Development Of Transgenic Swine Overexpressing Lp-PLA₂

Atherosclerosis is the common pathological foundation of hypertension, stroke,myocardial infarction and other cardiovascular diseases. It often appears in thecarotid, coronary and other arteries as one of the common diseases which is harmfulto human. Inflammation, which runs throughout the whole process of theatherosclerosis lesion, plays an important role in the formation of atherosclerosis.Some marker genes of inflammation are used to predict the risk of cardiovasculardisease, such as C-reactive protein （CRP）, tumor necrosis factor, interleukin and soon. Recently, more and more research shows that Lp-PLA₂plays a role in promotingthe atherosclerosis and is closely related to cardiovascular diseases. It has become anew potential target in the treatment of cardiovascular diseases.As a member of the phospholipase superfamily, Lp-PLA₂, also known as plateletactivating factor hydrolase （PAF-AH）, is a calcium-independent phospholipase in theformation of atherosclerosis, which is produced and secreted by a variety ofinflammatory cells （monocytes, macrophages, T lymphocytes and mast cells）. Inhuman blood circulation,80%of Lp-PLA₂is combined with the ApoB c-terminus ofLDL fragment and the remaining part is combined with HDL, lipoprotein （a） andsome of the VLDL. After LDL is oxidized, Lp-PLA₂acts on the water-solubleportion of phospholipids through the short sn-2ester bond, producing two kinds ofinflammatory mediators: lysolecithin （Lyso-LPC） and the oxidated non-esterifiedfatty acids （oxNE-FAS）, which have the strong pro-inflammatory characteristic. Thestudy shows that in the circulation of mice blood, by the combination of HDL,Lp-PLA₂hydrolyzes PAF, inhibits the formation of atherosclerosis. And Lp-PLA₂reduces the incidence of myocardial infarction and stroke. In the human bloodcirculation, by the combination of LDL, Lp-PLA₂acts on the oxLDL, producing two kinds of pro-inflammatory cytokines. And Lp-PLA₂promotes the occurrence anddevelopment of atherosclerosis. Therefore, mouse disease models of Lp-PLA₂cannot simulate the formation process of the human atherosclerosis.Currently, there is no unified standard of atherosclerosis animal models.However, due to the great similarities between swine and human in the anatomy anda number of physiological and biochemical indicators such as the cardiovascularsystem, digestive system, skin structure, nutritional needs and metabolism etc., theswine’s arterial circulation system is almost a replica of human’s arterial circulationsystem. In terms of cardiovascular diseases, swine excessive stress or excitement canstimulate the formation of atherosclerosis, myocardial infarction and othercardiovascular diseases. Therefore, the swine model is considered to be the bestchoice to simulate human atherosclerosis occurrence and development. Based on theswine Lp-PLA₂mRNA sequence which was published on the NCBI to designprimers. This experiment carried out PCR using the small swine lung cDNA asthe template, ligating the amplified products into the eukaryotic expression vectorpIRES2-EGFP with EF1α promoter, and Lp-PLA₂eukaryotic expression vector wasconstructed successfully. Then, the swine Lp-PLA₂expression vector was transfectedinto miniature swine fetal fibroblasts. Through G418resistance screening andidentification, swine fibroblast cell lines of Lp-PLA₂gene was obtained successfully.Followed by nuclear transfer and embryo transfer, three Lp-PLA₂transgenic swinewere finally born. After preliminary analysis of their phenotypes, there were threepositive Lp-PLA₂transgenic swine. The birth of Lp-PLA₂transgenic swine lays thefoundation for the study of Lp-PLA₂’s regulation mechanism of atherosclerosisoccurrence and development, which is beneficial to provide a new model ofdeveloping atherosclerosis drugs and is expected to achieve new breakthroughs inthe mechanism of atherosclerosis occurrence.