Correlations Of Human Apolipoprotein AⅤ With Blood Lipid And Inflammatory Cytokines And Mechanism Of TNF-a Down-Regulating Apolipoprotein AⅤ Expression

BackgroundCoronary heart disease (CHD) continues to be a major cause of morbidity and mortality worldwide. Besides reduced high density lipoprotein cholesterol (HDL-C) level, elevated plasma triglyceride (TG) level constitutes an independent risk factor for CHD. Apolipoproteins play a determinant role in lipoprotein metabolism, and the Apo A I /CIII/AIV apolipoprotein gene cluster is tightly related to plasma lipid homeostasis. A comparative genomic characterization of the Apo A I /CIII/AIV gene cluster flanking regions led to the identification of a new apolipoprotein gene, Apolipoprotein AV(Apo AV), present in both mice and humans. Previous studies have demonstrated that Apo AV plays a crucial role in modulating serum TG level, also there are reports that Apo AV might affect HDL-cholesterol metabolism. Most of these data had been gathered from mice, and many of the conclusions were not coincident with each other, so that it is important to have the study of Apo A V undertook on human self.Systemic inflammation is frequently associated with severe hypertriglyceridemia. Inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF-α) stimulate and modulate inflammatory response and correlate significantly with the concentrations of very low density lipoprotein (VLDL) triglyceride and cholesterol and negatively with HDL cholesterol. However, the mechanism involved is not completely understood. Whether inflammation affecting the expression of Apo AV is remained to be confirmed.ObjectivesTransform recombinated plasmid contained human Apo A V coding gene into E coli to establish prokaryotic expression system to express recombinated human ApoA V protein, further to purify the recombinated protein. Immunize mice to raise monoclone antibodies (McAbs) against recombinated human Apo AV, and then construct a double-antibodies sandwich enzyme-linked immunosorbentassay (ELISA) for human Apo A V measurement. To determine human serum Apo A V levels using this ELISA and study the correlation between Apo AV with lipid profiles, inflammatory cytokines high sensitive C reaction protein (Hs-CRP ) and TNF-αTo explore the effect of inflammatory cytokine TNF-αon Apo AV gene expression and protein production in HepG2 cell lines, and to study the transcription regulation of nuclear transcription factor-kappa B (NF-κB) on Apo A V gene expression reduced by TNF-α.Methods1.Human Apo AV coding gene and vector DNA were cut by restriction enzyme, and then linked to each other to construct recombinated plasmid. Transformed recombinated plasmid into E coli. Expression of interest protein in E coli was induced by IPTG, and the protein was extracted after clearage of E coli by ultrasonic wave, purified by Ni-NTA affinity chromatography, annealed by dialysis.2.Immunized Balb/c mice using recombinated human Apolipoprotein AV as antigen and then harvested immunizing splenocyte. Fused the immunizing splenocyte and SP2/0 myeloma cell through hybridoma technique, selectivly cultured the fused cells with HAT and HT, screened positive clone through limiting dilution assay and then established hybridoma cells which secreted McAbs. Infused hybridoma cell into mice abdominal cavity to prepare ascites. McAbs was purified by affinity chromatography and Labelled with HRP. The design of sandwich ELISA was performed by Square-titration method.3. Serum TG and total cholesterol (TC) concentrations were measured enzymatically, HDL-C and low-density lipoprotein cholesterol (LDL-C) were measured by Chemical Masked method, Hs-CRP was measured by immune transmission turbidity, TNF-αwas measured by method of ELISA, An ELISA performed by a couple of monoclonal antibodies was used to measure serum Apo A V.4.The HepG2 cells were treated with TNF-αof different concentration or different time or pretreated with Pytrolidine dithiocarbanate (PDTC, 50μmol/L) inhibitor for NF-κB 30 min followed by co-incubation with TNF-α. RT-PCR was used to detect the level of Apo AV mRNA. The concentration of Apo A V in culture supernatant was detected using Sandwich-ELISA. ELISA was also used to evaluate the activation of NF-κB p65 subunit.ResultsE coli transformed by recombinated plasmid pQE30-Apo A V expressed recombinated human Apo AV protein. The protein product was purified by affinity chromatograph. 2 McAbs (1E8,2G1) were prepared by hybridoma technique. The average Apo AV concentration was 182.7±104.7 ng/ml ranging from 5.4 to 455.6 ng/ml using the sandwich ELISA performed by the 2 McAbs. Serum Apo AV concentration was negatively correlated with TG (r=-0.225, P=0.031), body mass index (BMI)(r=-0.345, P=0.001), Hs-CRP and TNF-α(r=-0.300, P=0.004 for Hs-CRP and r=-0.424, P=0.001 for TNF-α), positively with HDL-C (r=0.453, P<0.001).Apo AV gene expression and protein production were down-regulated by TNF-αin a dose- and time- dependent manner (P<0.01); The expression of Apo A V and the activation of NF-κB were changed in reverse ways by decreasing or increasing significantly respectively(P <0.01). PDTC inhibited the process induced by TNF-α(P ConclusionsRecombinated human Apo AV protein was expressed by prokaryotic expression system. 2 McAbs were raised from mice. A double-antibodies sandwich ELISA for measuring recombinated human Apo AV was established. Human serum Apo AV level was very low and negatively correlated with TG and BMI, but positively correlated with HDL-C. Surem Apo AV was negatively correlated with inflammatory cytokines Hs-CRP and TNF-α. Apo A V gene expression with protein production in HepG2 cell lines was down-regulated by TNF-αand NF-κB activation was involved in the pathway.