| Hepatitis BHH is HHendemicHH in HHChinaHH. Of the 350 million individuals worldwide infectedwith the HHhepatitis B virusHH (HBV), one-third resides in China. As of 2006 China hasimmunized 11.1 million children in its poorest provinces as part of several programsinitiated by the Chinese government and as part of the HHGlobal Alliance for Vaccines andImmunizationHH (GAVI). However, the effects of these programs have yet to reach levels ofimmunization that would limit the spread of hepatitis B effectively. The reason for thisincreased HBV infection is unknown, because hepatitis B has no clear transmission routesin many people in China, although it has been widely accepted that HHneonatalHH HHinfectionHH andHHvertical transmissionHH during early childhood are still the most common routes. Also, mostresearchers divided the HHvertical transmissionHH to 3 different approaches: prenatal intrauterinetransmission, intrapartum transmission and postpartum transmission. As for the two of thelatter approaches, the transmission process can be effectively inhibited and blocked byusing of hepatitis B vaccine and hepatitis B immunoglobulin (HBIG), so a betterunderstanding of mechanism of intrauterine infection caused by intrauterine transmissionis critical, according to identify HBV transmission pathway(s).During the whole pregnancy, the placenta is a dynamic organ whose structure andfunction change. There is compelling evidence that the placenta plays an integral role in thevertical transmission of virus, such as HBV, HIV, CMV, and HCV, from mother to the fetus.Congenital viral infection is associated with fetal anomalies, postnatal infection, and fetaldemise. Although transplacental passage of these viruses is a critical aspect of theseinfections, few investigators have characterized placental viral infection and the role ofplacental barrier has in passage of virus from maternal to the fetal circulation. In spite ofintensive research of blood-brain barrier (BBB), a similar effort or interest in characterizingthe placental barrier does not exist yet. The emergence of in vitro models for the humanplacenta barrier will open the door for investigations of virus infection mechanisms andapply that knowledge to inhibiting, even blocking virus to pass through the placenta, thus reducing virus vertical transmission from mother to fetus. Therefore, it is necessary to seek a simple method of construction human placental barrier in vitro.Meanwhile, it has been proved that three different layers: trophoblastic cells, interstitial cells (mainly placental macrophage, i.e. Hofbauer) and human placental microvascular endothelial cells (HPMEC) of placenta are involved in intrauterine transmission from mother to fetus. And placental barrier consisted of trophoblastic cells, HPMEC and their basement membranes, is the unavoidable access of nutrients and some drugs, viruses and hormones to enter the fetus from mother. We use primary culture cytotrophoblast (CTB), human umbilical vein endothelial cells (HUVEC) and then chose three various ways to construct placental barrier: cytotrophoblast alone, HUVEC and CTB co-culture with or without contact. And we found that contacted HUVEC and CTB co-culture is the best model to mimic in vivo placental barrier construction and function. Then we used HBV copy more than 107 patients serum to infect the in vitro placental barrier, and examined the ability of infectious HBV to cross the placenta in this system.Basing on the research results on human immunodeficiency virus (HIV) and cytomegalovirus (CMV) can via receptor-mediated viral intrauterine infection and HBV is able to infect peripheral blood monocyte (PBMC) and then duplicate inside of the cells, we assumed that HBV probably through the following three approaches invading placental barrier:â’ˆmechanical injury: inflammation-caused direct or indirect trophoblastic cells and HUVEC injury or more leakage which lead to the increase of permeability of basement membrane so as to allow HBV to get through the first placental barrier (trophoblastic cells) to enter intervillus substance;â’‰cell transfer: the microfusion of HBV-infected PBMC and trophoblastic cells cause the transfer of HBV to the latter and further, the trophoblast cells release through HBV polarity into inervillus substance and then infect HUVEC;â’Šreceptor mediation: some receptors in the superior lateral membrane of trophoblastic cells might have mediated dissociative complete HBV particles into cells to duplicate and then release HBV into intervillus substance.In my project, I will focus on mechanical injury and receptor mediation hypothesis, because the receptor-mediated virus invasion is a very common invasion approach and it has been confirmed that HBV can damage the placenta in vivo. Compelling researches on HBV receptor have been carried out in the past twenty years and nearly 10 candidates of protein have been found. Currently, a high affinity receptor for HBV is still unknown. The dendritic cell specific C-type lectin DC-SIGN is involved in pathogen recognition through mannose and fucose containing carbohydrates leading to the induction of an anti-viral immune response. And DC-SIGN is exploited by many glycosylated viruses which subvert this immune surveillance function as a port of entry and for trans-infection of target cells. Also, it has been confirmed that DC-SIGN might have played an important role in the mother-to-infant transmission of HIV. Besides those distributed in premature dendritic cells (DC), DC-SIGN are also expressed in PBMC, placental vascular endothelial cells, placental macrophage and our group's preliminary data also showed that it is can be immunostained in trophoblastic cells. So far, reports have been found that DC-SIGN can serve as the receptor of HIV, CMV, Ebola virus, hepatitis C virus (HCV), SARS coronavirus, measles and etc ( no HBV-related reports have been found). The glycosylation pattern on HBV surface antigens (HBsAg) together with the tissue distribution of HBV would allow interaction between HBV and DC-SIGN. And because the N- high-mannose glycans also exists in the PreS2 antigen of HBV envelope protein, HBV has the structure base of combining with DC-SIGN. The HBV glycosylation pattern together with the cellular localization prompted us to investigate the possible role DC-SIGN in binding HBV. Also, according to the distribution of DC-SIGN in placenta, we hypothesize the possibility of DC-SIGN could be related with HBV intrauterine infection. Therefore, our project are mainly focus on the placenta barrier construction and HBV intrauterine infection model in vitro, then to further investigate the potential role DC-SIGN on HBV vertical transmission. Main results are as follows:1. Trypsin and DNAse sequential digestion method is used to separate human pregnant chorionic trophoblastic cells. By gradients of 35% and 45% Percoll noncontinuous density centrifugation method to purify separated cells to make the cell production exceed 106/ml. Then, Anti-HLA-DR Dynabeads were used for the purification and enrichment in the primary culture of human early pregnant chorionic trophoblastic cells. By using cell keratin and vimentin immunocytochemistry(ICC) staining, the positive staining rate of keratin exceeds 90% and that of vimentin is lower than 10%. The cell purity of over 90% can satisfy the purity requirements of the following experiments. Collagenase I is used for isolation of human umbilical vein endothelial cell. Then, both of the cells are contacted co-cultured to construct human placental barrier in vitro. From the functional and structural aspects, the barrier model has been identified and could be used for the following experiment.2. The direct serum infection method is used to simulate the natural HBV infection of trophoblastic cells or HUVEC separately, and then the possibility of HBV infection of human placental barrier is discussed. We find out that the HBV DNA in culture supernatant reaches the peak in 72-96 or 48-72 hours when the primarily cultured HBV infected trophoblastic cells or HUVEC is released. Furthermore, we use immunoenzyme double Staining method to determine and transmission electron microscope to identify that HBV can infect both trophoblastic cells/HUVEC and human placental barrier in vitro. Also, this barrier can block part of HBV transmission from mother side to fetal side, so HBV DNA in co-culture system outside supernatant only transiently can reach 103/ml after 24h infection. Try three different methods (traditional PCR method, Hirt method and plasmid extraction method) to detect the gold standard of infected HBV duplication---HBV covalently closed circular DNA (cccDNA). Because HBV cccDNA hasn't been detected in infected HBV human placental barrier in vitro, it is hard to conclude that HBV infection of trophoblastic cells is a transient infection or it can be duplicated within placental barrier. At least, the virus-like particle had been found in our in vitro model.3. Antibody DC-SIGN and siRNA DC-SIGN is carried out to preliminarily discuss the action of DC-SIGN in HBV intrauterine transmission. We find out that antibody DC-SIGN and siRNA DC-SIGN can significantly inhibit HBV infection in trophoblastic cells and HUVEC, but the blocking function of them are not prominent in placental barrier in vitro. Further reconstruct the receptor and use DC-SIGN plasmid transfection fetal hepatocyte to validate that DC-SIGN might have played an important role in HBV infection and it could be regarded as one of the potential receptors of HBV which is involved intrauterine transmission.In conclusion, our experiment successfully set up the primary culture system of human placental barrier in vitro and proves that HBV has the possibility of infecting through trophoblastic cells to HUVEC. Furthermore,although placental barrier can block HBV transmission from mother side to fetal side, HBV DNA still can be temporary induced in placental barrier in vitro. Also, high copies HBV can mechanical injury HUVEC, but in co-culture system this phenomena can be avoided. Preliminary research on the intrauterine transmission mechanism of trophoblastic cells and HUVEC invaded by DC-SIGN receptor-mediated HBV aims to lay down the foundation for further research on HBV intrauterine infection mechanism and to provide new guide for intrauterine intervention. Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI2, acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE2 and PGI2 in culture supernatant) in primary cultures of uterine smooth muscle cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF1-2, without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE2 acts through EP-2 to activate MAPK and increase COX2. These data provide further evidence in support of a dual role for cAMP in determining myometrial function.
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