Study On The Effects Of HBV Infection And Replication On Phospholipid Metabolism Of Host

Chronic hepatitis B Virus(HBV)infection has the characteristics of high morbidity,high pathogenicity and unclear pathogenesis.Previously,we showed that phospholipid metabolic pathway is important for HBV replication.However,the relationship between HBV replications and phospholipid metabolites remains to be further studied.Phospholipids are the main constituents of biological membranes and are involved in signal transduction and their biological function has been increasingly recognized.The structure of phospholipids is highly complex and diverse,and the levels of some important phospholipids in biological samples is very low.Therefore,there is an unmet need to develop methods capable of quantifying a wide range of phospholipids with high sensitivities and high throughput.In this thesis,the human plasma and cell models with HBV infection were used.We aim to develop a qualitative and quantitative analysis method of phospholipids based on liquid chromatography tandem mass spectrometry.Then the changes of the host phospholipid metabolites after HBV infection were studied with th is method and the relationship between HBV replications and the host phospholipid metabolites was comprehensively studied in combination with molecular biology research techniques.We employed an ultrahigh-performance liquid chromatography system coupled to a triple-quadrupole mass spectrometer(UHPLC-MS)and developed a method that can quantitatively analyze 10 major classes of phospholipid in biological samples in 10 mins.These are phosphatidic acid(PA),phosphatidylcholine(PC),phosphatidylethanolamine(PE),phosphatidylglycerol(PG),phosphatidylinositol(PI),phosphatidylserine(PS),sphingomyelin(SM),lyso-phosphaticdic acica(LPA),iyso-phosphatidylcholine(LPC)and lyso-phosphatidylethanolamine(LPE).The limit of detection(LOD)and limit of quantitation(LOQ)are 0.04-33 pmol/mL and 0.1-110 pmol/mL,respectively.The qualitative and quantitative analysis of phospholipids takes three steps:first and second steps identified phospholipid structures in a mixture containing aliquots of all the samples using the combinations of multiple reaction monitoring(MRM),product ion scan and retention time in the positive and negative ion mode.These steps enable identification of phospholipids presented in the samples and provided information for efficient sample analysis in the final step of sample quantitative analysis.We have developed simple relative response to take account ionization variations due to different acyl chain length to achieve more accurate quantification.The method developed was applied to analyze 6 different biological samples(2 kinds of plasma,3 kinds of cells and 1 tissue)for applicability validation,where a total of 308 phospholipid species across 10 phospholipid classes were identified and 295 phospholipid species were quantified.Therefore,our HPLC-MS method is highly efficient(especially for large number samples analysis),sensitive,and is universally applicable.To elucidate the relationship between the HBV replication and the host phospholipid metabolites,we detected the levels of a total of 10 main phospholipids in patients and cells with HBV infection.Then we found that the levels of phosphatidylcholine(PC),phosphatidylethanolamine(PE),and lyso-phosphatidic acid(LPA)were increased in HBsAg(+)group compared with HBsAg(-),while the levels of phosphatidylserine(PS),phosphatidylglycerol(PG),phosphatidylinositol(PI),and sphingomyelin(SM)were decreased.In addition,we confirmed this finding in HBV infected cell models,HepG2.2.15.The Receiver operating characteristic(ROC)analysis shows that,the levels of PCs were better prediction of HBV infection than other phospholipids,suggesting that the alterations of PCs may be a potential biomarker of HBV infection.Furthennore,we evaluated synthesis and metabolic pathways of PC and found that the relative mRNA and protein expression of key enzyme genes for PC synthesis,choline-phosphate cytidylyltransferase 1 A(PCYT1A)and lipid phosphate phosphatase 1(LPP1),were up-regulated after HBV infection.Moreover,the HBV replications was inhibited when the expression of PCYT1A and LPP1 were knocked down by transfection with small interference RNA(siRNA)in HepG2.2.15 cells.These results indicated that the PC synthesis in the HBV infected host are regulated by the activity of PCYT1A and LPP1,which suggests that PCYT1A,LPP1 could be new potential targets for HBV treatment.Our research provided new information for the pathogenesis of HBV infection,and shed potential new clues for the treatment of HBV infection.In summary,we developed a phospholipids quantitative analysis method with high efficiency,high sensitivity and high throughput based on UHPLC-MS,which is widely applicable to analyze large biological samples.Then the relationship between HBV replications and the host phospholipid metabolism was studied combined with this UHPLC-MS method and molecular biotechnology,providing new clues for the study of the pathogenesis and treatment of HBV infection and replication.