| Background According to the epidemiological data, there are about 220 million carriers with hepatitis B virus (HBV) in the world, and about 120 million, around 10% of total population in China. HBV possesses strong hepatotropism, but the scientists found the presence of HBV in some tissues beside liver such as pancreas, kidney, skin, heart, bone marrow, lymphocytes and male germ cells in free and integration types. The existence of HBV in cells may result in persistent infection. The integration of HBV DNA sequence can induce variety of chromosomal aberrations that may contribute to carcinogenesis by activating oncogenes. The governments and scientists in the world pay high attention to the transmission, mechanism and methodology for diagnosis and treatments of hepatitis B infection because of its high prevalence and harmful to human health.Purpose and Significance Cytotoxic T lymphocytes play a very important role in clearance of acute and chronic HBV infections. The integration of HBV DNA in chromosomes of spermatozoa and oocytes may result in the potential transmission of HBV from parents to offspring. So the further study on the presence and integration of HBV DNA in lymphocytes, spermatozoa and oocytes are very important in basic research and clinicalpractice for prevention and treatment of hepatitis B.Material and Methods Materials: 1. Lymphocytes and spermatozoa of the cases with chronic hepatitis B infection; 2. mouse oocytes; 3. pBR322-HBV DNA. Methods: 1. PCR and electrophoresis were performed to detect the presence of HBV DNA sequence in lymphocytes and spermatozoa. 2. Lymphocyte metaphases after peripheral blood cultures were prepared. Then FISH was carried out to detect the integration of HBV DNA sequence in lymphocyte chromosomes. 3. Co-cultures of mouse oocytes with pBR322 -HBV DNA plasmids were performed and the entering of HBV DNA into the oocytes and its integrating in host chromosomes were demonstrated using PCR, dot and Southern hybridizations and FISH techniques.Results 1. The positive bands of HBV DNA were observed in both of lymphocyte and sperm samples from seven of twenty cases with chronic hepatitis B infection. Two cases showed the positive results in lymphocyte samples and the negative in sperm. Three cases showed the negative results in lymphocyte samples and the positive in sperm. Eight cases showed the negative results in both of lymphocyte and sperm samples. 2. The integration signals of HBV DNA sequence were observed in 21 of 2,000 lymphocyte metaphases and its rate was 1.05%. 3. Each of oocyte samples washed eight times after co-cultures finished. Using dot hybridization with HBV DNA probe, the negative results were found in the samples of the washing solutions of the last three times in each experiment. The positive bands of HBV DNAwere seen in co-cultured oocyte samples using PCR and electrophoresis techniques. The positive bands were observed in all samples of co-cultured oocytes using Southern hybridization techniques. FISH of 638 metaphases of co-cultured oocytes with HBV DNA probe showed the clear signals, and its integrated rate was 0.8 % .Conclusion 1. HBV can enter into lymphocytes and sperm cells of the cases. When HBV DNA was tested by PCR in the same case, the lymphocyte sample showed positive result but the sperm sample may show negative, and vice versa. So the experimental tests in both lymphocyte and sperm samples are required for diagnosis, and the different strategies should be made for treatment according to differently tested results. 2. HBV DNA can integrate in the lymphocyte chromosomes. The HBV integration should be considered while clinical treatment because it could influence the functions of the lymphocytes. 3. HBV can pass through zona pellucida to enter into the cells and to integrate in the chromosomes during co-culture with oocytes. Our work established the experimental model in vitro for study on oocyte capturing viral genes. This model lays a foundation for investigating a possible vertical transmission of HBV from mother to in...
|
PDF Full Text Request