Human Cytomegalovirus Mediates The Damage Of Immature Biliary Epithelial Cells Through CD14 During Perinatal Period

Research Background: Human cytomegalovirus is a beta herpes virus.The infection rate is very high in the perinatal period.Due to the incomplete immune development of the newborn,HCMV infection can cause a variety of congenital diseases,such as neurological diseases,retinitis,Liver and splenomegaly and jaundice,about 40% of HCMV positive children are neonatal hepatitis,the symptoms of cholestasis are obvious,the duration of jaundice is long,and severe cholestasis can lead to many complications in children.Anti-viral treatment can significantly reduce the symptoms of cholestasis.Children with HCMV infection during the perinatal period have severe liver function damage,and intrahepatic bile duct destruction and fibrosis developed rapidly.It may be that HCMV damages the intrahepatic biliary epithelial cells and hepatocytes irreversibly,resulting in bile duct destruction and insufficient newly-increased small bile duct,Cholestasis further aggravates liver inflammation and damage.The study retrospectively compared the differences in liver function indicators between HCMV positive and negative children of different ages(congenital infection: less than 15 days,perinatal infection: 15 days to 3 months old,acquired infection: more than 3 months old)It was found that compared with HCMV-negative children,the enzyme metabolism index and bile acid metabolism index in liver function indexes of HCMV-positive children were significantly higher than those of HCMV-negative group;HCMV-positive group was obviously found by immunohistochemical staining of liver pathological tissue sections.The bile duct structure is defective,disordered,and the tubular structure disappears.In vitro experiments have found that HCMV has obvious invasion of immature biliary epithelial cells.HCMV-infected immature biliary epithelial cells show obvious cell shedding and morphological changes,while mature biliary epithelial cells,These changes in cell morphology and function were not obvious.It was further detected by cell immunofluorescence technology that biliary epithelial cells expressed cytomegalovirus markers PP65 and IE1,confirming that HCMV entered and replicated in biliary epithelial cells.Why are immature biliary epithelial cells susceptible to HCMV infection in the perinatal period and cause damage to immature biliary epithelial cells? To explain this problem,this study further analyzed the differential gene sequencing data of immature biliary epithelial cells and mature biliary epithelial cells by TMT-labeled proteomics technology.The results showed that immature biliary epithelial cells highly expressed CD14 gene and through the String database.The protein network interaction predicts that CD14 is the core protein.Compared with mature biliary epithelial cells,the immature biliary epithelial cells highly expressed CD14 as verified by cellular immunofluorescence.Silencing the CD14 gene of immature biliary epithelial cells by si RNA technology.After HCMV infection,compared with normal immature biliary epithelial cells,the morphological structure of immature biliary epithelial cells in the gene silencing group was normal.The immunofluorescence technology was used to detect the HCMV virus protein.The expression of PP65 and IE1 in the biliary epithelial cells of the gene silencing group was reduced,and the rate of IE1 positive cells was significantly lower than that of the normal group.In summary: Compared with acquired infection,human cytomegalovirus infection is more likely to enter the cell through the highly expressed CD14 receptor of immature biliary epithelial cells,and replicate in the cell,causing apoptosis during perinatal period.Research Objectives: This paper focuses on the analysis of the mechanism of perinatal HCMV injury to biliary epithelial cells and provides new research ideas for the clinical treatment of neonatal hepatobiliary diseases caused by HCMV infection.Research Methods: 1.Collect clinical data collection: This paper selects 10278 cases of children’s liver function data and other medical records from Guangzhou Women’s and Children’s Medical Center from January 2012 to January 2017 to reduce the impact of liver function caused by other diseases.We set the collection criteria and exclusion criteria for the collection of clinical liver function indicators: 1)real time PCR method was used to detect positive HCMV nucleic acid in children’s serum;2)liver function indicators were measured by centrifuged serum samples taken from whole blood before antiviral therapy Data;3)exclude other viral infections that cause abnormal liver function indicators,such as hepatitis B virus;4)exclude hemorrhagic diseases and hematopoietic system-related diseases;a total of 10278 liver function data were collected and divided into HCMV positive groups(1179 cases)And HCMV negative group(9099 cases).2.Human cytomegalovirus intervention in embryo and infant biliary epithelial cells in vitro,the expression of cytomegalovirus proteins PP65 and IE1 on the cell surface was detected by cell immunofluorescence,and the virus titer of the cell culture supernatant at different time points was determined;3.Through protein profiling technology,we predict the key molecular protein of cytomegalovirus into biliary epithelial cells,and verify the function of this key molecule through si RNA gene silencing technology.Research Results: 1.HCMV infection during the perinatal period affects the normal function of liver cells and biliary epithelial cells;2.HCMV intervention in embryo and infant biliary epithelial cell experiments found that HCMV can enter earlier and more easily replicate in embryo biliary epithelial cells;3.CD14 is a key protein for HCMV to enter embryo biliary epithelial cells.Conclusions In the perinatal period,HCMV may invade the BEC through the CD14 receptor highly expressed on the immature BEC,and replicate and proliferate within the BEC,causing BEC apoptosis.