【 Abstract 】 Objective To probe into the role of inositol 1, 4, 5-trisphosphate (IP3) and bax gene expression in apoptosis of HepG2 cells induced by genistein (Gen). Methods HepG2 cells were treated with different concentrations including 20, 40, 60 and 80 μ mol/L Gen as HepG2 cells cultured with 0 μmol/L Gen for 72 h was control; HepG2 cells were treated with 60 μmol/L Gen for 6, 12, 24, 48 and 72 h as HepG2 cells treated with 60 μmol/L Gen for 0 h was control. IP3 content, bax mRNA expression and apoptosis rate were assayed by IP3- ［ 3H ］ Birtrak assay, RT-PCR and flow cytometry, respectively. ResultsHepG2 cells incubated with each concentration of Gen for 72 h ， IP3 content was lower than that of control 〔 (17.7 ± 1.3), (11.2 ± 0.9), (4.9 ± 0.5), (4.8 ± 0.3) pmol/106 cells vs (29.4 ± 0.5) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression (RI which was the gray degree multiply area of bax/the gray degree multiply area of β -actin) was higher than that of control (0.26 ± 0.02, 0.33 ± 0.05, 0.35 ± 0.06, 0.38 ± 0.05 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate was higher than that of control 〔 (10.1 ± 0.9)%, (18.7 ± 1.6)%, (28.7 ± 2.5)%, (27.9 ± 2.0)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. HepG2 cells were incubated with 60 μ mol/L Gen for 6, 12, 24, 48 and 72 h ， IP3 content was lower than that of control 〔 (22.6 ± 0.9), (12.0 ± 1.4), (7.5 ± 0.8), (5.6 ± 0.5), (4.3 ± 0.6) pmol/106 cells vs (29.2 ± 0.6) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression was higher than that of control incubated with 60 μ mol/L Gen for above 12 h (0.25 ± 0.06, 0.29 ± 0.02, 0.30 ± 0.02, 0.35 ± 0.04 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate in groups incubated with 60 μ mol/L Gen for 24, 48 and 72 h was significantly higher than that in control 〔 (7.4 ± 0.5)%, (20.5 ± 2.0)%, (30.7 ± 1.6)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. ConclusionGen induces apoptosis of HepG2 cells by reducing IP3 production and increasing bax gene expression.
【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.
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.
Objective To investigate the effect of renal cell apoptosis induced by obstructive jaundice on the expression of bcl-2 in rats, and to explore the mechanism of renal impairment induced by obstructive jaundice. Methods Thirty-two male SD rats were randomly divided into 2 groups: SO group and BDL group. The rats in SO group received sham operation. Bile ducts of rats in BDL group were ligated. Pathology of kidneys was observed under the microscope. The levels of D-Bil, TBA, GOT, GPT, Cr and BUN in serum and β2-MG in urine were measured. The apoptotic rate of renal cells was calculated by flow cytometry and the forms of DNA fragmentation in renal cells were detected by agarose gel electrophoresis. The expression of inhibitory gene bcl-2 in the renal tissues was detected by immunohistochemistry. Results The color of urine in BDL group became dark yellow in day 2 after operation; The ears, tails and the muscle of abdominal wall and splanchnic organs, such as liver and kidney, also became yellow and swollen in day 7. The D-Bil, TBA, GOT, GPT, BUN of serum and β2 -MG of urine in BDL group were higher than those in SO group (P＜0.05, P＜0.01), and each value (except β2 -MG) in BDL group of 14 d was higher than that in BDL group of 7 d (P＜0.05, P＜0.01), respectively. The result of flow cytometry showed that the apoptotic rate of SO group and BDL (7 d and 14 d) group were (2.10±0.75)%， (18.17±0.86)% and (36.39±2.23)% respectively, there were significantly difference among them (P＜0.05). The expression rate of bcl-2 of renal cell in BDL group of 7 d was higher than that in BDL group of 14 d. Conclusion Obstructive jaundice could induce apoptosis of the renal cells, and activate the expression of bcl-2 of the renal tubular epithelial cells in feedback, which may regulate the process of apoptosis.
ObjectiveAfter establishing the rabbit brain death model, TUNEL, western blotting, and immuno-histochemical methods were used to detect hepatocyte apoptosis to study hepatocyte apoptosis level from rabbit donors after brain death. MethodsSixty healthy male New Zealand rabbits were divided into brain death group (n=30) and sham group (n=30). The rabbits of brain death group were established by increasing intracranial pressure in a modified, slow, and intermittent way, collecting liver tissues after corresponding treatment respectively, using TUNEL to detect apoptosis rate, western blotting and immunohistochemical methods to detect the expression of Cleaved-caspase 3. ResultsThe hepatocyte apoptosis rate at each time point of brain death group were higher than those of the corresponding time point of sham group (P<0.05), and the rate of hepatocyte apoptosis increased gradually with the extension of brain death time (P<0.05). The results of Western blot assay and immunohistochemistry assay showed that the relative expression amount of Cleaved-caspase 3 protein increased gradually with the extension of brain death time (P<0.05), and relative expression amount of Cleaved-caspase 3 protein at each time point of brain death group were higher than those of the corresponding time point of sham group (P<0.05). ConclusionsThe relationship between brain death donor liver and cell apoptosis is closely related. Along with the extension of the brain death time in rabbits, the level of apoptosis of liver cells gradually increased, which affects the quality of liver donors after brain death.
Objective To explore the effects of ulinastatin (UTI) on renal apoptosis and expression of bcl-2 in rats with severe acute pancreatitis (SAP). Methods Sixty rats weighing 250-300 g were randomized divided into 3 groups: pseudo-operation group (SO group, n=20), SAP group (n=20) and UTI treated group (UTI group, n=20). The model of SAP was established by retrograde injection of 5% sodium taurocholate solution into the biliopancreatic duct in the rats. Serum Cr and BUN were determined. The left kidneys were resected for light and electronic microscopic study. Renal cell apoptosis was determined by TUNEL. Expression of bcl-2 was detected by immunohistochemical staining of SABC. Results Serum Cr, BUN, renal cell apoptotic index and bcl-2 expression were markedly increased in SAP group compared with SO group （P＜0.05， P＜0.01）, Renal tissue injuries were aggravated in SAP group under light and electronic microscopic study as well. In UTI group, serum Cr, BUN and renal cell apoptotic index were decreased significantly while the expression of bcl-2 increased remarkably and renal tissue injuries relieved compared with SAP group （P＜0.05）. Positive correlations were found between the renal cell apoptotic index and BUN as well as Cr (r=0.807, P＜0.05; r=0.812, P＜0.05). Conclusion The protective effect of UTI on SAP renal injury is probably through increasing bcl-2 expression and decreasing apoptosis.
Objective To research the effect of γ-ray released from 103Pd radioactive stent on the expression of Fas gene and its relation with apoptosis of cholangiocarcinoma cell and the significances through the establishment of human cholangiocarcinoma model. Methods The model of nude mouse with implanted human cholangiocarcinoma was established. The mice were divided into study group and control group, 37 MBq 103Pd biliary stent was implanted in the study group and the ordinary metal biliary stent was implanted in the control group. The volume of tumor was measured, the cell apoptosis was detected by the TUNEL method and the expression of Fas gene of the cell apoptosis of the induced human cholangiocarcinoma was checked out by immunohistochemistry staining 10 d after the implantation. Results Compared with the control group, the growing speed of the volume of tumor in study group was significantly reduced (Plt;0.05), the expression positive rate of Fas gene was significantly higher (Plt;0.05), and the apoptotic rate of cancer cells was also higher (Plt;0.01). Conclusions The 103Pd radioactive stent can induce the cell apoptosis in nude mouse model with implanted human cholangiocarcinoma inhibit the cell growth of bile duct cancer and may promote the apoptosis of cancer cells by increasing the expression of Fas gene. It may be helpful for the further study of treatment for bile duct cancer using 103Pd radioactive stent.
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】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 summarize the role of survivin gene in tumor. MethodsThe research status on biological characteristics the role of survivin gene in tumor for gene therapy and clinical application was retrospectively analyzed after related domestic and foreign literatures were reviewed. ResultsSurvivin gene was by far found to be the best powerful apoptosis inhibition gene, which played antiapoptosis role mainly through inhibiting directly the activity of caspase-3 and caspase-7 in the downstream of cascade reaction. Survivin gene promoted tumor cell proliferation and differentiation through speeding up tumor cells transition of G1→S phase and eluding the recognition of tumor cells to the apoptosis in G2/M phase. Survivin gene played important role in the intermediate links of vasiformation through angiogenic factor (VEGF, bFGF, Ang-1, and COX-2). ConclusionSurvivin gene may inhibit the apoptosis, promote the proliferation and differentiation of tumor cells and tumor angiogenesis, suggested that survivin gene has potential to act as a novel tumor marker and become an indicator of malignant tumor.