AS;the rabbit model is esay to manipulte but is sensitive to dietary,and lack of hepatic enzymes which can lead to liver poision under a long-term high-cholesterol dietary.More importantly,rabbit has differen location of AS plaque compared to human.If we can construct an animal model,which has suitable size and similar genome and metabolic functions to human,will mean so much to study on human AS and its relation to apoE.Swine has similar genome and cardiac structure to human.Besides,swine is susceptible to get human diseases such as hypertension,hyperlipidemia and atherosclerosis.Furthermore,the atherosclerotic plaque on minipigs' vascular has the feature of human mature plaque in vascular.The size of minipig also benefits on manipulation and makes it easy to be produce.In this assay we used the newest genome engineering technology CRIPSR/Cas9,aimed to cut down a large fragment of apoE gene in bama minipig's fetal fibroblast cells.After screening the positive cells,we used somatic cell nuclear transfer technology and embryo transplantation technology to build apoE gene knockout pig models.We analyze the hepatic lipid profile,hepatic protein expression and related gene transcription levels of new born apoE gene knockout pigs.Compare to the wildtypes,we found that 1)the cholesterol and triglyceride contents are higher,free fatty acid,HDL-cholesterol and LDL-cholesterol contents did not change significantly;2)expression of PPAR did not change,expression of SRB? increased,expression of FADS1 and FADS2 decreased;3)expression of IL-4,IL-10 decreased,IL-6 and MCP-1 did not change,TNF-? and IFN-? increased.Based on these,we can see that the hepatics in apoE gene knockout pigs have higher levels of cholesterol and triglyceride and express higher levels of inflammatory factors such as TNF-? and IFN-?,also express lower levels of anti-inflammatory factors such as IL-4 and IL-10.The transcription levels of fatty acid enzyme FADS1 and FADS2,and SRB? which related to anti-atherosclerosis and cholesterol metabolism,are lower in apoE gene knockout pigs.From these,we can deduce the apoE gene knockout pigs enhance the inflammatory response and accumulation of lipids,and inhibit lipid metabolism,which can develop to atherosclerosis and accelerate the process.ApoE gene knockout pigs are ideal models to study on AS and its relation to apoE,also have directive function on new therapy and prevention treatment.