Objective To investigate the protective effects of antitumor necrosis factor-α antibody （TNF-αAb） on lung injury after cardiopulmonary bypass （CPB） and their mechanisms. Methods Forty healthy New Zealand white rabbits，weighting 2.0-2.5 kg，male or female，were randomly divided into 4 groups with 10 rabbits in each group. In groupⅠ，the rabbits received CPB and pulmonary arterial perfusion. In group Ⅱ，the rabbits received CPB and pulmonary arterial perfusion with TNF-αAb. In group Ⅲ，the rabbits received CPB only. In group Ⅳ，the rabbits only received sham surgery. Neutrophils count，TNF-α and malondialdehyde （MDA） concentrations of the blood samples from the left and right atrium as well as oxygenation index were examined before and after CPB in the 4 groups. Pathological and ultrastructural changes of the lung tissues were observed under light and electron microscopes. Lung water content，TNF-α mRNA and apoptoticindex of the lung tissues were measured at different time points. Results Compared with group Ⅳ，after CPB，the rabbitsin group Ⅰ to group Ⅲ showed significantly higher blood levels of neutrophils count，TNF-α and MDA（P＜0.05），higherTNF-α mRNA expression，apoptosis index and water content of the lung tissues （P＜0.05），and significantly lower oxyg-enation index （P＜0.05） as well as considerable pathomorphological changes in the lung tissues. Compared with group Ⅱ，after CPB，the rabbits in groups Ⅰ and Ⅲ had significantly higher blood concentrations of TNF-α （5 minutes after aortic declamping，220.43±16.44 pg/ml vs.185.27±11.78 pg/ml，P＜0.05；249.99±14.09 pg/ml vs.185.27±11.78 pg/ml，P＜0.05），significantly higher apoptosis index （at the time of CPB termination，60.7‰±13.09‰ vs. 37.9‰±7.78‰，P＜0.05；59.6‰±7.74‰ vs. 37.9‰±7.78‰，P＜0.05），significantly higher blood levels of neutrophils count and MDA （P＜0.05），significantly higher TNF-α mRNA expression and water content of the lung tissues （P＜0.05），and significantly loweroxygenation index （P＜0.05） as well as considerable pathomorphological changes in the lung tissues. Compared with groupⅠ，rabbits in group Ⅲ had significantly higher above parameters （P＜0.05） but lower oxygenation index （P＜0.05） only at 30 minutes after the start of CPB. Conclusion Pulmonary artery perfusion with TNF-αAb can significantly attenuate inflammatory lung injury and apoptosis of the lung tissues during CPB.
Objective To observe the outcomes of using different concentrations of arsenic trioxide at varying phases on the breast cancer cell line MCF-7 and to study the mechanism of this effect. Methods The effect of arsenic trioxide on the growth of breast cancer cell line MCF-7 was observed after applying arsenic trioxide of different concentrations （0.5-16 μmol/Ｌ）. The inhibitory effect of arsenic trioxide on the cell proliferation was investigated with 3-(4，5-dimethyl-thizazol-2-yl)-2， 5-diphenyl tetrazolium bromide (MTT) and the induction of arsenic trioxide on cell apoptosis was detected by DNA ladder and terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Results The effect of arsenic trioxide on breast cancer cell line MCF-7 depended on the phase and the dose. The number of cell decreased significantly and there were conspicuously typical morphological changes of apoptosis after the use of arsenic trioxide， including membrane blebbing， chromatin pyknosis, nuclear fragmentation and the formation of apoptotic body. The typical DNA ladders were observed in the MCF-7 cells after 48 h administration of arsenic trioxide at concentrations 1-8 μmol/Ｌ. Significant elevations of apoptosis index at 24 h, 48 h and 72 h were all detected by TUNEL after incubating with 4 μmol/L arsenic trioxide. Conclusion Arsenic trioxide may inhibit the growth of breast cancer cell line MCF-7 significantly by inducing the apoptosis of breast cancer cell.
ObjectiveTo explore apoptosis of acinar cells during pancreatic allograft rejection in rats.MethodsGroups of Wistar rats underwent heterotopic pancreaticoduodenal transplantation from syngenic Wistar of allogenic SD rats. The grafts were harvested on postoperative day 3, 5 and 7. All graft samples were subjected to histological examination and apoptotic cells of graft acinar cells using in situ terminal deoxynucleotidy1 transferasemediated dUTP nickend labeling (TUNEL). Histopathological rejection score and apoptotic index (AI) were analyzed. ResultsThe incidence of apoptotic cells was increased steadily over time in allografts, in contrast with syngenic grafts. The apoptotic cells in allografts were mainly acinar cells and few infiltrating lymphocytes. A significant correlation between apoptotic index and histopathological rejection score was noted.ConclusionTUNEL can display apoptosis of single cell in situ. Apoptosis is an important mechanism of tissue injury in acute pancreatic allograft rejection in rats. Acinar cell apoptosis can be used as a valuble index to estimate the injury of grafts and to monitor the acute rejection.
【Abstract】 Objective To investigate the effect of verapamil on apoptosis, calcium and expressions of bcl-2 and c-myc of pancreatic cells in ischemia-reperfusion rat model. Methods Wistar rats were randomly divided into three groups: control group （n=10）; ischemia-reperfusion group （n=10）; verapamil treatment group （n=10）. The anterior mesenteric artery and the celiac artery of rats in both ischemia-reperfusion group and verapamil treatment group were occluded for 15 min followed by 12-hour reperfusion. Verapamil （1 mg／kg） was injected via caudal vein to the rats in verapamil treatment group 15 min before occlusion and 1 hour after the initiation of reperfusion, respectively; and ischemia-reperfusion group was given the same volume of salient twice intravenously. Pancreatic tissues were collected from the dead rats after twelve hours since the reperfusion. The pathologic characters of pancreatic tissue were observed under light microscope; The level of calcium in the tissue was measured by atomic absorption spectrometer; TUNEL was used to detect apoptosis of pancreatic cells; and the expressions of c-myc and bcl-2 in the cells were also analyzed by immunohistochemistry technique and flow cytometry. Results The pathologic change in verapamil treatment group was less conspicuous than that of ischemia-reperfusion group. Both the calcium level and the number of apoptotic cells in verapamil treatment group were less than those of ischemia-reperfusion group 〔（411.1±55.8） μg/g dry weight vs （470.9±31.9） μg/g dry weight, P＜0.05 and （9.5±2.9）% vs （18.4±3.1）% 〕, P＜0.05. After taking verapamil, the number of apoptotic cells decreased, whereas the expressions of bcl-2 and c-myc increased. The fluorescent indexes of bcl-2 and c-myc in verapamil treatment group were significantly higher than those of ischemia-reperfusion group (1.72±0.11 vs 1.41±0.07, P＜0.05; 1.76±0.19 vs 1.55±0.13, P＜0.05. Conclusion Ischemia-reperfusion injury can induce apoptosis of pancreatic cells. Verapamil could protect the injured pancreatic tissue by reducing the level of calcium, stimulating the expressions of bcl-2 and c-myc and inhibiting apoptosis of pancreatic cells.
【Abstract】ObjectiveTo study the effect of preoperative gastric arterial chemoembolization on apoptosis of lymph node metastasis of gastric cancer. MethodsForty patients with gastric cancer and lymph node metastasis underwent curative resection, among which there were 20 patients who received the preoperative gastric arterial chemoembolization, and they constituted the treatment group. The rest of the patients were included in the control group. The expressions of p53, CD95 and bcl-2 were examined by immunohistochemistry and apoptosis in the lymph node metastasis was examined by in situ terminal transferasemediated dUTP nick end labeling （TUNEL）. ResultsThe expression intensity of p53 and CD95 in lymph node metastasis of treatment group increased more significantly than that of control group, whereas the expression intensity of bcl-2 decreased in treatment group. There was a significantly positive correlation between the expressions of p53 and CD95 and the apoptosis.ConclusionPreoperative gastric arterial chemoembolization may affect the expressions of p53, CD95 and bcl-2 and may induce the apoptosis of lymph node metastasis. It may be helpful to improve the effect of curative resection of gastric cancer.
ObjectiveTo introduce the relationship between the apoptosis hepatocyte and its genic mediation and the ischemia of portal vein. MethodsThe combination of related literatures and our research findings were made.ResultsPortal vein ischemia may induced hepatocyte apoptosis, p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. Expression of p53 gene is enhanced in hepatic tissue when hepatocyte apoptosis is not obvious, but after 24-72 h of portal vein ischemia, when hepatocyte apoptosis is obvious, enhanced expression of p53 gene or reduced expression of bcl2 gene occur. There exists close relationship between portal vein ischemia and hepatocyte apoptosis. Conclusion Apoptosis hepatocyte is involved in organic atrophy after ischemia of portal vein, and p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. At present, the mechanism of apoptosis of hepatocyte induced by ischemia of portal vein is not clear, which needs further study.
ObjectiveTo investigate the effects of cinobufagin on the apoptosis in U-2OS osteosarcomas cells (U-2OS cells) and explore its potential mechanism. MethodsThe cytostatic effects of cinobufagin (10, 20, 50, 100, 200, and 400 nmol/L) on U-2OS cells were evaluated by MTT assay at 24, 48, and 72 hours after culture; simple U-2OS cells served as control group. The impact of cinobufagin (100 nmol/L) on the apoptosis in U-2OS cells was determined by flow cytometry at 48 hours after culture, which were treated with cinobufagin (experimental group) or with cinobufagin plus Z-VAD-FMK (control group), and simple U-2OS cells served as blank control group. The Caspase-3 activity was measured by Caspase-3 activity assay kit at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group.The expression of apoptosis signal pathway related proteins in U-2OS cells treated with cinobufagin were detected by Western blot at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. ResultsThe results of MTT assay showed that cinobufagin inhibited the proliferation of U-2OS cells in a dose- and time-dependent manners. At each time point, the growth rate of U-2OS cells was significantly reduced with the increasing cinobufagin concentration, and as time prolonged, the growth rate of U-2OS cells behaved the same way in the same group. There were significant differences among different time points and groups (P<0.05). The apoptotic rate of experimental group (46.87%±11.23%) was significantly higher than that of the control group (2.34%±0.98%) and blank control group (1.04%±0.25%) (P<0.05). The Caspase-3 activity in 20, 50, and 100 nmol/L groups were 1.14±0.32, 1.31±0.41, and 1.92±0.54, respectively, which were significantly higher than that in control group (P<0.05). Compared with 20 and 50 nmol/L groups, 100 nmol/L group significantly increased the Caspase-3 activity in U-2OS cells (P<0.05). Compared with the control group, the expressions of cleaved Caspase-3, cleaved Caspase-9, and Bax were obviously up-regulated; the Bcl-2 expression was down-regulated; and the ratio of Bax/Bcl-2 was increased in different cinobufagin-treated groups (P<0.05). The same tendency was seen in different cinobufagin-treated goups, showing significant differences among groups (P<0.05). ConclusionCinobufagin can inhibite the proliferation of U-2OS cells, and induce cell apoptosis. The potential mechanism of cinobufagin-induced apoptosis may be related to the mitochondria-mediated pathway.
Objective To investigate the mechanism of the resistance of pancreatic cancer cells to tumor necrosis factor related apoptosis inducing ligand (TRAIL)mediated apoptosis. MethodsThe expression of TRAIL receptor-4 (TRAIL-R4) in normal pancreas tissue and pancreatic cancer was analyzed by using Northern blotting, Western blotting and immunohistochemistry.ResultsTRAIL-R4 mRNA and protein were expressed at moderate to high levels in human pancreatic cancer, but demonstrated weak to negative in the normal pancreas. Moreover, pancreatic cancer cells showed b TRAIL-R4 immunostaining throughout the tumor mass. Conclusion TRAIL-R4 levels are significantly different in pancreatic cancer in comparison to the normal pancreas. These findings give new insights into the resistance mechanisms of pancreatic cancer cells towards TRAILmediated apoptosis.