| The aortic vascular endothelium is easily damaged because of faster blood flow, bigger blood pressure and other factors. Metabolites of cholesterol are easily adhere to the injury vascular intima, leading to lipid deposition and blocking formation, developing to atherosclerosis (AS). Arterial lesions are mainly caused by abnormal cholesterol metabolism. ATP-binding cassette protein A1 (ABCAl), a speed limiting protein of reverse cholesterol transport (RCT), is very important for cholesterol metabolism. The relationship and regulated function between ABCAl and arterial lesions remain unclear, many microRNAs have been discovered participate in the regulation process of RCT and atherosclerosis.Guangxi Bama mini-pigs were chosen as experimental animals in this study. Four pigs which fed with normal diet as the control group, Nine pigs which fed with high fat and high sugar diet as the experimental group 1 (Ex-1), the experimental group 2 (Ex-2) has 5 pigs which fed with high fat, high sugar and high cholesterol diet. The feeding experiment continued for three months. The blood physiological and biochemical indexes of the pigs were detected every month and the arteries pathological changes were inspected at the experiment end. The ABCAl gene coding sequences (CDs) of Guangxi Bama mini-pig was cloned and analyzed with the biological information. ABCAl gene mRNA and protein of the aorta, carotid artery and aortaventralis were detected in the experiment pigs, the expression of miRNA-33a and miRNA-758 which targeting to ABCAl gene in pigs liver also were detected. An eukaryotic expression vector was constructed to verify the function of ABCAl gene. The experiments aimed to discuss the changes between ABCAl and cholesterol metabolism on the process of atherosclerosis and the microRNAs acted on ABCAl, as well as laid a foundation for producing transgenic Guangxi Bama mini-pigs. Here are the results:(1) The serum lipid level of pigs in Ex-2 tends to rise, while it has no significantly change in ex-1 and control group.(2) The results of HE staining indicated that the vascular endothelium of the aorta, carotid artery and aortaventralis were injured in Ex-1 and Ex-2. The three arteries in ex-1 showed intima thickening and smooth muscle cells disordering. In ex-2, the arteries showed more serious lesions of unsmooth intimas and macrophages and foam cells on thickening medial membrane.(3) The ABCAl gene CDs of Guangxi Bama mini-pigs was 2856 bp, encoded 961 amino acids, the protein without a signal peptide, the ATP transporter center located in the 602nd to 834th amino acid.(4) The results of qRT-PCR indicated that the level of ABCAl gene mRNA of aorta and carotid artery in Ex-2 were significantly higher than those of Ex-1 and control group (P<0.05) and it was significantly higher in aortaventralis of Ex-2 and Ex-1 than control group (P<0.05). The immunohistochemical result indicated that the level of ABCAl protein of the aorta was Ex-2>Ex-1>control group with no significant difference and those in carotid artery of Ex-1 and Ex-2 were significantly higher than control group (P<0.05) while in aortaventralis of Ex-2 were significantly higher than Ex-1 (P<0.05) and those of Ex-1 were significantly higher than control group (P<0.05). There were more ABCAl protein expression in the lesion areas of arterial, and the expression level tended to increase with rising of the lesion severity.(5) The results of qRT-PCR showed that the expression level of ABCAl gene in liver of Ex-2 was significantly higher than those of Ex-1 and control group (P<0.05). The expression levels of miR-33a in liver of Ex-1 and Ex-2 was significantly lower than that of control group (P<0.05). The miR-758 expression level in liver of Ex-1 was significantly higher than that of control group (P<0.05), but the miR-758 expression level in liver of Ex-2 was significantly lower than that of control group (P<0.05).(6) The green fluorescence could be detected in the PK-15 cells after transfected with pEGFP-Nl-ABCAl vectors, and the expression of ABCAl mRNA was significantly increased. The green fluorescence was observed in embryos of pigs at the oocytes cleavage and blastocyst stage after cytoplasm injection pEGFP-Nl-ABCAl vectors. The results indicated that pEGFP-N1-ABCAl vector was constructed successfully.Conclusion:After fed with high fat and high sugar diet, or add cholesterol diet, could lead to arterial lesions in Guangxi Bama mini-pigs, and the latter diet made more serious damage. The lesion areas showed more ABCA1 expression. It showed that the high level cholesterol in diet could speed up the arterial pathological process, and the ABCA1 mainly worked in the lesions. The eukaryotic expression vector pEGFP-N1-ABCAl could be used to produce transgenic Guangxi Bama mini-pigs. |
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