Identification Of Cultured Human Trophoblasts In Vitro And Investigation Of Trophblasts Infected By HBV

Hepatitis B is a worldwide serious disease caused by hepatitis B virus (HBV). Statistics show that there is a total of more than 120 million chronic asymptomatic HBV carriers (AsC) in China today. The virus can be transmitted from mother to child (mathernal-fetal transmission, MFT) and the AsC infected in this way account for 70% of the total infected. Intrauterine infection is one of the main routes of MFT infection, caused by the HBV through the placental barrier. Its transmission mechanism has been unknown yet. Only a few studies have been conducted in this field abroad and home. The two hypotheses made by the Department of Epidemiology of the Fourth Military Medial University: "hematogenous transmission"and "transplacental cellular traffic". According to the transplacental cellular traffic hypothesis, the HBV is transmitted from the mathernal uterus mucous membrane to the fetal villous capillary endothelial cells through the villous trophoblasts and thevillous interstitial cells, respectively. Therefore, it is crucial to make clear the infection mechanism of HBV in the fetal villous trophoblasts for the recognization of the mechanism of intrauterine infection. It is impossible to investigate the dynamic course of virus infection in vivo, so human villous trophoblasts should be cultured in vitro. It involves an adequate culture method and the establishment of an in vitro culture system, which are needed to obtain sufficient and pure trophoblasts. No satisfactory trophoblasts have been obtained in China so far. In the present study, the purified and subcultured early villus cells were observed and identified by their general cellular morphology, cytoskeleton and hormone secretion. The identified trophoblasts were infected with HBV positive serum and the morphological changes were observed.Methods:1. The purified and subcultured villus tissue, which had been isolated from the hosts of a 6 to 10 weeks' pregnancy by the trypsin digestion method, was cultured. The 50th generation of the cultured cells was employed in the present study. The general cellular morphology was observed with an inverted phase contrast microscope. The nucleus and cytoplasm structure was observed with haematoxylin and eosin (HE) staining. The cells' surface structure was observed with scanning electron microscope (SEM). The cellular ultrastructure was also observed with transmission electron microscope (TEM). The immunofluorescence histochemistry combined with laser scanning confocal microscopy (LSCM) was employed to observe the expression of cytokeratin 18 (CK18), vimentin (Vim), β -human chorionic gonadotrophin (β -hCG) and human placental lactogen (hPL) in the cultured cells.2. The identified cells were incubated in HBV positive serum. The incubation was stopped after different periods of time (Oh, 15min, 30min,1h, 2h, 6h, 12h, 24h, 48h), the cellular morphology was observed , the ultrastructural changes cytoskeleton expression were detected with TEM and LSCM.Results:1. The cellular morphology:1) Light microscopyAs shown by inverted phase contrast microscope, the cells appeared irregular polygonal in shape and grew in a monolayer flaky way. They have abundant cytoplasm with one or more large oviform nuclei. HE stainning showed that the cells appeared polygonal in shape with blue-violet nucleus, pink cytoplasm and sometimes multinucleate giant cells. Mitotracker stain combined with LSCM showed that abundant mitochondria were present in cytoplasm.No obvious morphological changes were observed in cells being infected with HBV, the cells also appeared irregular polygonal. The shrinkage was found in the cells by HE stainning , the secretion substances were observed in a lot of cells and increased as the time of incubation of the cells in HBV positive serum.2) Electron microscopyAs shown by SEM, the cells grew in a spread monolayer way with abundant microvilli on the surface. The desmosomes were found between the cells when observed with TEM, There were a great number of lipid droplets, mitochondria, developed Golgi complex, glycogen and dissociative ribosome in cytoplasm.No obvious change was observed with SEM in the cellular morphology of the cells being infected with HBV. The cells still gathered in flakes with a few of microvilli on their surface and some of them present as an early apoptosis-like change. As shown by TEM, the cells...