Immunity Studies Of HSP 70 Complex On Anti-human Hepatocellular Carcinoma

Primary liver cancer was one of malignant tumors in the world.The patients were often found in moderate or late stage, and lost the chance for treatment. Even early diagnosed and treated, it still remained a high rate of recurrence and metastasis. Thereafter, a general therapy for hepatocellular carcinoma was to carry out a plan including surgery, chemotherapy, radiotherapy and biotherapy. Heat shock proteins (HSPs) were extremely conserved family proteins which were expressed in various living organisms. Many HSPs were constitutively expressed at a certain content and played an essential role in assisting the refolding of damaged proteins or facilitating its degradation. It has been verified that either viral transformed or chemically induced tumor cells could express HSPs in certain levels. When the cells were stressed, its HSPs expression increased obviously. The high expression of I-ISPs was not only the demand for tumor cells?unlimited proliferation and metabolisms but also the results of oncogene products functioned corordinarily each other. It was showed that HSPs could interact with oncogene protein mainting the tumors proliferation and malignant characters. Owing to the 搈olecule chaperone?effect, HSPs from high expression tumor cells or HSPs-peptides complex purified from tumors could in vivo and in vitro induce specific CTL response, engendering tumor rejection effect. Therefore,there are many experimental or clinical application of HSPs in the immune therapy of tumors.A few examples of HSPs vaccination against animal liver cancer were found. No work has been found concerning hepatocellular carcinoma of human origins. In order to study the different expression of HSP7O in human hepatocarcinoma cell lines, HSP7O was detected by imniunocytochemistry, immunofluorescence and FCM. The results showed that different hepatocarcinoma cell lines expressed HSP7O in minimal amount and increased -5- expression after heat treatment. Kinetic study revealed that in different period of heating, HSP7O moved from cytoplasms to nucleus and then from nucleus to cytoplasms again. Based on the finding, HSP7O of SMMC-7721 was purified through chromatographies at its peak of increase about 1 6h after heat treatment. The quality of HSP7O was identified by SDS-PAGE and western Blot. In vitro human splenic cells were sensitized by purified I-ISP7O complex and then co-cultured with human hepatocarcinoma cell lines. The sensitized human splenic cells showed a definite cytotoxity to human hepatocarcinoma cell lines at different effector: target ratios. Blocking test revealed that anti-HSP7O almost completely and anti-CD4, CD8 partially blocked the cytotoxity of the sensitized splenic cells. Then, 2 hepatocarcinoma patients were recruited. The HSP7O in their cancer tissues were proved positive by immunohistochemistry. By way of immuno- precipitation and chromatographies, purified HSP7O was obtained and injected intradermally on the forearm of the patients. The results showed, comparing the cytotoxicity of the PBL of the patients to their own tumor cells before and after immunization, the cytotoxicity of PBL after vaccination increased markedly. To further understand the 揵uilt in adjuvant?effect of I-ISP7O, in vitro AFPIHSP7O eukaryotic expression vector was constructed, which was verified could secret fusion protein temporarily in SP2/O cell line. By way of naked gene DNA immunization in Balb/c mice, a definite cytotoxicity effect...