Research On The Mechanism Of Cannabinoids HPV Infection Of Cervical Cancer And Establishment Of Hepatitis B Virus Chronic Infection Mouse Model

Background and ObjectiveVirus is composed of a nucleic acid molecμle (DNA or RNA) and proteins or proteins only. Generally speaking, According to different viral genetic material, Virus can be divided into DNA virus, RNA virus and protein virus, DNA viruses can only infect an animal (a few exceptions). It is found that virus has not only the pathogenic but also the carcinogenic in Modern Medicine. By experimental study of modern, It is shown that the occurrence of cervical cancer has a relationship with HPV infection, HCC has a relationship with HBV infection. Cervical cancer is second only to breast cancer in women of common malignant disease. According to available statistics, the number of cervical cancer deaths is nearly 200,000 in worldwide each year. About the number of cervical cancer, There are up to 510,000 new cases in Global and more than 100,000 in China each year. It has more than 100 million carriers of hepatitis B virus while liver cancer deaths about 30 million people every year in China. In patients with chronic hepatitis, apart of the patient’s liver fibrosis will occur, and development of cirrhosis, liver cancer in further.Cannabinoids are a group of terpene phenolic compounds from the Cannabis indica, and found in animal nervous system and the immune system. Cannabinoid receptors are Basis of biological effects of cannabinoids. The study found that cannabinoids can inhibit breast cancer, prostate cancer, glioma, etc. Increasing the secretion of cannabinoid receptors may selectively inhibit the proliferation of malignant cells in tumor tissue. Other studies found that cannabinoids can influence the process of liver pathological changes in patients of chronic HBV infection.Present study aims to construct a eukaryotic expression vector containing CB1 and CB2 gene and select the human kidney embryonic cells (HEK293) as a biological carrier and research the biological characteristics of cannabinoids. Explore its influence apoptosis of human cervical cancer and its related mechanisms. To establish a mouse model of a chronic viral hepatitis induced by HBV cccDNA and study the influence of cannabinoid on the hepatitis pathological changes and its mechanism. For the study about biological activity of the CB gene and its influence of on cervical cancer and mechanisms, it provides the experimental evidence. It provides an animal model to study the effects of methanol to HBV induced liver injury in further.Methods1. The target gene was amplified by PCR.2. The CB eukaryotic expression vector was constructed by double enzyme digestion, transformation, plasmid extraction. And identify recombinant plasmid by double digestion method.3. The CB eukaryotic expression vector was transfected into HEK293 cells by liposomes and the expression and cellular localization of CB Protein was detected by cellμlar Immunofluorescence laser scanning confocal microscop.4. The CB eukaryotic expression vector was transfected into CaSki cells by liposomes. The apoptosis rate of CaSki cells was detected by flow cytometry.5. The mRNA and protein expression of CBR, Bcl-2, Bax and Bad was examined by real-time fluorescent quantitative PCR (qRT-PCR), and to study the mechanisms about CB on cervical cancer.6. The HBV gene was amplified by PCR. HBVcccDNA was obtained by enzyme digestion, purification, cyclization and other biological methods.7. HBV cccDNA was injected in C57BL/6 mice by hydrodynamic injection. After injection, Serum samples and liver tissues of the mice were collected in different time points(1d、3d、1w、2w、3w、4w、5w、6w、 7w、8w、9w、10w), and liver tissues of the mice were collected in 3w、6w、10w after the mice were sacrificed by cervical dislocation.8. Radioimmunoassay was used to examine the levels of HBsAg and HBeAg. Real time PCR was used to determine the copy numbers of HBV DNA in both serum and the liver. Immunohistochemistry was used to examine the levels of HBsAg and HBcAg in the liver. HE staining was used to checked pathological analysis of the liver tissues.Results1. The fragment of rCB1 (1.5Kb), CB1 (1.5Kb) and CB2 (1.1Kb) were obtained after the PCR products were electrophoresed.2. By double enzyme digestion and electrophoresis, The fragment of 5.3 Kb and 1.5 Kb was obtained in pCDNA3.1(+)-CB1 plasmid, the fragment of 5.3 Kb and 1.5 Kb was obtained in GV230-CB1 plasmid, the fragment of 5.3 Kb and 1.1 Kb was obtained in GV230-CB2 plasmid.3. After transfected into HEK293 cells, the expression of CB protein in cytoplasm and cellμlar membrane.4. After transfected into CaSki cells, the apoptosis rate of CB group was increased compared with the blank group.5. By real-time fluorescent quantitative PCR(qRT-PCR), it was shown that bad and bax was upregμlated, Bcl-2 was down-regμlated.6. The fragment of 3.2Kb HBV was obtained by PCR, the cyclization of HBVcccDNA was got by BspQ I enzyme digestion, purification and T4 DNA ligase.1. By reflection immunoassay with the mice blood serum of different time points, the evels of expression of HBsAg and HBeAg all showed four increasing-decreasing patterns in experimental groups. By the real time PCR, it sμggested that the copies of HBV DNA were reduced with the growth of the time. By immunohistochemistry, the expression of HBsAg and HBeAg were positive in the liver tissue of mice. By HE staining, the liver tissue of mice were found the symptom of liver cell inflammation, fibrosis and necrosis.Conclusion1. The CB eukaryotic expression vector is constructed successfμlly, it was confirmed that the expression of CB in the cytoplasm and cell membrane.2. CB can promote the apoptosis of cervical cancer CaSki cells.3. The mechanism of CB in promotes cancer cell apoptosis is up-regulating the expression of Bax and Bad, and down-regμlating the expression of Bcl-2.4. A HBV chronic infection mice model was successfμlly established.