The Study Of ApoA-I Cysteine Mutants With ApoA-V On Atherosclerosis In Vitro

Objective:The research aimed to study the anti-atherosclerosis effect of apoA-I cysteine mutant and apoA-V constructed recombinant liposomes in vitro.Methods:1.The Escherichia coli prokaryotic culture system of apoA-Iwt was optimized from three aspects:temperature,IPTG induction concentration and IPTG induction time.2.Enough apoA-Iwt,apoA-IM and apoA-I?N74C?are cultured by the Escherichia coli?E.coli?prokaryotic system and they are purified by a Ni-chelating affinity chromatographic column(Ni²⁺column).After dialysis and ultrafiltration concentration,apoA-V was added in according to experimental proportions of 1:0,100:1,1:1,and 0:1,and DPPC was added in according to the mass fraction of 1:3.35.Besides,rHDL liposome was constructed through the cholate dialysis method.3.DMPC turbidity clearance assay:Lipid binding capacities of mixtures of apoA-Iwt,apoA-IM,apoA-I?N74C?and apoA-V under different mixing ratios were analyzed by a DMPC turbidity clarification experiment.4.Antioxidant assay against LDL in vitro:The in vitro LDL oxidizability of rHDL liposome with different mixing ratios was analyzed by LDL oxidative system mediated by in vitro copper ion and TBARS colorimetric method.5.Intracellular lipid accumulation in vitro:Influences of rHDL liposome with different mixing ratios on macrophage induced by THP-1 cell absorption and intracellular lipid accumulation of oxLDL were analyzed by macrophages.Dyeing areas in different rHDL groups were tested by Image Proplus through oil red O specificity dyeing lipid.And TC content in different rHDL groups was tested by the TC colorimetric test kit to verify dyeing areas.Results:1.SDS-page?12%?result showed that The best conditions are concluded by optimizing prokaryotic culture conditions of apoA-I,which are composed of 37?,1mmol/LIPTG and3h induction.apoA-Iwt,apoA-IM and apoA-I?N74C?are expressed and purified successfully in E.coli prokaryotes.they are purified by a Ni²⁺column successfully,The purified protein was verified by 12%SDS-page which staining with coomase brilliant blue,and the molecular weight was at 28 KD.2.Thirteen rHDL liposomes were constructed by sodium cholate dialysis including apoA-Iwt-rHDL,apoA-IM-rHDL,apoA-I?N74C?-rHDL,apoA-V-rHDL,apoA-Iwt:apoA-V?100:1?-rHDL,apoA-IM:apoA-V?100:1?-rHDL,apoA-I?N74C?:apoA-V?100:1?-rHDL,apoAIwt-:apoA-V?1:1?-rHDL,apoA-IM:apoA-V?1:1?-rHDL,apoA-I?N74C?:apoA-V?1:1?-rHDL,apoA-Iwt:apoA-V?1:100?-rHDL,apoA-IM:apoA-V?1:100?-rHDL and apoA-I?N74C?:apoA-V?100:1?-rHDL?3.DMPC turbidity clearance experiment results that turbidity of DMPC significantly decreased with the addition of mixed rHDL comparing with Negative group,and rHDL mixtures could promote the turbidity clearance rate of DMPC to increase,when rHDL contained more apoA-V,among which the turbidity clearance rate of apoA-V-rHDL was the strongest.With the increase of the proportion of apoA-V in rHDL,k value gradually increased,turbidity clearance rate increased,and lipid binding ability gradually increased.4.A ntioxidant assay against LDL in vitro results that MDA,the oxidation products in the copper-mediated LDL oxidation system,significantly reduced?P<0.05 vs negative group?,when rHDL joined in systems,and The antioxidant capacity of apoA-V was stronger than that of apoA-Iwt,apoA-IM and apoA-I?N74C?.With content of apoA-V in rHDL increasing,the amount of MDA decreased,that is the antioxidant capacity increased5.The intracellular lipid accumulation modulated by rHDL using differentiated human THP-1 macrophages as a cell model,and the results show that the addition of rHDL significantly reduces the intracellular lipid accumulation?P<0.05,vs Normal group?,and apoA-Iwt,apoAIM and apoA-I?N74C?significantly inhibited the accumulation of lipid,among which apoa-im had the strongest effect.The TC content determined by total cholesterol colorimetry was consistent with the result of oil red O staining.Conclusion:The best conditions are concluded by optimizing prokaryotic culture conditions of apoA-Iwt,which are composed of 37?,1 mmol/L IPTG and 3 h induction.apoA-Iwt,apoA-IM and apoA-I?N74C?are expressed and purified successfully in E.coli prokaryotes.A total of 13 rHDLs were constructed by a sodium cholate dialysis.It was proved that compared with the control group,rHDL indeed improved lipid binding ability,resistance to intracellular lipid accumulation and oxidation resistance.Moreover,rHDLs with different proportions also presented different improvements.Since lipid binding,intracellular lipid accumulation and oxidation have been documented to have an effect on AS,it is speculated that the involvement of rHDL will affect the course of AS.