Induction of apoptosis via Fas system and enhancement of the induction in bile duct carcinoma cells

结果1.结果：1.流式细胞仪检测胆管癌细胞Fas表达情况：HuCCT1和HuH28细胞Fas表达分别为21.7%和30.9%。诱导细胞凋亡的抗Fas单抗CH-11处理后的细胞存活率：在浓度为0.01~1.0μg/ml范围内，CH-11可剂量依赖性地使HuCCT1和HuH28细胞数分别减少16.4%和71.7%。CH-11或干扰素-γ处理的BDC细胞的DNA片段化：在CH-11或干扰素-γ处理的HuCCT1和HuH28细胞中，观察到与阳性对照细胞相对应的峰。CH-11或干扰素-γ处理的BDC细胞的形态变化：以细胞皱缩、染色质固缩和核碎裂为特征的细胞凋亡的形态学改变。不诱导细胞凋亡的抗Fas单抗4B4对CH-11诱导的细胞凋亡的抑制作用：4B4可抑制细胞数量减少41…在HuH28细胞中高达0.7%。干扰素-γ对BDC细胞Fas表达的增强作用：在HuCCT1细胞中，Fas表达增强了17.6%。然而，在HuH28细胞中，干扰素-γ并不影响Fas的表达。干扰素-γ预处理对CH-11诱导的BDC细胞活力的影响：干扰素-γ作用第3天后，HuCCT1和HuH28细胞计数分别减少41.2%和8.2%。IL-2预处理对CH-11处理的BDC细胞活力的影响：在HuCCT1细胞中，单独IL-2对第3天的细胞数无影响，IL-2预处理也不影响细胞数的减少。在HUH28细胞中，IL-2单独作用第3天细胞数下降11.2%，IL-2预处理也不影响细胞数的减少。9.经INF-γ处理的BDC细胞的存活率：在浓度为0.1~100U/ml范围内，INF-γ在第3天可使HuCCT1和HuH28细胞数分别减少45.1%和87.8%。CH-11或INF-γ对BDC细胞半胱氨酸天冬氨酸酶活性的影响在HuCCT1细胞中未检测到半胱氨酸氨基转移酶。在CH-11作用8小时的HuH28细胞中可检测到caspase-3的表达。结论1.Fas在人BDC细胞中有表达。抗Fas单抗和干扰素-γ诱导人BDC细胞凋亡。4B4可抑制CH-11诱导人BDC细胞凋亡。干扰素-γ增强人外周血树突状细胞Fas的表达和诱导细胞凋亡。在抗Fas单抗作用下，BDC细胞中caspase-3被激活。较少

Results1. Fas expression in bile duct carcinoma (BDC) cells, analyzed by flow cytometry : Fas was expressed at 21.7% in HuCCT1 cells and at 30.9% in HuH28 cells, respectively.2. Viability of BDC cells treated with anti-Fas monoclonal antibody (mAb), CH-11 which induces apoptosis : CH-11 dose-dependently reduced HuCCT1 and HuH28 cell counts at the concentration range from 0.01 to 1.0 μg/ml by up to 16.4% and 71.7% on day 3, respectively.3. DNA fragmentation in BDC cells treated with CH-11 or IFN-γ : In HuCCT1 and HuH28 cells treated with CH-11 or IFN-γ, the peak corresponding to that of positive control cells was observed.4. Morphological changes of BDC cells treated with CH-11 or IFN-γ : The shrinkage of cells, chromatin condensation and nuclear fragmentation were observed as morphological changes characteristic of apoptosis.5. Inhibitory effect of anti-Fas mAb 4B4, which does not induce apoptosis, on the induction of apotosis by CH-11 : 4B4 inhibited the reduction of cell counts by 41 … More .7% in HuH28 cells.6. Enhancement of Fas expression in BDC cells treated with IFN-γ : In HuCCT1 cells, the Fas expression was enhanced by 17.6%. In HuH28 cells, however, IFN-γ did not affect the Fas expression.7. Effect of pretreatment with IFN-γ on the viability of BDC cells treated with CH-11 : Pretreatment with IFN-γ increased the reduction of HuCCT1 and HuH28 cell counts by 41.2% and 8.2% on day 3, respectively.8. Effect of pretreatment with IL-2 on the viability of BDC cells treated with CH-11 : In HuCCT1 cells, IL-2 alone did not affect the cell number on day 3. Pretreatment with IL-2 also did not affect the reduction of cell counts. In HuH28 cells, IL-2 alone reduced the cell counts by 11.2% on day 3. Pretreatment with IL-2 also did not affect the reduction of cell counts.9.Viability of BDC cells treated with INF-γ : INF-γ dose-dependently reduced HuCCT1 and HuH28 cell counts at the concentration range from 0.1 to 100 U/ml by up to 45.1% and 87.8% on day 3, respectively.10. Caspases in BDC cells treated with CH-11 or INF-γ. Any caspase was not detected in HuCCT1 cells. Caspase-3 was detedeted in HuH28 cells treated with CH-11 for 8 hr.Conclusions1. Fas was expressed in human BDC cells.2. Apoptosis was induced by anti-Fas mAb and IFN-γ in human BDC cells.3. Induction of apoptosis by CH-11 was inhibited by 4B4 in human BDC cells.4. The expression of Fas and the induction of apoptosis was enhanced by IFN-γ in human BDC cells.5. Caspase-3 was activated in BDC cells treated with anti-Fas mAb. Less