Expresstion Of DC-SIGN, DC-SIGNR In The Placenta Chorionic Villi And Primary Trophoblast Cells And Its Significance

At present, several viruses, such as human immunodeficiency virus (HIV), heptitis B virus (HBV), heptitis C virus (HCV), cytomegalovirus (CMV), herpes simplex virus (HSV), are the cause of intrauterine infection that not only impact prepotency but also involved in public health.Placenta is a main factor of intrauterine infection. The placental barrier are constituted by trophoblast cells, human placental microvascular endothelial cells (HPMEC) and their basement membranes. Placenta plays an impotant role in regulating the exchange of various materials between the maternal and the fetal circulations. Trophoblast cells, as the outermost covering of placental barrier, are bathed in maternal blood and contact with viral particle directly, might be the key location for virus invasion into placental. Therefore, we focus on the trophoblast cells.DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin, also termed DC-SIGN) and its homologue DC-SIGNR (DC-SIGN-related molecule, also termed DC-SIGNR), are thought to play an important role in establishing the initial contact between dendritic cells (DCs) and resting T cells, as well as facilitating intrauterine infection of HIV. DC-SIGN and DC-SIGNR can bind to various carbohydrate, by which we can anticipate the pathogen relevant to both of them. DC-SIGN and DC-SIGNR can serve as receptor of HIV, HCV, CMV and etc. so far, there are no reports about DC-SIGN and DC-SIGNR related to HBV. Interestingly, PrS2 antigen of HBV envelope protein posses N-high-mannose glycans-the common structure base by which virus can combine to DC-SIGN and DC-SIGNR. It suggested that DC-SIGN and DC-SIGNR might be novel receptor of HBV. The expressiones of both DC-SIGN and DC-SIGNR in the placenta have important implication for intrauterine transmission of virus (no reports about their expression on trophoblast cells). Whether virus invade tropholast cells through the receptor-mediated pathway? Receptor existing in the cell surface is the basic factor of receptor-mediated infection, and is the problem we focused on .In the experiment, we primarily cultured chorionic trophoblast cells and observe their biological characteristics; studied the expression of DC-SIGN and DC-SIGNR in human placenta and trophoblast cells cultured in vitro; and provided a basis for experiment in vitro aiming to investigate the mechanism of receptor-mediated intrauterine transmission.Main methods and results are as follows:1. Trypsin and Dnase I sequential digestion method is used to isolate human chorionic trophoblast cells . By gradients of 35% and 45% Percoll noncontinuous density centrifugation method and changing culture plate step to purify isolated cells. The isolated cells are resuspended in DMEM containing 20% fetal bovine serum. The cells are plated at density of 5×10⁵/ml on coverslips in 12-well plates, gassed with 5% CO₂ in air at 37℃. By using trypan blue staining method, the survival rate of cells exceeds 95%. By using cell keratin and Vimentin immunocytochemistry (ICC) stain, about 90% cytokeratin positive cells obtained from our method. The cell purity satisfies the requirement of following experiments.2. The expression of DC-SIGN and DC-SIGNR in the placenta and cells cultured in vitro is measured by Immunohistochemistry(IHC) stain, reverse transcription polymerase chain reaction (RT-PCR), Western-blot. DC-SIGN is mainly expressed in the nucleus of placental trophoblast cells and placental vascular endothelial cells, in the membrane of Hofbauer cells; DC-SIGNR is mainly expressed in the membrane and plasm in placental trophoblast cells, Hofbauer cells and placental vascular endothelial cells. DC-SIGN, DC-SIGN mRNA and protein are detected in the placental trophoblast cells.In conclusion, primary culture system of human chorionic trophoblast cells are established. The expression of DC-SIGN and DC-SIGNR is determined by IHC, RT-PCR and Western-blot analysis. The study provides a cellular basis for experiment in vitro aiming to investigate the mechanism of receptor-mediated intrauterine transmission. Hurther investigation is required to prove whether DC-SIGN and DC-SIGNR mediate virus invading into trophoblast cell and infecting the fetus ultimately.