ABSTRACT
Objective To observe the transport of hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC)through placental barrier set up by choriocarcinoma trophoblast cells (Bewo cells),and to explore the biological role of PBMC as a carrier for HBV transport.Methods Bewo cells and PBMC were cultured and their proliferation and activity were detected by cell counting kit (CCK)-8.One hundred μL serum containing 5 ×10 6 copy/mL HBV DNA was used to infect PBMC,and cells infected with HBV were labeled by fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE).A co-culture model of Bewo cells and HBV-infected PBMC was set up by transwell chamber. The migration of HBV-infected PBMC was detected by flow cytometry.Realtime fluorescence quantitative polymerase chain reaction method was used to detect HBV DNA contents of PBMC under transwell chamber.Results PBMC and Bewo cells proliferated at around 24 h and entered into growth stagnation at around 120 h.The contents of PBMC labeled by green fluorescent at 0,12,24 and 48 h during co-culture under chamber were (0.445 ±0.021)%,(21 .180 ± 4.653 )%,(34.830 ± 7.156 )% and (64.185 ± 3.161)%,respectively.The amount of PBMC marked green fluorescence increased over prolonged incubation time (F =68.983,P =0.001 ).PBMC HBV DNA contents at 24 and 48 h of co-culture under chamber were (1.925±0.431)×103 copy/mL and (2.565 ±0.361)×103 copy/mL,respectively,indicating that PBMC under chamber were infected with HBV.Conclusions PBMC may be a target for HBV infection in extrahepatic tissues.Placental trophoblastic barrier built by transwell chambers may provide new ideas to investigate HBV transmission across the placenta in vitro .
ABSTRACT
Objective To study the relationship between placenta HBsAg in HBsAg positive pregnant women and serum hepatitis B virus (HBV) markers,HBV DNA levels in newborns.Methods Placenta HBsAg was detected by immunohistochemical affinity hormone-biotin complex (ABC) method in 155 HBsAg positive pregnant women.Serum HBV markers in newborns were detected by enzyme-linked immunosorbent assay (ELISA).Serum HBV DNA levels of newborns were detected by real-time fluorescence quantitative polymerase chain reaction (PCR).The positive rates were compared using x2 test.Results HBsAg was expressed with different levels in various types of cells of placenta in 155 pregnant women.The total placenta HBsAg positive rate was 37.4％ (58/155),and those in decidual cells,trophoblastic cells,villous mesenchymal cells and villous capillary endothelial cells were 37.4％ (58/155),25.8％ (40/155),18.7％ (29/155) and 7.1％ (11/155),respectively.The HBsAg positive rates of placenta gradually decreased from decidual cells of the maternal surface to villous capillary endothelial cells of the fetal surface (tendency x2 =43.01,P=0.00).The positivity of placenta HBsAg was associated with both HBsAg and HBeAg in newborns (x2 =4.88,P＜0.05 and x2 =3.86,P＜0.05,respectively),while that was not associated withanti-HBe and anti-HBc in newborns (x2 =3.36,P＞0.05 and x2 =0.00,P＞ 0.05,respectively).The risk of HBsAg positive in newborns was higher when HBsAg was positive in villous capillary endothelial cells and villous mesenchymal cells (OR=5.31,95 ％CI=1.38-20.40 and OR=3.33,95％CI=1.16-9.52,respectively).The risk of HBeAg positive in newborns was higher when HBsAg was positive in trophoblastic cells and villous mesenchymal cells (OR=3.04,95 ％CI=1.45-6.39 and OR=3.05,95 ％ CI=1.32-7.03,respectively).However,placenta HBsAg positive was not associated with HBV DNA positive in newborns (x2 =0.09,P＞0.05).Conclusion The risk of neonatal HBV serological markers positive is higher when the HBsAg positive placental cells are closer to fetal surface,which indicates that HBsAg enters fetal blood circulation by means of cell transferring layer by layer.