Objective： To block the apoptosis of T lymphocytes induced by Fas/FasL in order to establish a method of the large-scale preparation of large amounts of tumor-specific cytoxic T-lymphocytes （CTL）. Methods： Liver c...Objective： To block the apoptosis of T lymphocytes induced by Fas/FasL in order to establish a method of the large-scale preparation of large amounts of tumor-specific cytoxic T-lymphocytes （CTL）. Methods： Liver cancer cells and tumor infiltrating lymphocytes （TIL） were isolated from FasL positive fresh specimens, and co-cultured. Specific CTL were activated and prepared in the presence of the co-stimulation of monoclonal antibody CD28. Then the blocking and activation of apoptosis of T lymphocytes was activated by soluble Fas receptor, which was detected by cytometry and DNA ladder test simultaneously. The apoptosis-blocking effect was compared with the control group. Furthermore, the changes of T cell proliferation and killing activity were detected by the method of ^3H thymidine incorporation and ^51Cr release test. Results： There was a significant increase in apoptosis rate in unblocking group compared with blocking group and quiescent group, with the unblocking group of 47.82%±0.13%, quiescent group of 3.76%±0.25%, and the blocking group of 8.22%±0.26% respectively （P〈0.01）. T cell-ladder appeared in unblocking group by DNA ladder test. Both the killing ability and proliferation rate of T cells were significantly increased after blocking. There was significant difference among blocking group, unblocking group and quiescent group （P〈0.01）. Conclusion： With this method we obtained large amounts of tumor-specific T lymphocytes, which was able to kill liver cancer cells effectively.展开更多
Objective:To further illuminate a possibme mechanism of Fas/FasL in the treatment of psoriasis, the expression of it and apoptosis of KC were investigated. Methods: With cell culture,immunocytochemistry, the expressio...Objective:To further illuminate a possibme mechanism of Fas/FasL in the treatment of psoriasis, the expression of it and apoptosis of KC were investigated. Methods: With cell culture,immunocytochemistry, the expression of Fas/FasL protein after the treatment with etretin was observed in cultured human normal keratinocytes. Then, the state of apoptosis in cultured keratinocyte after stimulation with etretin was detected with FACS(Fluorescence Activited Cell Sortor). Results:① There was no expression of Fas/FasL protein in the cultured normal human KC. ②After the treatment with etretin, the strongest expression of FasL protein appeared after 16h,so did Fas after 40h.③ The higher the concentration of etretin,the stronger the expression of Fas/FasL protien.Under the same condition,the expression of Fas is stronger than that of FasL.④ Keratinocytes' apoptosis occurred after stimulation with etretin. Conclusion: Fas/FasL system wasn't involved in apoptosis in cultured normol human keratinocytes.But during limited period, the apoptosis of KC could be induced by (etretin),thus it can antagonize benign proliferate of keratinocytes.Our data showed up-regulation of the expression of Fas/FasL and apoptosis in cultured human keratinocytes stimulated by etretin, and its function may be involved in the therapeutic machanism of psoriasis.展开更多
The host immune system functions as a guardian against tumor development. It has been demonstrated that cytotoxic T lymphocyte (CTL)-mediated cytotoxic pathways function to inhibit or delay human colorectal cancer dev...The host immune system functions as a guardian against tumor development. It has been demonstrated that cytotoxic T lymphocyte (CTL)-mediated cytotoxic pathways function to inhibit or delay human colorectal cancer development. However, the host anti-tumor immune responses also 'edit' the tumor and select for more aggressive variants, resulting in immune evasion and tumor escape. Fas is a death receptor that mediates one of the major cytotoxic effector mechanisms of the CTLs. Fas is highly expressed in normal human colon epithelial cells but is frequently silenced in colorectal carcinoma, especially in metastatic colorectal carcinoma, suggesting that loss of Fas expression and function may be an immune evasion and tumor escape mechanism. In addition, recent studies indicated that Fas also mediates cellular proliferation signaling pathways to promote tumor development. Therefore, the death receptor Fas may not only transduce death signals to suppress tumor development but also activate cellular proliferation and the migration process to promote tumor growth and progression. Thus, understanding the mechanisms by which the Fas receptor and its associated protein complex transduces the death and survival signals may identify molecular targets for the development of therapeutic strategy to enhance the Fas-mediated death signals to increase the efficacy of cancer immunotherapy.展开更多
To study the significance of FasL expression in immune escape of colorectal carcinoma, FasL protein expression and the number of tumor infiltrating lymphocytes （TILs） in 80 specimens of colorectal carcinoma were det...To study the significance of FasL expression in immune escape of colorectal carcinoma, FasL protein expression and the number of tumor infiltrating lymphocytes （TILs） in 80 specimens of colorectal carcinoma were detected by immunohistochemitry. The mRNA of FasL was measured by in situ hybridization in the consecutive tissue slices of 80 colorectal carcinomas respectively. Using terminal deoxynucleotidyl transferase-mediaed dUTP nick end labeling （TUNEL）, apoptotic cells were detected in 80 specimens of colorectal carcinoma. The expression of FasL was detected in all 80 specimens, but it was not even in the same or among different tissues. In the consecutive tissue slices, the location of expression of FasL protein corresponded with that of FasL mRNA. In those with FasL extensive expression, the number of TILs was less than that of FasL weak expression （P〈0. 05）, and the apoptotic index （AI） of TILs was higher and that of tumor cells was lower than that of FasL with weak expression respectively （P〈0. 01）. The AI of TILs was correlated with that of tumor cells （r=-0. 631, P〈0. 01）. It was suggested that colorectal carcinoma cells can induce the apoptosis of TILs through the expression of FasL, which can counterattack the immune system. This may be one of the mechanisms of immune evasion in colorectal carcinoma.展开更多
AIM: To investigate the potential role of cytokines in promoting Fas ligand (FasL)-expressing colon cancer cells. METHODS: Immunohistochemical SABC method was used to observe the expression of Fas receptor and ligand ...AIM: To investigate the potential role of cytokines in promoting Fas ligand (FasL)-expressing colon cancer cells. METHODS: Immunohistochemical SABC method was used to observe the expression of Fas receptor and ligand in SW620 colon cancer cell line and Jurkat T cells in order to provide the morphological evidence for the functions of Fas receptor and ligand. To examine the cytotoxicity of effector cells, CytoTox96 non-radioactive cytotoxicity assay was adopted to measure the lactate dehydrogenase-releasing value after SW620 cells were co-cultured with Jurkat T lymphocytes. RESULTS: The FasL of colon cancer SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm. The Fas receptor of colon cancer SW620 cells was negative. The Fas receptor and ligand of Jurkat T lymphocytes turned out to be positive. The positive substances were distributed in the cell membrane. After phytohemagglutinin (PHA)-stimulated Jurkat T lymphocytes were co-cultured with phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin-stimulated (for 48 h) SW620 cells or tumor necrosis factor-alpha (TNF-α)-stimulated (for 48 h) SW620 cells or unstimulated SW620 cells for 4 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 10:1, 5:1, 2.5:1, and 1.25:1 was 74.6%, 40.8%, 32.4%, and 10.9% (F= 8.19, P<0.05); or 54.9%, 35.3%, 22.0%, and 10.3% (F= 11.12, P<0.05); or 14.9%, 10.5%, 6.9%, and 5.8% (F = 3.45, P<0.05). After PHA-stimulated Jurkat T lymphocytes were co-cultured with unstimulated SW620 cells for 8 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 5:1, 2.5:1, and 1.25:1 from the experiment was 83.9%, 74.1%, and 28.5% (F=137.04, P<0.05) respectively. Non-radioactive cytotoxicity assay showed that the apoptotic rate of Jurkat cells remarkably increased with the increase of planting concentration of SW620 cells and co-culture time after the SW620 cells were co-cultured with the Jurkat T lymphocytes. The cytotoxicity was significantly enhanced by PMA+ionomycin or TNF-α. CONCLUSION: The FasL expressed in human colon cancer cells may be regulated by endogenous factors in the microenvironment of the host and facilitate the escape of tumor cells from the host immune system.展开更多
基金This project was supported by a grant from the National Natural Science Foundation of China (No. 03030302).
文摘Objective： To block the apoptosis of T lymphocytes induced by Fas/FasL in order to establish a method of the large-scale preparation of large amounts of tumor-specific cytoxic T-lymphocytes （CTL）. Methods： Liver cancer cells and tumor infiltrating lymphocytes （TIL） were isolated from FasL positive fresh specimens, and co-cultured. Specific CTL were activated and prepared in the presence of the co-stimulation of monoclonal antibody CD28. Then the blocking and activation of apoptosis of T lymphocytes was activated by soluble Fas receptor, which was detected by cytometry and DNA ladder test simultaneously. The apoptosis-blocking effect was compared with the control group. Furthermore, the changes of T cell proliferation and killing activity were detected by the method of ^3H thymidine incorporation and ^51Cr release test. Results： There was a significant increase in apoptosis rate in unblocking group compared with blocking group and quiescent group, with the unblocking group of 47.82%±0.13%, quiescent group of 3.76%±0.25%, and the blocking group of 8.22%±0.26% respectively （P〈0.01）. T cell-ladder appeared in unblocking group by DNA ladder test. Both the killing ability and proliferation rate of T cells were significantly increased after blocking. There was significant difference among blocking group, unblocking group and quiescent group （P〈0.01）. Conclusion： With this method we obtained large amounts of tumor-specific T lymphocytes, which was able to kill liver cancer cells effectively.
文摘Objective:To further illuminate a possibme mechanism of Fas/FasL in the treatment of psoriasis, the expression of it and apoptosis of KC were investigated. Methods: With cell culture,immunocytochemistry, the expression of Fas/FasL protein after the treatment with etretin was observed in cultured human normal keratinocytes. Then, the state of apoptosis in cultured keratinocyte after stimulation with etretin was detected with FACS(Fluorescence Activited Cell Sortor). Results:① There was no expression of Fas/FasL protein in the cultured normal human KC. ②After the treatment with etretin, the strongest expression of FasL protein appeared after 16h,so did Fas after 40h.③ The higher the concentration of etretin,the stronger the expression of Fas/FasL protien.Under the same condition,the expression of Fas is stronger than that of FasL.④ Keratinocytes' apoptosis occurred after stimulation with etretin. Conclusion: Fas/FasL system wasn't involved in apoptosis in cultured normol human keratinocytes.But during limited period, the apoptosis of KC could be induced by (etretin),thus it can antagonize benign proliferate of keratinocytes.Our data showed up-regulation of the expression of Fas/FasL and apoptosis in cultured human keratinocytes stimulated by etretin, and its function may be involved in the therapeutic machanism of psoriasis.
文摘The host immune system functions as a guardian against tumor development. It has been demonstrated that cytotoxic T lymphocyte (CTL)-mediated cytotoxic pathways function to inhibit or delay human colorectal cancer development. However, the host anti-tumor immune responses also 'edit' the tumor and select for more aggressive variants, resulting in immune evasion and tumor escape. Fas is a death receptor that mediates one of the major cytotoxic effector mechanisms of the CTLs. Fas is highly expressed in normal human colon epithelial cells but is frequently silenced in colorectal carcinoma, especially in metastatic colorectal carcinoma, suggesting that loss of Fas expression and function may be an immune evasion and tumor escape mechanism. In addition, recent studies indicated that Fas also mediates cellular proliferation signaling pathways to promote tumor development. Therefore, the death receptor Fas may not only transduce death signals to suppress tumor development but also activate cellular proliferation and the migration process to promote tumor growth and progression. Thus, understanding the mechanisms by which the Fas receptor and its associated protein complex transduces the death and survival signals may identify molecular targets for the development of therapeutic strategy to enhance the Fas-mediated death signals to increase the efficacy of cancer immunotherapy.
文摘To study the significance of FasL expression in immune escape of colorectal carcinoma, FasL protein expression and the number of tumor infiltrating lymphocytes （TILs） in 80 specimens of colorectal carcinoma were detected by immunohistochemitry. The mRNA of FasL was measured by in situ hybridization in the consecutive tissue slices of 80 colorectal carcinomas respectively. Using terminal deoxynucleotidyl transferase-mediaed dUTP nick end labeling （TUNEL）, apoptotic cells were detected in 80 specimens of colorectal carcinoma. The expression of FasL was detected in all 80 specimens, but it was not even in the same or among different tissues. In the consecutive tissue slices, the location of expression of FasL protein corresponded with that of FasL mRNA. In those with FasL extensive expression, the number of TILs was less than that of FasL weak expression （P〈0. 05）, and the apoptotic index （AI） of TILs was higher and that of tumor cells was lower than that of FasL with weak expression respectively （P〈0. 01）. The AI of TILs was correlated with that of tumor cells （r=-0. 631, P〈0. 01）. It was suggested that colorectal carcinoma cells can induce the apoptosis of TILs through the expression of FasL, which can counterattack the immune system. This may be one of the mechanisms of immune evasion in colorectal carcinoma.
基金Supported by the Science and Technology Commission Foundation of Tianjin, No. 003119711
文摘AIM: To investigate the potential role of cytokines in promoting Fas ligand (FasL)-expressing colon cancer cells. METHODS: Immunohistochemical SABC method was used to observe the expression of Fas receptor and ligand in SW620 colon cancer cell line and Jurkat T cells in order to provide the morphological evidence for the functions of Fas receptor and ligand. To examine the cytotoxicity of effector cells, CytoTox96 non-radioactive cytotoxicity assay was adopted to measure the lactate dehydrogenase-releasing value after SW620 cells were co-cultured with Jurkat T lymphocytes. RESULTS: The FasL of colon cancer SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm. The Fas receptor of colon cancer SW620 cells was negative. The Fas receptor and ligand of Jurkat T lymphocytes turned out to be positive. The positive substances were distributed in the cell membrane. After phytohemagglutinin (PHA)-stimulated Jurkat T lymphocytes were co-cultured with phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin-stimulated (for 48 h) SW620 cells or tumor necrosis factor-alpha (TNF-α)-stimulated (for 48 h) SW620 cells or unstimulated SW620 cells for 4 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 10:1, 5:1, 2.5:1, and 1.25:1 was 74.6%, 40.8%, 32.4%, and 10.9% (F= 8.19, P<0.05); or 54.9%, 35.3%, 22.0%, and 10.3% (F= 11.12, P<0.05); or 14.9%, 10.5%, 6.9%, and 5.8% (F = 3.45, P<0.05). After PHA-stimulated Jurkat T lymphocytes were co-cultured with unstimulated SW620 cells for 8 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 5:1, 2.5:1, and 1.25:1 from the experiment was 83.9%, 74.1%, and 28.5% (F=137.04, P<0.05) respectively. Non-radioactive cytotoxicity assay showed that the apoptotic rate of Jurkat cells remarkably increased with the increase of planting concentration of SW620 cells and co-culture time after the SW620 cells were co-cultured with the Jurkat T lymphocytes. The cytotoxicity was significantly enhanced by PMA+ionomycin or TNF-α. CONCLUSION: The FasL expressed in human colon cancer cells may be regulated by endogenous factors in the microenvironment of the host and facilitate the escape of tumor cells from the host immune system.
