Derivatization Of Fusion Protein And Establishment And Identification Of Animal Model On Human Metastatic Hepatocelluar Carcinoma

The metastasis is one of the biological characteristics of malignant tumor, and it is also difficult to be cured. It is reported that, 60% of malignant tumor has metastasized before the initial diagnosis. In the clinic, about 80%-90% die from invasion and metastasis. That is to say, metastasis may be the main reason for the death of most malignant tumor patients. Because of the characteristic of the liver tumor, it is apt to metastasize. So it is one of the reasons for the unfavorable prognosis. Atuopsy showed that there is 50%-84.6% metastasis existed in the hepatic cellular cancer's body. The main therapies for the liver cancer include operation, radiotherapy, chemotherapy and Chinese traditional medicine etal.With further knownledgement in tumor immunity theory and emerge of new experiment technology,tumor vaccines have become one of the most attractive methods to prevent the metastasis of liver cancer. The Melanoma Antigen (MAGE) is the earliest discovered human specific Antigen. The Melanoma associated antigen-1 (MAGE-1) was the first reported immunologic tumor rejection antigen discovered by boon in 1991, which is one of important numbers in the MAGE family.It is expressed in most types of malignant tumor except testicle and placenta. Furthermore, MAGE-1 can cohere to human leukocytes antigen (HLA) molecules to form antigen compounds and this antigen compounds can be recognized by the tumor cell receptor (TCR) of cytotoxic T lymphocytes (CTL). The recognition will lead to specific CTL to kill tumor cells. So MAGE-1 has been used as an ideal target molecule for tumor immunotherapy. As molecular chaperone, heat shock protein (HSP)take part in processing and presentation of tumor antigen and plays an important role in arousing antitumor immunity .Our researches have demonstrated that fusion protein of MAGE-1 and HSP70 can enhance tumor vaccines efficiency.In order to offer the animal model for the protective effects against liver cancer metastasis by Nanoliposome-encapsulated Tumor Specific Antigen Protein Vaccine of fusion protein of MAGE-1 and HSP70, this research used Human peripheral blood lymphocyte(huPBL)and high Metastatic hepatocellular carcinoma cell 97 (MHCC97) established immune reconstruction human metastatic hepatocelluar carcinoma model and indeficated it. The research was divided into three parts as following:Experiment 1: The MAGE-1 protein expression in the MHCC97-H cells Objective: To detect MAGE-1 antigen expression in the MHCC97-H cells,lay a basis for the experiment of protective effects against liver cancer metastasis. Methods: Using the methods of Immunohistochemistry, immunofluorescence, and western blotting to detect the MAGE-1 antigen expression in the MHCC97-H cells. Results: The MAGE-1 antigen expresses in the MHCC97-H cells. The result of the Immunohistochemistry, immunofluorescence, and western blotting are coherent. Conclusion: The MAGE-1 antigen's expression in the MHCC97-H cells offers the basis for the experiment of MAGE-1 and HSP70 fusion protein (M1H) protective effects against liver cancer metastasis. Experiment 2: The derivatization of MAGE1-HSP70 fusion protein Objiective: To make the macromolecule protein carries more dosage.Methods: The recombinant fusion protein was cross linked with Palmitic acid by chemical methods. The purified recombinant fusion protein was esterified and then was wrapped up with nano-liposome.Results: The derivatization of MAGE1-HSP70 fusion protein was prepared successfully.Conclusion: The derivatization of MAGE1-HSP70 fusion protein lays a basis for the experiment of protective effects against liver cancer metastasis.Experiment 3: Establishment and identification of HU-PBL-SCID mice model of human high metastatic hepatocelluar carcinomaObjective: To explore the effective methods of establishing a human metastatic hepatocelluar carcinoma model in human peripheral blood lymphocyte (hu-PBL) engrafted to severe combined immunodeficient (SCID) mice. Methods: the immunological features of mice were evaluated after intra-peritoneal injection of hu-PBL and subcutaneous implantation of human high metastatic hepatocelluar carcinoma cells (MHCC97-Hs).Results: (1) subcutaneous tumors developed in all the mice given hu-PBL and MHCC97-Hs. The latency period was significantly prolonged, and the tumor size was marked depressed, as compared with mice given human MHCC97-Hs alone (p<0.05). The tumor's live metastatic rate in the mice given human MHCC97-Hs alone was 82.5 %(5/6) .While in the mice given hu-PBL and MHCC97-Hs was 67%(4/6). The tumor's metastatic time needed in the mice given hu-PBL and MHCC97-Hs was longer than the mice given human MHCC97-Hs alone(p<0.05). (2) In the 2nd, 4th and 6th week, the amount of the human IgG in the mice given hu-PBL and MHCC97-Hs was lager than the mice given MHCC97-Hs alone(p<0.05). (3)In the 6th week, the number of the human CD3+ T lymphocytes and CD20+ B lymphocytes in the mice given hu-PBL and MHCC97- Hs was lager than the mice given MHCC97-Hs alone(p<0.05). (4)Immunohistochemical staining revealed presence of remarkable human CD3+ T lymphocytes and CD20+ B lymphocytes in SCID mice's spleen. Conclusion: A human high metastatic hepatocelluar carcinoma model has been established hu-PBL engrafted to SCID mice, which is an ideal animal model for preclinical research and treatment for metastatic of hepatocelluar carcinoma.In this study, the Nanoliposome-encapsulated Tumor Specific Antigen Protein Vaccine NL (M1H) could protect hepatocelluar carcinoma metastasis which express MAGE-1.It may develop a new route to design a more efficient and safe tumor vaccine. And this study will lay a basis for the further development of the vaccine.