目的:观察大鼠酒精性肝病组织病理形态学改变,探讨细胞凋亡与细胞色素P4502E1的表达以及和氧化应激的关系.方法:用酒精灌胃法制备酒精性肝病大鼠模型,模型组给予酒精8 g/kg,每天分2次灌胃连续8 wk,对照组给予等量的生理盐水灌胃.实验8 w...目的:观察大鼠酒精性肝病组织病理形态学改变,探讨细胞凋亡与细胞色素P4502E1的表达以及和氧化应激的关系.方法:用酒精灌胃法制备酒精性肝病大鼠模型,模型组给予酒精8 g/kg,每天分2次灌胃连续8 wk,对照组给予等量的生理盐水灌胃.实验8 wk末,观察肝组织的病理形态学改变,用原位末端标记法(TUNEL)检测肝细胞凋亡,用免疫组化法检测肝组织中Caspase-3蛋白表达,用全自动生化仪检测ALT和AST的含量,用PCR法测定肝细胞色素P4502E1的基因表达,分别用硫代巴比妥酸法(TBA法)和黄嘌呤氧化酶法测定肝组织丙二醛(MDA)的含量和超氧化物歧化酶(SOD)的活力.结果:模型组凋亡的肝细胞明显增多,主要分布在中央静脉周围、点状和灶状坏死区;Caspase-3主要分布于中央静脉及肝细胞坏死灶周围细胞的胞质中.模型组肝细胞凋亡指数(AI)和Caspase-3蛋白表达强度明显高于对照组(AI:6.2%±1.7% vs 1.7%±0.8%;Caspase-3:66.7% vs 9.5%,P<0.05,P<0.01).CYP2E1表达:对照组c1基因频率为91.6%,c2基因频率为8.4%;模型组c1基因频率为53.4%,c2基因频率为46.6%,均有显著性差异(P<0.05).长期酒精摄入大鼠血清MDA含量增加(41.53±7.43μmol/L vs 15.72±2.06μmol/L,P<0.05),SOD活力下降(353.12±61.02 kU/L vs 636.82±138.60 kU/L,P<0.05),与酒精性肝病肝细胞凋亡程度有相关性(r=0.644,r=-0.511).结论:长期酒精摄入可引起大鼠酒精性肝病及及肝功能损伤,肝细胞凋亡明显增加.CYP2E1基因PstⅠ及RsaⅠRFLPs与酒精性肝病有关,其中c2基因可能与大鼠酒精性肝病的发生有关.MDA含量和SOD活力在酒精性肝病的肝细胞凋亡过程及脂质过氧化反应中发挥重要作用.展开更多
目的:研究缺血再灌流时异丙酚对肠上皮细胞凋亡的影响及可能机制.方法:96只成年♂Wistar大鼠,随机分为假手术组、缺血再灌流+生理盐水组(I/R+NS)和I/R+异丙酚组(I/R+Pr).采用夹闭肠系膜上动脉(SMA)的方法制作肠缺血再灌流模型.以上各组...目的:研究缺血再灌流时异丙酚对肠上皮细胞凋亡的影响及可能机制.方法:96只成年♂Wistar大鼠,随机分为假手术组、缺血再灌流+生理盐水组(I/R+NS)和I/R+异丙酚组(I/R+Pr).采用夹闭肠系膜上动脉(SMA)的方法制作肠缺血再灌流模型.以上各组分别在再灌流后0,30,60,120和240 min(每时间点8只)处死动物取肠袋组织.采用病理学方法观察肠上皮细胞损伤指数;原位DNA末端标记法(TUNEL)检测肠上皮细胞凋亡率的变化;免疫组化法检测肠上皮细胞Caspase-3,bcl-2表达的变化.结果:I/R+Pr组与I/R+NS组相比,肠上皮细胞病理变化较轻,肠上皮细胞的凋亡率明显下降(P<0.01),再灌流后0,30,60,120和240 min肠上皮细胞中Caspase-3阳性细胞数明显减少(104.4±5.3 vs 146.4±7.6;97.4±6.2 vs 130.4±7.4;134.4±5.1 vs 170.4±8.1;125.4±6.2 vs 160.4±9.5:101±5.8 vs 120.4±8.2,均P<0.01),而bcl-2阳性细胞数明显增加(13.34±4.12 vs 6.72±2.59;14.96±4.85 vs 8.24±3.13;15.29±5.28 vs 9.63±2.89;10.39±3.61 vs 9.63±2.89;10.39±3.61 vs 5.96±1.93;11.08±4.83 vs 6.87±2.43,均P<0.01).结论:异丙酚能抑制缺血再灌流时肠上皮细胞Caspase-3表达,而增加bcl-2表达,减少肠上皮细胞的凋亡.展开更多
Objective and background： Although p21 ras has been reported to be upregulated in hepatocellular carcinoma complicating chronic hepatitis C type I, p21 ras has a different role in advanced stages, as it has been foun...Objective and background： Although p21 ras has been reported to be upregulated in hepatocellular carcinoma complicating chronic hepatitis C type I, p21 ras has a different role in advanced stages, as it has been found to be downregulated. The goal of this study was to investigate the status of p21 ras in early-stage/low-grade and late-stage/high-grade hepatocellular carcinoma and its possible link to apoptosis. Material and methods： Thirty-five cases each of chronic HCV hepatitis type 4 （group I） and cirrhosis with hepatocellular carcinoma （HCC） complicating chronic HCV hepatitis （groups Ⅱ and Ⅲ） were immunohistochemically evaluated using a p21 ras polyclonal antibody. The apoptotic index was determined in histologic sections using the terminal deoxynncleotidyl transferase-mediated d-UTP biotin nick end labeling （TUNEL） assay. Results： Significant differences （P=0.001） were detected in p21 ras protein expression between the three groups. A near 2-fold increase in p21 ras staining was observed in the cirrhotic cases compared to the hepatitis cases, and p21 ras expression was decreased in the HCC group, p21 ras expression correlated with stage （r=0.64, P--0.001） and grade （r=-0.65, P=0.001） in the HCC group and grade in the HCV group （r=0.44, P=0.008）. Both p21 ras expression and TUNEL-LI were significantly lower in large HCCs compared to small HCCs （P=0.01 each）. The TUNEL values were negatively correlated with stage in the HCC group （r=-0.85, P=0.001）. The TUNEL values were also negatively correlated with grade in both the HCV and HCC groups （r=0.89, P=0.001 and r=0.53, P=0.001, respectively）. The p21 ras scores were significantly correlated with the TUNEL-LI values in the HCC group （r=0.63, P=0.001） and HCV group （r=0.88, P=0.001）. Conclusions： p21 ras acts as an initiator in HCC complicating type 4 chronic HCV and is downregulated with HCC progression, which most likely promotes tumor cell survival because it facilitates the downregulation of apoptosis with tumor progression.展开更多
AIM: To investigate the effect of gambogic acid(GA) on apoptosis in the HT-29 human colon cancer cell line. METHODS: H-29 cells were used for in vitro experiments in this study. Relative cell viability was assessed us...AIM: To investigate the effect of gambogic acid(GA) on apoptosis in the HT-29 human colon cancer cell line. METHODS: H-29 cells were used for in vitro experiments in this study. Relative cell viability was assessed using MTT assays. Cell apoptosis was detected by terminal deoxynucleotidyl transferase d UTP nick end labeling and Hoechst 33342 staining, and quantified by flow cytometry. Cellular ultrastructure was observed by transmission electron microscopy. Real-time PCR and Western blot analyses were used to evaluate gene and protein expression levels. For in vivo experiments, BALB/c nude mice received subcutaneous injections of HT-29 cells in the right armpit. When well-established xenografts were palpable with a tumor size of 75 mm3, mice were randomly assigned to a vehicle(negative) control, positive control or GA treatment group(n = 6 each). The animals in the treatment group received one of three dosages of GA(in saline; 5, 10 or 20 mg/kg) via the caudal vein twice weekly, whereas animals in the negative and positive control groups were given equal volumes of 0.9% saline or 10 mg/kg docetaxel, respectively, via the caudal vein once weekly. RESULTS: The cell viability assay showed that GA inhibited proliferation of HT-29 cells in a dose- and time-dependent manner after treatment with GA(0.00, 0.31, 0.62, 1.25, 2.50, 5.00 or 10.00 μmol/L) for 24, 48 or 72 h. After 48 h, the percentage of apoptotic cells in cells treated with 0.00, 1.25, 2.50 and 5.00 μmol/L GA was 1.4% ± 0.3%, 9.8% ± 1.2%, 25.7% ± 3.3% and 49.3% ± 5.8%, respectively. Ultrastructural analysis of HT-29 cells treated for 48 h with 2.5μmol/L GA revealed apoptotic bodies and condensed and fragmented nuclei. Levels of caspase-8,-9 and-3 m RNAs were significantly increased after treatment with GA(1.25, 2.50 or 5.00 μmol/L) for 48 h(P < 0.05 for all). Protein levels of apoptosis-related factors Fas, Fas L, FADD, cytochrome c, and Apaf-1 were increased in GA-treated cells, whereas levels of pro-caspase-8,-9 and-3 were significantly decreased(P < 0.05 for all). Furthermore, GA significantly and dose-dependently inhibited the growth of HT-29 tumors in a mouse xenograft model(P < 0.05).CONCLUSION: GA inhibits HT-29 proliferation via induction of apoptosis. The anti-cancer effects are likely mediated by death receptor(extrinsic) and mitochondrial(intrinsic) pathways.展开更多
文摘目的:观察大鼠酒精性肝病组织病理形态学改变,探讨细胞凋亡与细胞色素P4502E1的表达以及和氧化应激的关系.方法:用酒精灌胃法制备酒精性肝病大鼠模型,模型组给予酒精8 g/kg,每天分2次灌胃连续8 wk,对照组给予等量的生理盐水灌胃.实验8 wk末,观察肝组织的病理形态学改变,用原位末端标记法(TUNEL)检测肝细胞凋亡,用免疫组化法检测肝组织中Caspase-3蛋白表达,用全自动生化仪检测ALT和AST的含量,用PCR法测定肝细胞色素P4502E1的基因表达,分别用硫代巴比妥酸法(TBA法)和黄嘌呤氧化酶法测定肝组织丙二醛(MDA)的含量和超氧化物歧化酶(SOD)的活力.结果:模型组凋亡的肝细胞明显增多,主要分布在中央静脉周围、点状和灶状坏死区;Caspase-3主要分布于中央静脉及肝细胞坏死灶周围细胞的胞质中.模型组肝细胞凋亡指数(AI)和Caspase-3蛋白表达强度明显高于对照组(AI:6.2%±1.7% vs 1.7%±0.8%;Caspase-3:66.7% vs 9.5%,P<0.05,P<0.01).CYP2E1表达:对照组c1基因频率为91.6%,c2基因频率为8.4%;模型组c1基因频率为53.4%,c2基因频率为46.6%,均有显著性差异(P<0.05).长期酒精摄入大鼠血清MDA含量增加(41.53±7.43μmol/L vs 15.72±2.06μmol/L,P<0.05),SOD活力下降(353.12±61.02 kU/L vs 636.82±138.60 kU/L,P<0.05),与酒精性肝病肝细胞凋亡程度有相关性(r=0.644,r=-0.511).结论:长期酒精摄入可引起大鼠酒精性肝病及及肝功能损伤,肝细胞凋亡明显增加.CYP2E1基因PstⅠ及RsaⅠRFLPs与酒精性肝病有关,其中c2基因可能与大鼠酒精性肝病的发生有关.MDA含量和SOD活力在酒精性肝病的肝细胞凋亡过程及脂质过氧化反应中发挥重要作用.
文摘目的:研究缺血再灌流时异丙酚对肠上皮细胞凋亡的影响及可能机制.方法:96只成年♂Wistar大鼠,随机分为假手术组、缺血再灌流+生理盐水组(I/R+NS)和I/R+异丙酚组(I/R+Pr).采用夹闭肠系膜上动脉(SMA)的方法制作肠缺血再灌流模型.以上各组分别在再灌流后0,30,60,120和240 min(每时间点8只)处死动物取肠袋组织.采用病理学方法观察肠上皮细胞损伤指数;原位DNA末端标记法(TUNEL)检测肠上皮细胞凋亡率的变化;免疫组化法检测肠上皮细胞Caspase-3,bcl-2表达的变化.结果:I/R+Pr组与I/R+NS组相比,肠上皮细胞病理变化较轻,肠上皮细胞的凋亡率明显下降(P<0.01),再灌流后0,30,60,120和240 min肠上皮细胞中Caspase-3阳性细胞数明显减少(104.4±5.3 vs 146.4±7.6;97.4±6.2 vs 130.4±7.4;134.4±5.1 vs 170.4±8.1;125.4±6.2 vs 160.4±9.5:101±5.8 vs 120.4±8.2,均P<0.01),而bcl-2阳性细胞数明显增加(13.34±4.12 vs 6.72±2.59;14.96±4.85 vs 8.24±3.13;15.29±5.28 vs 9.63±2.89;10.39±3.61 vs 9.63±2.89;10.39±3.61 vs 5.96±1.93;11.08±4.83 vs 6.87±2.43,均P<0.01).结论:异丙酚能抑制缺血再灌流时肠上皮细胞Caspase-3表达,而增加bcl-2表达,减少肠上皮细胞的凋亡.
文摘Objective and background： Although p21 ras has been reported to be upregulated in hepatocellular carcinoma complicating chronic hepatitis C type I, p21 ras has a different role in advanced stages, as it has been found to be downregulated. The goal of this study was to investigate the status of p21 ras in early-stage/low-grade and late-stage/high-grade hepatocellular carcinoma and its possible link to apoptosis. Material and methods： Thirty-five cases each of chronic HCV hepatitis type 4 （group I） and cirrhosis with hepatocellular carcinoma （HCC） complicating chronic HCV hepatitis （groups Ⅱ and Ⅲ） were immunohistochemically evaluated using a p21 ras polyclonal antibody. The apoptotic index was determined in histologic sections using the terminal deoxynncleotidyl transferase-mediated d-UTP biotin nick end labeling （TUNEL） assay. Results： Significant differences （P=0.001） were detected in p21 ras protein expression between the three groups. A near 2-fold increase in p21 ras staining was observed in the cirrhotic cases compared to the hepatitis cases, and p21 ras expression was decreased in the HCC group, p21 ras expression correlated with stage （r=0.64, P--0.001） and grade （r=-0.65, P=0.001） in the HCC group and grade in the HCV group （r=0.44, P=0.008）. Both p21 ras expression and TUNEL-LI were significantly lower in large HCCs compared to small HCCs （P=0.01 each）. The TUNEL values were negatively correlated with stage in the HCC group （r=-0.85, P=0.001）. The TUNEL values were also negatively correlated with grade in both the HCV and HCC groups （r=0.89, P=0.001 and r=0.53, P=0.001, respectively）. The p21 ras scores were significantly correlated with the TUNEL-LI values in the HCC group （r=0.63, P=0.001） and HCV group （r=0.88, P=0.001）. Conclusions： p21 ras acts as an initiator in HCC complicating type 4 chronic HCV and is downregulated with HCC progression, which most likely promotes tumor cell survival because it facilitates the downregulation of apoptosis with tumor progression.
文摘AIM: To investigate the effect of gambogic acid(GA) on apoptosis in the HT-29 human colon cancer cell line. METHODS: H-29 cells were used for in vitro experiments in this study. Relative cell viability was assessed using MTT assays. Cell apoptosis was detected by terminal deoxynucleotidyl transferase d UTP nick end labeling and Hoechst 33342 staining, and quantified by flow cytometry. Cellular ultrastructure was observed by transmission electron microscopy. Real-time PCR and Western blot analyses were used to evaluate gene and protein expression levels. For in vivo experiments, BALB/c nude mice received subcutaneous injections of HT-29 cells in the right armpit. When well-established xenografts were palpable with a tumor size of 75 mm3, mice were randomly assigned to a vehicle(negative) control, positive control or GA treatment group(n = 6 each). The animals in the treatment group received one of three dosages of GA(in saline; 5, 10 or 20 mg/kg) via the caudal vein twice weekly, whereas animals in the negative and positive control groups were given equal volumes of 0.9% saline or 10 mg/kg docetaxel, respectively, via the caudal vein once weekly. RESULTS: The cell viability assay showed that GA inhibited proliferation of HT-29 cells in a dose- and time-dependent manner after treatment with GA(0.00, 0.31, 0.62, 1.25, 2.50, 5.00 or 10.00 μmol/L) for 24, 48 or 72 h. After 48 h, the percentage of apoptotic cells in cells treated with 0.00, 1.25, 2.50 and 5.00 μmol/L GA was 1.4% ± 0.3%, 9.8% ± 1.2%, 25.7% ± 3.3% and 49.3% ± 5.8%, respectively. Ultrastructural analysis of HT-29 cells treated for 48 h with 2.5μmol/L GA revealed apoptotic bodies and condensed and fragmented nuclei. Levels of caspase-8,-9 and-3 m RNAs were significantly increased after treatment with GA(1.25, 2.50 or 5.00 μmol/L) for 48 h(P < 0.05 for all). Protein levels of apoptosis-related factors Fas, Fas L, FADD, cytochrome c, and Apaf-1 were increased in GA-treated cells, whereas levels of pro-caspase-8,-9 and-3 were significantly decreased(P < 0.05 for all). Furthermore, GA significantly and dose-dependently inhibited the growth of HT-29 tumors in a mouse xenograft model(P < 0.05).CONCLUSION: GA inhibits HT-29 proliferation via induction of apoptosis. The anti-cancer effects are likely mediated by death receptor(extrinsic) and mitochondrial(intrinsic) pathways.
