Establishment Of An Immunodeficient Mouse Model With Hyperlipidemia Syndrome

Background and Purpose:Hyperlipidemia,also known as dyslipidemia,refers to elevated levels of one or more of total cholesterol,triglycerides,and low-density lipoproteins in plasma;clinically,it can be divided into two categories,congenital and acquired,depending on the cause^[1].Most congenital hyperlipidemias are due to familial inherited LDL receptor gene defects and are classified as monogenic hyperlipidemias and sporadic or polygenic hyperlipidemias;the main causative factors of acquired hyperlipidemias are poor lifestyle habits of high-fat and high-calorie diets and some diseases.The pathophysiological process of hyperlipidemia is complex,and the most important consequence of hyperlipidemia is that it leads to lipid deposition in the vascular endothelium,causing atherosclerosis and cardiovascular and cerebrovascular diseases^[2].Hyperlipidemia has caused a significant impact on our national health,and according to statistics,about 90 million people suffer from hyperlipidemia in China.Therefore,it is important to carry out research on the pathological mechanisms of hyperlipidemia for the effective treatment and prevention of hyperlipidemia and related diseases.Patients with hyperlipidemia experience chronic inflammation and abnormal activation of the immune system in the body,which is closely related to the occurrence and development of concomitant diseases such as diabetes mellitus and atherosclerosis^[3].Early reports found high expression of TNF-αin the visceral adipose tissue of obese mice,revealing a link between hyperlipidemia,diabetes and chronic inflammation induced by obesity^[4].Early studies focused on the role of abnormal activation of natural immune cells in disease processes associated with abnormal lipid metabolism^[5].At the molecular level,hyperlipidemia causes cholesterol accumulation in macrophages as well as other immune cells,leading to Toll-like receptor（TLR）and inflammatory vesicle activation;at the cellular level,activation of TLR signaling causes reduced cholesterol efflux,which can lead to more cholesterol accumulation and enhance the body’s inflammatory response^[6].These chronic activations of the natural immune system have been shown to be closely related to the pathomechanism of atherosclerosis and insulin resistance.Recent studies have shown that chronic activation of the adaptive immune system also plays an important role in the pathology of hyperlipidemia and associated co-morbidities^[7].For example,T cells that accumulate in atherosclerotic plaques mostly exhibit the Th1 subtype and can produce inflammatory factors such as IFN-γand TNF-α,while activated T and B lymphocytes are present around necrotic adipocytes and can induce insulin resistance.Animal models are important tools to study the immunopathological mechanisms of hyperlipidemia and to develop relevant new drugs.In recent years,humanized mouse models with human immune system have emerged,so the pathological process of some human immune system-related diseases can be studied directly on the basis of this model,which is widely used in the fields of infection,tumor,transplantation,autoimmune diseases,and regenerative medicine,and has become a bridge between basic research and clinical applications^[8].Researchers have used the inbred LDLR-KO C57BL/6J mouse as the gold standard,however,this mouse has a strong T-cell subpopulation bias and is not fully applicable to the study of the more complex human immune system^[9,10].Moreover,the immune systems of mice and humans are very different,which leads to the fact that research results obtained from common mouse models often fail to explain human physiological or pathological problems.Humanized mouse models of the immune system have provided a new way to study the role of high cholesterol on human immune cell function.After about 30 years of development,there are two main types of immune system humanized mouse models that are most widely used.One is the immune system humanized mouse model established by injecting immunodeficient mouse pups with human-derived cord blood CD34⁺cells;the other immune system humanized mouse model is the tail vein injection of CD34⁺human embryonic hepatocytes transplanted with human embryonic thymus transplanted to immunodeficient mice,and this immune system humanized mouse model is also called BLT mice^[11].The second immune system humanized mouse is considered to be one of the best in vivo models for studying human immune-related diseases^[12,13].Recent studies^[14]have shown that by high-fat feeding of NSG mice and preprotein convertase bacillus subtilisin/kexin type 9-adeno-associated virus 8（AAV8-PCSK）9,AAV8-PCSK9)infection can be used to simulate abnormal human immune function under hyperlipidemic conditions.However,the method is more complex and not easy to be applied in a generalized manner.Therefore,in this study,we chose to perform LDLR gene knockdown in NOD/SCID mice to reduce LDLR expression and increase LDL-C content in mice as a basis for later humanization of the immune system.This method is simple and long-lasting compared to the strategies reported so far.Methods:1.Nod SCID mice were selected as the basis of gene editing.Exons 2-18 of mouse LDL receptor gene were knocked out by microinjection and Crispr/Cas9 technology.Heterozygous offspring mice with LDLR gene defect were identified by PCR.2.Detect the blood lipid and body w eight of offspring wild-type and heterozygous mice.3.The embryo absorption of offspring of heterozygous mice was detected and determined by pathological HE staining.Results:1.Heterozygous mice with LDLR gene defect in offspring were obtained.2.The blood lipid of heterozygous mice was higher than that of wild-type mice.3.The absorption of offspring embryos was found.Conclusions:The LDLR gene of nod SCID mice was knocked out by Crispr/Cas9 technology,and the blood lipid of the heterozygous offspring was higher than that of the wild-type.