| Objective: To understand the risk factors of neonatal HBV intrauterine infection in HBs Ag-positive pregnant women, and to provide references for preventing and controlling neonatal HBV intrauterine infection. While investigating HBV infection in peripheral blood mononuclear cells and using in situ PCR technique to detect the HBV DNA and HBV ccc DNA in PBMC, to study the role of PBMC in neonatal HBV intrauterine infection.Contents and methods: 1. Analysis of risk factors in neonatal HBV intrauterine infection(1) Objects Continuously collected the HBs Ag-positive pregnant women and their newborns(in total 312 pairs) in various hospitals Beijing from August, 2008 to September, 2012 as a cohort, collected their clinical epidemiological data and blood samples.(2) Methods 1) Epidemiological survey: Approach the method of nested case-control study, utilize the customized survey to enquire the objects and record, and also write down the clinical examination results. 2) Serological detection: ELISA has been used to detect the HBs Ag and HBe Ag in the blood samples of the study subjects. The conserved sequence near the gene promoter and terminator of S gene in HBV gene promoter and terminator has been selected to detect the DNA of HBV in serum and PBMC. 3) Statistical analysis: Check the data and enter it into Epi Data 3.1, create a database, use SPSS 22.0 to analyze the data. Use ±S to represent the result, approach t-text test method. The result of count data would be represented by percentage or proportion, utilize t-text test or χ2 detection during comparison. Use Excel 2013 and Graph Pad Prism5.0 to make figures. 2. PBMC cell transfection experiments and in situ PCR detection of HBV DNA and ccc DNA in PBMC(1) PBMC cell transfection experiment Get the full-length of HBV DNA fragments by PCR amplified, through the connection built into T vector into competent cells,then into competent cells; Coated plate an screening positive by blue-white,then expanding culture; extracted the plasmid by extraction kit containing endotoxin removal; digested the amplified product to obtain a high concentration of HBV DNA genome plasmid. Separation of healthy human PBMC, then electroporation manner HBV DNA transfected into healthy human PBMC, after culture to detect the expression of HBV in PBMC.(2) In situ PCR detection of HBV DNA and ccc DNA in PBMC Centrifuge the blood plasma of HBV infectors and using Ficoll density gradient centrifugation method to obtain PBMC, fixed PBMC to the adhesion slides by cell rejection machine. Using in situ PCR technology and combined with rolling circle and across-gap to detect the HBV DNA and ccc DNA in PBMC. Observe the expression and location in the cells by microscope.Results 1. Risk factors of neonatal HBV intrauterine infection(1) The laboratory test results of HBs Ag positive pregnant momen and their infants 312 cases of HBs Ag positive pregnant women and their newborns laboratory test results showed that a total of 142 cases of pregnant women whose newborns occurred HBV intrauterine infection, intrauterine infection rate was 45.5%(142/312).In 142 cases of intrauterine infection of newborns, the positive rates of serum HBs Ag and HBV DNA and HBV DNA in PBMC were 7.7%(24/312), 19.6%(61/312) and 26.0%(81/312). 15 cases of neonatal simultaneously detected serum HBs Ag and HBV DNA both positive, nine cases of neonatal all test results were positive.(2) Risk factors of Neonatal HBV intrauterine infection with HBs Ag positive pregnant women Comprehensive 312 cases of HBs Ag-positive mothers and their newborns clinical epidemiological data, demographic survey data and laboratory test results show that: in intrauterine infection, 100 cases PBMC HBV DNA positive of mothers`(32.1%, 100 / 312 cases); the number of HBe Ag positive detected of mothers is 78 cases, the positive rate of 25%;137 cases of HBV DNA positive in serum, the positive rate was 43.9%; the three factors above are the risk factors of neonatal HBV intrauterine infection, after the chi-square test, p values were less than 0.001, less than 0.001 and 0.0147; Other factors were associated with HBV intrauterine infection unrelated. Further analysis of the relationship between maternal serum HBV DNA levels and HBV intrauterine infection of newborns found that serum HBV content into a high risk group(> 106 copies / ml), low-risk group(103copies / ml ~ 106 copies / ml) and very low risk group(<103copies / ml), showed that based on very low risk group, the risk of neonatal HBV intrauterine infection of high risk group and low risk group were 5.33 times and 2.61 times, the trend chi-square test shown that the difference of three groups intrauterine infection rate was statistically significant, χ2 value is 28.24, p <0.001. 2. HBV DNA electroporation PBMC experiments Extracted HBV DNA genome sequence By PCR, after a 1.5% agarose gel electrophoresis showed that amplification product bands consistent with the expected results; target gene connected transformed after extraction of the ligation product digested, its products were 1.5% agarose gel electrophoresis; measured the purpose plasmid concentration of 20 ng by UV spectrophotometer; plasmid transfected to healthy PBMC by electroporation, cultured 6 hours and 24 hours after the extraction of DNA, the use of quantitative fluorescence PCR method to detect copy number of HBV DNA in the cell were 5.67 × 103 copies / ml and 7.4 × 104 copies / ml 3. Detection of PBMC of the neonatal HBV intrauterine infection by cells ISPCR To detect the expression of HBV DNA in PBMC of hepatitis B patients by the use of in situ PCR, the hepatitis B liver tissue paraffin sections as a positive control. Nuclear fast red staining as the background, blue-purple mass was found in nuclei of liver tissue and PBMC. Detected HBV ccc DNA by rolling circle and across-gap with in situ PCR technique, blue-purple mass was found in nuclei of liver tissue and PBMC. After the ISH detect HBV DNA and ccc DNA in PBMC of pregnant women, the positive rate of in situ detection correlated with serum HBV DNA level. By comparing 74 cases, higher levels of HBV DNA in peripheral blood, the higher the positive rate detected ccc DNA in PBMC. Summary 62 cases of infant PBMC situ detection results showed that, 22 cases detected ccc DNA in PBMC of 25 cases of infants intrauterine infection, the positive rate is 88%(22/25).Fisher exact chi-square calculations shows p < 0.001.Conclusions The study found that maternal serum HBs Ag, HBV DNA and PBMC HBV DNA positive were the risk factors of neonatal HBV intrauterine infection, detection of HBV DNA in PBMC helps the determine of intrauterine infection; by transfection hepatitis B virus to human peripheral blood mononuclear cells. Fluorescence quantitative PCR and cell in situ PCR technology to detect can confirm that PBMC infected HBV. Useing rolling circle and across-gap in situ PCR technique to confirm the relation between PBMC HBV ccc DNA and intrauterine infection. Pregnant PBMCs are a hiding and replication places of HBV besides liver tissue, infected peripheral blood mononuclear cells is one of the factors causing neonatal HBV intrauterine infection. |
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