| Throughout the world, there are about 170,000,000 people afflicted ?with hepatitis C virus, and most of them are chronic carriers. And moreover, hepatitis C uausually leads to cirrhosis and carcinoma of the liver. 3. 2% of people in our country are afflicted this disease. The typical transmission routes of HCV is through blood or blood products. After 1991, screening of donators greatly helps decrease the transmission of HCV through blood. However, the route of HCV transmission is changing now. An increasing number of children who sufferfrom HCV are not inffected through blood. The transmission of HCV in uterus , during delivery or after delivery has became an important issue which deserves further exploration. Recently, many studies has shown that HCV can be transmitted to the foetus through the placenta, and infection in uterus is one important route of HCV transmission.The trophoblast is the first line of defence aganist HCV transmitting to the foetus. In recent years, some studies have introduced the conception of antibody dependent enhancement of virus infection. This phenomenon has been found in many kinds of viruses, such as human immunodeficiency virus (HIV), influenza virus A, coxsackie virus, feline infection peritonitisvirus, and Dengue virus (DV). The antibody dependent enhancement (ADE) also plays an important role in the mother-to-infant transmission of HIV. At present, there is no report about whether HCV can be infected through ADE.In order to investigate the mechanisim of HCV transmitted in uterus, we conducted following studies:Firstly, we isolate and cultured trophoblasts in vitro. To isolate cytotrophoblasts from Villus and to observe their biological characteristics, we prepared cytotrophoblasts from human Villus decomposed by trypsin, and centrifuged it on two layers (35% and 45%) of Percoll gradient. Biological characteristics were observed by immunohistochemistry and transmission electron microscope. More than 90% trophoblasts were obtained by improved means. Under the transmission electron microscopy, we observed three different mature stages of cytotrophoblasts. Higher purity trophoblasts could be obtained by this improved method. We observed positive signal of keratin and CD16(FcrR III) and negative signal of vimentin in cytotrophoblasts. This is the cytologyic experimental basis for studying the mechanism of mother-to-infant transmission of HCV.Secondly, the cultured cytotrophoblasts were incubated with a HCV RNA positive serum. Under the immunoelectron microscopy ,we observed the HCV viral particles, whose envelope protein labeled by SPA-colloidal gold, in those cells. The size of the particles was similar to that of the hepatitis C virus. The ultrastructure of the infected throphotoblast cells differed obviously from that of normal cells, and characterized by hyperplasia of lysosomes and rough endoplasmic, appearance of vacuoles, and virus-like particles, and decreased lipid droplets. The study provided direct morphological evidence for the mather-to-infant transmission of HCV and academic basis for the explanation that HCV enters cytotrophoblasts by ADE mechanism.Thirdly, we incubated the cytotrophoblasts with four different methods. Cells group A were infected by HCV RNA positive serum and those of group B by the same serum which pre-incubated under 56℃ for 30 min ; cells of group C were incubated with CD16 McAb under 37℃ for 60min by the same serum, and cells of group D by normal serum. These cells were cultured for 21 days. Qualitative RT-PCR method and immunohistochemistry were employed to assay the expressions of HCV NS5 and NS3 and HCV-C antigen and HCV RNA in cells or suspendant. The HCV RNA and reverse RNA were only intermittently detected in the culture medium and the cells. The expressions of HCV NS5 and NS3 and HCV-C antigen only was observed in group A. HCV entered the cytotrophoblasts through both FcR-ADE and complememnt-ADE paths.Anti-HCV and/or complement probably enhanced the replication of viral particles.
|
PDF Full Text Request