Study On Lipid-lowering Mechanism Of Polysaccharides From Ganoderma Lucidum And Application Of Magnetic Fluorescent Nanoprobe Synthesis In Separation Membrane Proteins

Hyperlipidemia is the primary risk factor for atherosclerosis.Although the western medicine used in clinical practice is effective in reducing lipid,it has relatively large toxic and side effects and should not be taken for a long time.Therefore,it is of great significance to find and develop nontoxic and effective lipid-lowering drugs.This study is divided into two parts.We first explored the molecular mechanism of the inhibition of cholesterol synthesis in HepG2 cells induced by polysaccharides from pines and ganoderma lucidum.In the process of screening the receptors of polysaccharides on the cell membrane,the corresponding receptors were not screened in the currently known polysaccharide binding membrane proteins.Based on the limitations of the research methods of receptors on the polysaccharide cell membrane,we carried out the research work of the second part and constructed the Fe₃O₄@SiO₂/CdTe/CdS-SBT dual-functional magnetic fluorescent nano-probe,and successfully verified its operability?.molecular mechanism of inhibition of cholesterol synthesis in human hepatocellular carcinoma cells induced by polysaccharides from ganoderma lucidum1.In previous studies,we found that SBT can induce the decrease of HMGCoA expression of cholesterol synthesis rate-limiting enzyme in hepatocytes.In order to further study the pharmacological mechanism,we induced HepG2 cells by SBT at different concentrations for 48h,and measured the expression of various proteins in the physiological synthesis pathway of cholesterol.Various proteins were found to be regulated by SBT.The expression of SREBP2 protein increased gradually,while the expression of S2P protein decreased gradually.After the induction of HepG2 cells at fixed concentration?0,3,6,12,24 hours?,it was found that the expression of SREBP2 protein showed a slight decline with the increase of induction time.SCAP protein expression was first increased and then decreased.The expression of S2P protein decreased first and then increased.The expression of Insig-1 protein decreased.Although there is a certain change in the expression level of each protein,the amount of change is small.Therefore,we conclude that the physiological synthesis pathway of SBT for hepatocyte intracellular cholesterol regulation is not associated with the SREBP-SCAP pathway.2.To further explore the molecular mechanism of polysaccharide SBT inhibiting cholesterol synthesis in hepatocytes.Screening of SBT polysaccharide binding proteins on cell membrane was carried out.Cell fluorescence immunoassay was used to study HepG2 and L02 of human normal hepatocytes,and the results showed that the expression of dectin-1 receptor protein on the surface of hepatocytes was very low,while dectin-1 protein was expressed,but it was not the receptor protein of SBT on the liver cell membrane.?.Construction of bifunctional magnetic fluorescence nano-probe and its application in membrane protein separation1.Firstly,Fe₃O₄ magnetic nanoparticles were synthesized by co-precipitation method,and a layer of SiO₂ was modified on the surface to obtain Fe₃O₄@SiO₂magnetic nanoparticles.Fe₃O₄@SiO₂-SA magnetic nanoparticles were prepared by ring opening reaction of succinic anhydride and modification of sioh on its surface.The results show that it has good magnetic separation ability.Secondly,water-soluble CdTe QDs was synthesized by one-pot method,CdS was coated on the surface of CdTe QDs with thiourea,and core-shell CdTe/CdS fluorescence quantum dots were synthesized,with a large number of amino groups on the surface.Its fluorescence intensity is high and its stability is good.The magnetic fluorescence complex Fe₃O₄@SiO₂/CdTe/CdS was further synthesized by amideation reaction.The characterization results showed that Fe₃O₄@SiO₂/CdTe/CdS composite had better magnetic property and stronger fluorescence intensity,and had potential application value in the biomedical field.2.After SBT is dissolved in water,its reduced end is exposed to condensation reaction with amino group.By condensation reaction,CdTe/CdS and Fe₃O₄@SiO₂-SA were coupled with SBT to construct Fe₃O₄@SiO₂/CdTe/CdS-SBT magnetic fluorescent nanoprobe.It was also used to detect SBT membrane protein receptor on HepG2 cell surface,and the distribution of magnetic fluorescence nano-probe was clearly seen on the cell surface,and the presence of SBT receptor protein on HepG2 cell surface was identified.SBT receptor proteins specifically bound by magnetic fluorescence nanoprobe were rapidly enriched by ultrasonic cleavage and magnetic separation.This study provides new ideas and methods for the future study of membrane proteins.