| For solid tumor patients undergoing high-dose chemotherapy sustained by peripheral blood stem cell transplantation (PSCH), it is crucial to improve immune reconstitution and get rid of remaining cancer. Gene therapy and irnmuotherapeutic approaches hold great promise as effective weapons against the recurrence of tumor. The Fas/Fas ligand (FasL) system plays an important role in the transduction of apoptotic signal into the cells. Within several hours after the engagement of target cells Fas by effector cells FasL, target cells undergo cell death by way of the Fas-mediated apoptosis. In recent years, numerous studies have demonstrated that Fas is expressed on the surface of cells of various types, whereas FasL expression is restricted to a small number of cell types, such as lymphocytes, cells of the immune-privileged organs and many types of malignant tumor cells. Meanwhile, evidence has pointed to an abnormal increase in apoptosis among activated Fas-positive lymphocytes, mainly in the periphery of the FasL-expressing tumors. On the other hand, to a great extent, the occurrence of tumor is due to the fact that the converted cells can not undergo a normal process of apoptosis. Therefore, a research about how to disarm the tumor's Fas counterattack and how to promote tumor cell apoptosis will provide new approaches to the therapy of tumors. A better understanding of FasL function will help to fully realize the potential of the FasL gene therapy and even offer a potential therapeutic intervention.According to earlier literature, resistance to apoptosis through the Fas receptor pathway coupled with expression of the Fas ligand might enable many cancers to deliver a pre-emptive strike or counterattack against the immune system. Disarmingthe Fas counterattack is a conceptually appealing and exciting potential goal for tumor immunotherapy. Our research is about the interaction in vitro between T cells that express Fas and tumor cells that express FasL. The approach of this project is to block the expression or function of FasL on the tumor cell by antisensing FasL, with a view to abrogate the Fas counterattack and prolong TIL's life span, which will ultimately regulate immune recovery and enhance antitumoral efficacy.Part I The study on tumor cells escaping from Fas-mediated apoptosisTo investigate the possibility of colon cancer cells escaping from immune surveillance through the expression of FasL on tumor cells, a study was undertaken by using immunohistochemical SABC method. The expression of Fas receptor and Fas ligand in SW620 colon carcinoma cell line and Jurkat T cells was observed so as to supply morphological evidence for the functions of Fas receptor and Fas ligand. It was shown that the Fas ligand of colon carcinoma SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm while the nuclei of the cells were negative, and the Fas ligand of Jurkat T lymphocytes turned out to be positive. Meanwhile, it was also shown that the Fas receptor of Jurkat T cells was positive. The positive substances were distributed in the cell membrane and cytoplasm while the nuclei of the cells were negative, and the Fas receptor ofcolon carcinoma SW620 cells was negative. In an effort to examine the cytotoxicity of effective cells, we adopted CytoTox 96 Non-Radioactive Cytotoxicity Assay to measure LDH releasing value after the SW620 cells were co-cultured with the Jurkat T lymphocytes. At the same time, we also measured the values of Effector Cell Spontaneous LDH Release, Target Cell Spontaneous LDH Release, Target Cell Maximum LDH Release, Volume Correction Control and Culture Medium Background. Then the following formula was applied in the calculation of percentcytotoxicity: Cytotoxicity (%) = (Experimental- Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous) X 100. The result indicates that FasL expression in the colon carcinoma SW620 cells could inversely induce apoptosis of Fas-expressing Jurkat T lymphocytes.Part...
|
PDF Full Text Request