Effect Of Apolipoprotein M On Expression Of Inflammatory Factor And Its Susceptibility To Coronary Artery Diseases

Part I Preparation and identification of high affinity monoclonalantibodies against Apo MObjective: Preparation and identification of specific and high-affinity monoclonal anti bodies against human apo M.Methods: Each female BALB/c mice about 6 to 8 weeks old primarily immunized using Freund’s adjuvants and recombinant apo M protein 50mg. Then the mice immunized with recombinant apo M protein every 15 days. Four to five days before the scheduled fusion give a final injection 50mg of apo M recombinant protein wirth Freund’s incomplete adjuvant. The spleen cells from antibdy-positive mouse were fusing with myeloma cells SP2/0. The hybridomas specifically secreting mouse anti-human apo M monoclonal antibody were generated. After cloning and ascites fluid inducing, the apo M monoclonal antibody was purified with Protein G column chromatography. The quantitative and specific of antibody were detected by Western blot assay.Results: After screening, fusion, selective culture and sub cloning, 4 strains of specific mouse anti human apo M monoclonal antibody hybridoma cell lines were successfully obtained. Western-blotting analysis showed that these four antibodies can specifically recognize the proteins of apo M. And then we select the hybridoma 1368CT871.72 to induce ascites fluid. After protein G column chromatography, the concentration of monoclonal antibody of apo M protein content was 1mg/ml. The purified apo M mc Ab were gradient diluted from 1:1000 to 1:4000 to verify the sensitivity. Result shown the apo M mc Ab could also detect the target protein until 1:4000.Conclusion: The mouse anti human apo M monoclonal antibody has good sensitivity and specificity.Part II Purification of Apo M+ HDL and its abilityto bind to SR-BI in vitroObjective Isolated apo M+ HDL and apo M- HDL particles from plasma human high density lipoprotein(HDL). And detected different affinity of apo M+ HDL and apo M-HDL binding to SR-BI, respectively.Methods Mouse anti human apo M monoclonal antibody was coupled to Hi Trap NHS-activated HP column by covalent binding mode. Two components in HDL protein, apo M+ HDL and apo M– HDL particles, were separated by affinity chromatography method. And then uses the BCA protein quantitative assay to analysis the concentration of the separation. After induced by PMA and ox-LDL in turn, human monocytic leukemia cell line, THP-1 cells were incubated with apo M– HDL, apo M+ HDL and the recombinant human apo M protein, respectively. Cells were collected and total proteins were extracted. The interaction between apo M+ HDL and SR-BI was confirmed by co-immunoprecipitation.Results By the Hi Trap NHS-activated HP column purification, the two components in HDL protein, apo M+ HDL and apo M– HDL particles, were separated. The effluent liquid and elution liquid were detected by dot blot and western blot with apo M antibody, respectively. Results showed the apo M protein could be detected in unpurified HDL protein and elution liquid, but not in the effluent liquid. A final concentration of apo M– HDL and apo M+ HDL particles were 3.23 mg/ml and 0.34 mg/ml, respectively. Treatment with PMA and ox-LDL, the expression of SR-BI on surface of THP-1 cells increased about 10%. The results of co-immunoprecipitation showed recombinant human apo M, apo M– HDL and apo M+ HDL particles could interact with SR-BI in the surface of THP-1 cell line.Conclusion It is successful to separated HDL into two kinds of protein components of apo M– HDL and apo M+ HDL particles. The interaction between SR-BI to apo M– HDL, apo M+ HDL and recombinant human apo M were confirmed, respectively.Part III Different Effects of apo M– HDL and apo M+ HDL onmacrophage: Influence of inflammatory factors secretion and itspossible mechanismObjective To Analyze the different of inflammatory factors secretion induced by apo M– HDL or apo M+ HDL on macrophage, and find the possible signal transducer using Gene Chip® Prime View? Human Gene Expression Array.Methods(1) THP-1 cells were induced to differentiate into macrophages by PMA and ox-LDL. The lipids in cells were observed by oil red O stain.(2) Induced THP-1 cells were divided into four groups(each group had 6 duplicates): group 1, culture medium(containing apo M– HDL 20μg/ml); group 2, culture medium(containing apo M+ HDL 20μg/ml); group 3, control group. After induction of 24 hr in the culture medium above, the cell supernatant were collected and detected the expression of TNF- α, IL-10, IL-6, IL-1β and MCP-1 by Luminex 200, respectively.(3) Analyzed the difference of the signal pathway between three groups treated above using Gene Chip® Prime View? Human Gene Expression Array.Results(1) Compared with the control group, cellular lipid accumulation significantly in THP-1 cells after induced with PMA and ox-LDL. Foam cell model was induced successfully.(2) Apo M– HDL and apo M+HDL could down regulate the secretion of TNF-α, IL-6, IL-1β and MCP-1, respetively. And apo M+ HDL down-regulation of IL-6 and IL-1β is more significant(P=0.0411,0.0002 and 0.0101, respectively), compared with apo M– HDL treatment group. It was also found that compared with the control group the expression of IL-10 was significantly up-regulated in the apo M+ HDL treatment grouop(P=0.0066).(3) Compared with control group the up-regulated genes in apo M– HDL and apo M+HDL treatment groups are 309 and 287, respectively. According to the information of pathway KEGG and BIOCARTA, signal pathways stimulated by apo M– HDL and apo M+HDL were basically the same. Compared with the control group, cytokines/cytokine receptor signaling pathway, MAPK signaling pathway, adhesion molecular signal pathway were activated. Compared with apo M+HDL, the cell treated with apo M– HDL up-regulated the expression of MMP-12 and MMP-7. Due to differences in gene number is too small, unable to carry on the analysis of pathway.Conclusion Apo M+ HDL has more significant anti-inflammatory effect. And it may have certain effect in promoting plaque stability.Part IV Regulation of apolipoprotein M and its susceptibility tocoronary artery diseasesObjective The present study investigated the correlation among genetic polymorphisms of proximal promoter region of apolipoprotein M(apo M) gene, the polymorphisms in relation to apo M expressions and the susceptibility of coronary artery diseases(CAD) in Han Chinese population.Methods We analyzed the genotypes of apo M proximal promoter region in 206 CAD patients diagnosed by angiography and 209 healthy controls by directly sequencing. The levels of serum lipid profiles were also studied by biochemical methods. Furthermore the wide-type and mutant promoter region of apo M were cloned into the luciferase expression vector p GL3, respectively. Luciferase reporter assay was used to detect the activity of apo M promoter.Results Four common polymorphic sites, i.e., T-1628 G, C-1065 A, T-855 C and T-778 C, were confirmed, and a new deletion mutation C-724 del was found, in 206 CAD patients and 209 non-CAD patients with direct DNA sequencing analyses. Occurrences of alleles T-1628 G, T-855 C and C-724 del were significantly statistically higher in the CAD patients than in non-CAD patients. Moreover we examined all these polymorphisms in relation to the apo M expressions by the luciferase reporter assay. It demonstrated that constructs-855 C and 724 del showed obvious decreased luciferase activities, i.e.,(0.93±0.15 vs. 2.11±0.15; P=0.012) and(1.13±0.25 vs. 2.11±0.15; P=0.009), respectively, which indicates these two polymorphisms could confer decreased apo M expressions. Meanwhile the occurrences of these two SNP were also significantly higher in the CAD patients than in non-CAD patients.Conclusion It is therefore reasonable to speculate that down-regulated apo M expressions in relation to these polymorphisms may affect HDL and cholesterol metabolism in vivo and further influence the susceptibility of CAD, although detailed mechanism needs further investigation.