| Hepatitis B is one of most severe worldwide diseases. There are about 120 millions HBsAg carriers in China. Although some vaccines and many drugs have been developed for nearly 20 years, vaccination is not effective for about 5%~10%, and attempts at treatment of chronic infections have had only limited success. Recently the public sanitation improvement has reduced the risk of horizontal and perinatal transmission. However, effective methods for prevention of hepatitis B intrauterine infection are still not available. Once the intrauterine infection is installed, the neonate will develop to chronic hepatitis probably and become a harmful carrier. Research HBV infection molecular mechanism is the primary foundation to find more effective vaccines and drugs.It is widely accepted HBV invade into host cells by "receptor(s)" on cell surface. Although 10 or more candidate proteins have been found in nearly 20 years, but the exact property of HBV receptors has not been affirmed. This fact suggests, on the one hand, strategy to looking for receptors need improvement; on the other hand, there are possible more than one molecule concerned with HBV invade. Intrauterine infection of HBV need specific receptors too and fetal hepatocytes is the major target cells. It has been found intrauterine infection is rare in the first trimester, but infectious rate rise significantly in the second and third trimester. We suppose early fetal hepatocytes do not express HBV receptors due to low differentiation. With pregnancy prolong, fetal hepatocytes differentiate to adult hepatocytes phenotype and express HBV receptors. HBV then invade by these receptors and replicate. Based on above-mentioned assumption, we design 10 kinds of culture condition with adding differentiation-induce ingredients to serum-free medium. These ingredients includes epidermal growth factor, hepatocyte growth factor, dimethyl sulfoxide, phenobarbital sodium and all trans-retinoic acid. We isolate and culture primary 10-weeks-old human fetal hepatocytes, observe hepatocytes form and its function markers. Inoculate hepatocytes with HBV Dane particles to observe if HBV susceptibility of fetal hepatocytes can be induced in vitro. To detect HBV covalently closed circular DNA(cccDNA) within hepatocytes, weimprove the existent PCR method. Based on previous experiments, we create double-subtraction strategy and compare the difference between hepatocytes mRNA populations which extracted before and after HBV susceptibility occurrence. Screen the different genes to get the HBV infection-associated molecules.Major results:1. Combination of mung bean nuclease(MBN) selective digestion and PCR specific amplification is a simple, fast, sensitive and specific method for detection of HBV cccDNA.To design PCR primers striding over single chain region of HBV relaxed circular DNA(rcDNA) with reference to abroad reports. Experiments show the primers cannot distinguish cccDNA and rcDNA with more than 104 templates molecules. To digest templates with single chain nuclear acid specific MBN, we can amplify correspondent bands with 105~102 cccDNA templates after digestion. On the other hand, we cannot amplify any bands with 105 rcDNA templates after digestion. The results suggest MBN pretreatment can improve the PCR specificity significantly.2. Phenobarbital sodium induces differentiation and susceptibility to HBV in primary culture of early human fetal hepatocytes. Isolate 10-weeks-old human fetal hepatocytes with non-perfusion method and the cells viability is good. The Ricin A subunit treatment can purify hepatic parenchymal cell effectively. Based on serum-free medium designed by our self, we create 10 culture conditions. we find 2.5mM phenobarbital sodium(PB) can induce typical hepatocytes polygon form after 10 days culture. PB inhibit the fetal-specific gene such as alpha fetoprotein(AFP) and gamma glutamyl transferase(GGT), and facilitate expression of the adult hepatocytes-specific genes such as albumin(ALB) and alanine aminotransferase(ALT). The results... |
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