Effects Of Transcription Factor FoxO3 On The Maturation And Activation Of Hepatic NK Cell In Biliary Atresia

PART ONE NK cell activity in peripheral blood and expression of transcription factor Fox O in NK cell and liver of biliary atresia patientsObjective: To study NK cell activity in peripheral blood and the expression of transcription factor Fox O1 and Fox O3 in peripheral blood NK and liver of patients with BA and CBD.Methods: Peripheral blood was collected from 33 BA patients and 10 CDB patients.Peripheral blood lymphocytes were separated and NK cells were obtained by using Human NK cell isolation kit.Flow cytometry was applied to determine the purity of NK cells.Use LDH release assay to detect NK activity.Transcription factor Fox O1 and Fox O3 expression of peripheral blood NK were conducted by means of Westen blot and RT-PCR.Meanwhile,liver specimens from patients with BA and CDB were collected.HE staining was used to evaluate the pathological changes of liver tissue.Fox O1 and Fox O3 m RNA and protein levels in liver tissue were detect by methods of Western-blot and RT-PCR.Results: The purity of isolated NK cells by methods of flow cytometry were as high as96%.LDH release assay was performed to detect the activity of NK cell in peripheral blood,and the result showed the activity of NK from BA was significantly enhanced compared to that from CDB.The protein and m RNA levels of transcription factor Fox O1 and Fox O3 in isolated NK cells were significantly decreased in BA patients.Results of HE staining showed that in the BA group,hepatocyte swelling,cytoplasm rarefaction,spotty necrosis scattering in hepaticlobule and inflammatory cell infiltrating in portal duct areas were found.In group CDB,the hepatic lobule of liver was normal,and the liver cells werenot denatured and necrotic.The results of Western blot showed that the expression of Fox O3 protein in the liver of group BA was significantly decreased than that in the CDB group.The results of RT-PCR were consistent with those of protein expression.Conclusion: The activity of isolated NK cells in BA patients is enhanced,and the expression of Fox O1 and Fox O3 is obviously decreased.The decrease of Fox O3 expression is found in the BA liver.In the course of BA,the decrease of Fox O3 expression may lead to the change of immune cell function.PART TWO Effect of Fox O3 on the maturation and activation of hepatic NK cells in RRV induced BA miceObjective: To study NK cell maturation and activation in RRV induced BA mice overexpressing of Fox O3 in vivo,and to explore the effect of Fox O3 on the maturation and activation of NK cells.Methods: Mice at E17.5d were injected of Fox O3 gene over expression of adenovirus vector through vena caudalis.The newborn mice were divided into 4 groups: saline group,intraperitoneal injection of normal saline within 12 h after the birth;RRV group,intraperitoneal injection of 2*106PFU RRV within12 h after the birth;Fox O3 over expression of adenovirus vector + RRV group,intravenous injection of 2*106PFU RRV on the mice whose mather were injected of Fox O3 gene over expression of adenovirus vector through vena caudalis at E17.5;Fox O3 blank vector + RRV group,intraperitoneal injection of 2*106PFU RRV on the mice whose mather were injected of Fox O3 gene in blank vector through vena caudalis at E17.5.Record the incidence of BA,the development and the survival rate of 21 d.Detect Fox O3 expression in liver after birth by methods of Western-blot and RT-PCR.HE staining was used to evaluate the pathological changes of liver at 14 d.Obtain NK cells from liver at the third day,seventh day and fourteenth day respectively and the expression of CD69,TNF-α and IFN-γ of NK cells were detected by flow cytometry.Protein of granzyme B and perforin were detected by methods of Western-blot.CD69,TNF-α,IFN-γ,granzyme B and perforin m RNA expression were detected by methods of RT-PCR.Results: Mice whose mather were injected of Fox O3 gene over expression of adenovirus vector through vena caudalis at E17.5 showed a Fox O3 protein overexpression in liver at the time of born.The results of HE staining were shown there was no obvious necrosis and infiltration of inflammatory cells around the portal area in Fox O3 over expression of adenovirus vector + RRV group compared with RRV group.Overexpression of Fox O3 in vivo could reduce the incidence of BA,promote the growth of body weight and improve the survival rate of 21 d.Flow cytometry,Western blot and RT-PCR results showed expression of CD69,TNF-α,IFN-γ from NK cells in Fox O3 over expression of adenovirus vector + RRV group at 3d decreased compared with RRV group.The m RNA expression of perforin and granzyme B significantly decreased,and the difference was statistically significant.The markers of activated NK cell and perforin and granzyme B in Fox O3 over expression of adenovirus vector + RRV group were significantly lower than those in the RRV group at 7d,and the difference was statistically significant.On the 14 d,the expression of markers of activated NK cell and perforin and granzyme B decreased in Fox O3 over expression of adenovirus vector + RRV group compared with RRV group.Perforin detected by methods of RT-PCT and CD69 b detected by methods of flow cytometry is significantly lower in Fox O3 over expression of adenovirus vector + RRV group than RRV group.Conclusion: Fox O3 is involved in the function of NK cells in the pathogenesis of BA.Overexpression of Fox O3 in vivo can inhibit the activation of NK cells,reduce the incidence of BA and increase 21 d survival rate.PART THREE Mechanism of Fox O3 affecting the activation of NK cellsObjective: To study Fox O3 regulate the activation of NK92 cells by affecting the expression of T-bet by bioinformatics analysis,Chromatin immunoprecipitation assays,si RNA transient transfections and plasmids construction.Methods: We analyzed whether there are foxo binding sequences in human Tbx21 promoter by bioinformatics analysis,and Chromatin immunoprecipitation assays were performed to study whether Fox O3 can bind to Tbx21 promoter region.The Fox O3 si RNA and Fox O3 overexpression plasmid transfection technology was used to interfere and over express the expression of Fox O3 in NK92 cells.Fox O3 and T-bet protein and m RNA levels in NK92 cells intervened by Fox O3 were detected by methods of Western-blot and RT-PCR.The expression of T-bet,Granzyme B,perforin,IFN-γ and TNF-α m RNA in NK92 cells intervened by T-bet si RNA and T-bet overexpression plasmid transfection technique were detected by methods of RT-PCR.Results: Bioinformatics analysis found that there was a potential forkead binding sequence TTGTTTT in the human Tbx21 promoter region from 1187 to-1181 bp.The chromosome immunoprecipitation assays showed that Fox O3 could combine with the promoter region of Tbx21.The expression of T-bet m RNA in NK92 cells was inhibited compared with the control group after Fox O3 si RNA treatment.In turn,Fox O3 overexpression increaseed the m RNA level of T-bet in NK92 cells.The protein expression of T-bet showed the same trend at the level of m RNA.The expression of granzyme B and IFN-γ m RNA in NK92 cells interfered by T-bet si RNA increased.When T-bet was overexpressed in NK92 cells,the expression of granzyme B and IFN-γ m RNA in NK92 cells decreased.Conclusion: The interference and overexpression of Fox O3 can regulate the activation of NK92 cells,which may be achieved by promoting or inhibiting the expression of T-bet.