| Objective: Hepatitis B virus（HBV）infection and diabetes are serious public health problems worldwide.Our previous work found that HBV transregulated proteins pre-S2 and HBx up-regulate the expression of a phospholipase autotaxin（ATX）and enhance lysophosphatidic acid（LPA）signal transduction.Recent studies have shown that ATX/LPA signal transduction is associated with insulin resistance and impaired glucose homeostasis.However,it is still unclear whether HBV infection affects insulin secretion and glucose homeostasis through LPA signal transduction.This thesis investigated the effects of HBV infection on insulin secretion and glucose homeostasis as well as the role of LPA in insulin secretion and glucose homeostasis in the mice under HBV infection by using the NIT,a mouse insulin-secreting cell line,stable transfection cells expressing pre-S2 and HBx,respectively and an HBV-infected mouse model.Methods: This study includes two aspects: cell model research and animal model research.In the cell model research:（1）construction of the recombinant lentiviral plasmids of pre-S2 and HBx.（2）pre-S2 and HBx lentiviral plasmids were transfected into NIT to establish NIT stable transfection cells expressing pre-S2 and HBx,respectively.（3）glucose-stimulated insulin secretion（GSIS）test in NIT stable transfection cells of pre-S2 and HBx.（4）test of the effect of Ki16425,an antagonist of LPA receptor,on insulin secretion of the NIT stable transfection cells of pre-S2 and HBx in GSIS.In the animal model research:（1）establishment of the HBV-infected mouse model by injecting the recombinant deno-associated virus 8 harboring 1.3 length of HBV genome（r AA8-1.3 HBV）into mouse tail vein.（2）detection of the serum HBV DNA of the mice by PCR;（3）Detection of covalently closed circular DNA（ccc DNA）in liver tissue by rolling-loop amplification of（RCA）combined with gap PCR,and the PCR products were ligated,transformed,positive clone selection and DNA sequencing.（4）measurement of the levels of blood glucose and plasma insulin of the mice in the intraperitoneal glucose tolerance test（IPGTT）;（5）assay of plasma LPA of the mice by liquid chromatography-mass spectrometry（LC-MS）.Results: The results of cell model research were as follows:（1）PCR identification showed that the constructed recombinant lentiviral plasmids of pre-S2 and HBx were in agreement with the design.（2）stable-transfected NIT cells of LV5,LV5-pre-S2 and LV5-HBx stably expressed fluorescence;（3）the insulin secretion of the NIT stable transfection cells of pre-S2 and HBx was significantly decreased in GSIS test（P < 0 01）;（4）the insulin secretion of the NIT stable transfection cells of pre-S2 and HBx was significantly increased in GSIS test（P < 0 01）by adding LPA receptor antagonist Ki16425 into the culture media.The results of animal model research were as follows:（1）PCR assay of the serum HBV DNA of the mice injected with r AAV8-1.3HBV showed positive result;（2）the DNA sequencing of the PCR product amplified by RCA combined with gap PCR indicated that the livers of the mice injected with the r AA8-1.3HBV were infected.（3）the plasma LPA of HBV-infected mice was 1.579 times higher than that of control group（P < 0.01）;（4）the blood glucose of HBV-infected mice increased significantly at the time point of 15 min of GTT（P< 0.05）.Conclusion: In this study,the pre-S2 and HBx,the HBV trans-regulatory proteins,stable-transfected NIT,a mouse insulin-secreting cell line,cell models have been established.The two HBV trans-regulatory proteins were found to down-regulate the insulin secretion of NIT cells.Ki16425,a LPA receptor antagonist,blocked the down-regulation of insulin secretion by pre-S2 and HBx.The model of HBV-infected mice has been established.The plasma LPA of HBV-infected mice was higher than that of control group.The level of blood glucose of the HBV infected mice was significantly higher than that of control group in GTT,suggesting that HBV infection impairs glucose homeostasis,and the mechanism needs to be further studied. |
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