疏肝健脾安神和胃法对D-IBS结肠HO-1和iNOS的影响

目的观察疏肝健脾安神和胃法的康泰方对肝郁脾虚型D-IBS患者治疗前、后结肠HO-1和iNOS的表达的影响,探讨康泰方治疗D-IBS的可能机制。方法采用随机法将63例受试对象分为治疗组32例、对照组31例,观察治疗前、后结肠HO-1和iNOS表达的变化,并分别与健康对照组进行比较。结果治疗组治疗前、后HO-1和iNOS的表达有显著性差异(P0.05),对照组治疗前、后的HO-1和i-NOS的表达亦有显著性差异(P0.05);结论结肠HO-1和iNOS表达可能是其临床表现的致病机理,康泰方可通过上述机理改善临床症状。     

Expression and significance of HIF-1α and VEGF in rats with diabetic retinopathy

Objective:To investigate the expression of hypoxia inducible iaclor-1α(HIF-1α)and vascular endothelial growth factor(VECF)in diabelic retinopathy(DR)rats and its effect on the DR occurrence and development.Methods:A total of 120 SD rats were randomly divided into trial group and control group with 60 in each.STZ.i.p.was used in the trial group to establish the DM model,citrate buffer salt of same amount was used up.to the control group.1,3 and 6 months after injection,respective 20 rats were sacrificed in each group to observe expression of HIF-1αand VEGF in the rat retina tissue at different lime points.Results:Expression of HIF-1αand VEGF were negative in the control group;expression of HIF-1αand VKGF protein in retinal tissue were weak after 1 month of DR mold formation.It showed progressive enhancement along with the progression in different organizations,differences between groups were significant(P0.05).Conclusions:Expressions of HIF-1αand VF.GF were;correlated with disease progression in early diabelic relinopathy.Retinal oxygen can induce over-expression of HIF-1αand VEGF.It shows that HIF-1αand VEGF play an important role in the pathogenesis of DR.     

HIF-1α、iNoS在溃疡性结肠炎中的表达及临床意义

目的探讨低氧诱导因子-1α（（HIF-1α）、诱导型一氧化氮合酶（iNOS）在溃疡性结肠炎（UC）组织中的表达及与疾病活动性的关系。方法采用免疫组织化学方法检测51例UC患者（活动期38例、缓解期13例）和20例正常对照组中的HIF-1α、iNOS表达。结果UC患者活动期HIF-1α表达显著高于正常人，差异有显著性（153．29±15．26 vs 193．28±16．65，P〈0．01），缓解期HIF-1α表达与正常人差异无显著性（185．45±12．08 vs 193．28±16．65，P〉0．05），HIF-1α的表达与Walmsley评分标准呈正相关；iNOS在溃疡性结肠炎患者活动期的表达显著高于正常人，差异有显著性（151．32±14．62 vs 196．67±17．43，P〈0．01），缓解期的表达与正常人差异无显著性（189．93±15．20 vs 196．67±17．43，P〉0．05），iNOS的表达与Walmsley评分标准也呈正相关；HIF-1α和iNOS在溃疡性结肠炎中表达水平呈正相关（r=0．627，P〈0．05）。结论HIF-1α和iNOS均参与了溃疡性结肠炎的发病且与疾病的活动性有关，HIF-1α作为一种转录因子可能是iNOS基因表达中的一个重要调节者。     

HO-1诱导对大鼠急性肝损伤的保护作用及其机制

目的研究诱导HO-1高表达对大鼠急性肝损伤的保护作用及其机制探讨。方法随机将大鼠分成4组,即正常对照组、四氯化碳染毒组（CCl4）、血晶素（hemin）组、hemin＋CCl4组。采用western blot法测定HO-1蛋白的诱导表达情况：测定各组大鼠血清ALT、AST水平,肝组织MDA浓度和SOD活性以及Caspase-3活性和TNF-α水平变化;HE染色观察肝组织病理形态学改变。结果CCl4成功诱导了大鼠急性肝损伤,表现为染毒24 h后血清ALT、AST水平以及肝组织MDA浓度、Caspase-3活性和TNF-α水平均显著升高,SOD活性下降,和正常对照组相比差异有统计学意义（P〈0.01）;病理学检测结果显示肝脏有严重损伤,大量肝细胞发生凋亡。给予hemin预处理能显著诱导大鼠肝脏HO-1的表达,并对肝脏产生明显的保护作用,表现为大鼠血清ALT、AST水平和MDA浓度明显降低,SOD活性升高,组织TNF-α水平和Caspase-3活性明显低于CCl4组;此外,hemin预处理亦使染毒大鼠的病理学指标得到明显改善。结论HO-1诱导表达对急性肝损伤大鼠产生明显的保护作用,提示HO-1在防治急性肝损伤的病理生理过程中占有重要地位;HO-1的保护机制可能与其减轻脂质过氧化反应,抑制Caspase-3活性和降低TNF-α水平有关。     

肝泡状棘球蚴组织中HIF-1α、VEGFA的表达及对血管生成的作用

目的 探讨HIF-1α、VEGFA在沙鼠肝泡状棘球蚴组织的表达及血管生成过程中的作用。方法 126只沙鼠随机分成空白组(6只)、假手术组(60只)、模型组(60只),采用开腹直视肝脏穿刺法建立泡状棘球蚴动物模型,假手术组接种同体积的PBS,术后第3 d、7 d、14 d、28 d、42 d、56 d、70 d、84 d、98 d、112 d随机取6只沙鼠,取临近病变的边缘区组织及正常肝组织。采用HE观察病理改变;qRT-PCR、原位杂交、免疫组化检测HIF-1α、VEGFA表达,CD34标记微血管进行MVD计数。结果 HE染色根据病理特点将病程分为早期(14 d内)、中期(14 d~56 d)、晚期(56 d后)。模型组泡状棘球蚴组织随感染时间不同,其HIF-1αmRNA随感染时间呈动态改变,术后第14 d其表达量高于空白组、低于假手术组(F=82.732,P< 0.001);术后第42 d、112 d其相对表达量均高于空白组与假手术组(χ²=11.536,χ²=15.189,P< 0.01);模型组术后第14 d VEGFA mRNA相对表达量低于空白组及假手术组(χ²=15.174,P< 0.01);术后第42 d、112 d,VEGFA mRNA表达量均高于空白组与假手术组(χ²=15.158,χ²=15.158,P< 0.01)。模型组术后第14 d、42 d、112 d HIF-1α表达均高于空白组、假手术组(χ²=8.627,χ²=9.000,F=15.690,P< 0.01);模型组术后第14 d、42 d VEGFA表达高于空白组、假手术组(F=11.250, F=70.059,P< 0.001);模型组术后第14 d、42 d、112 d,其MVD-CD34均高于空白组、假手术组(χ²=12.517,P< 0.01,χ²=13.157,P< 0.01;χ²=13.220,P< 0.01)。结论 沙鼠感染肝泡状棘球蚴中期,病变边缘区HIF-1α、VEGFA表达均升高,同时伴有大量微血管生成,可能存在HIF-1α转录因子激活并上调VEGFA的表达,促进肝泡状棘球蚴组织边缘区的血管新生。;     

Hypoxia-inducible factor 1 alpha and vascular endothelial growth factor overexpression in ischemic colitis

AIM: To examine the etiology and pathophysiology in human ischemic colitis from the viewpoint of ischemic factors such as hypoxia-inducible factor 1 alpha (HIF-1 alpha and vascular endothelial growth factor (VEGF). METHODS: Thirteen patients with ischemic colitis and 21 normal controls underwent colonoscopy. The follow-up colonoscopy was performed in 8 patients at 7 to 10 d after the occurrence of ischemic colitis. Biopsy samples were subjected to real-time RT-PCR and immunohistochemistry to detect the expression of HIF-1 alpha and VEGF. RESULTS: HIF-1 alpha and VEGF expression were found in the normal colon tissues by RT-PCR and immunohistochemistry. HIF-1 alpha and VEGF were overexpressed in the lesions of ischemic colitis. Overexpressed HIF-1 alpha and VEGF RNA quickly decreased to the normal level in the scar regions at 7 to 10 d after the occurrence of ischemic colitis. CONCLUSION: Constant expression of HIF-1 alpha and VEGF in normal human colon tissue suggested that HIF-1 alpha and VEGF play an important role in maintaining tissue integrity. We confirmed the ischemic crisis in ischemic colitis at the molecular level, demonstrating overexpression of HIF-1 alpha and VEGF in ischemic lesions. These ischemic factors may play an important role in the pathophysiology of ischemic colitis.     

Expression of NOS and HIF-1α in human colorectal carcinoma and implication in tumor angiogenesis

AIM: To study CD34, CD105, inducible nitric oxide synthase (iNOS), endogenous nitric oxide synthase (eNOS), and hypoxia-inducible factor 1 (HIF-1) alpha expression in human colorectal carcinomas. METHODS: The tissue microarrays (TMAs) were made up of 80 cases of colorectal carcinoma and 80 cases of non-neoplasm colorectal mucosa. The expression of CD34, CD105, NOS and HIF-1alpha was detected by immunohistochemistry (S-P). RESULTS: iNOS and HIF-1alpha expression in colorectal carcinoma was significantly higher than in non-neoplasm colorectal mucosa (c2 = 43.166, P < 0.01; c2 = 10.4278, P < 0.01); eNOS expression in colorectal carcinoma was significantly lower than in non-neoplasm colorectal mucosa (c2 = 11.354, P < 0.01). The expression of iNOS correlated with differentiation (c2 = 18.141, P < 0.01), invasive depth (c2 = 4.748, P < 0.01), and Micro vessel density (MVD) (t = 2.327, P < 0.05). The expression of HIF-1alpha was correlated with infiltrating depth (c2 = 4.397, P < 0.05), Dukeos staging (c2 = 4.255, P < 0.05), and MVD (t = 2.272, P < 0.05). No correlation was found in eNOS expression. CONCLUSION: Over-expression of iNOS and HIF-1alpha in colorectal carcinoma is correlated with the biological character MVD.     

The role of calorie restriction and SIRT1 in prion-mediated neurodegeneration

A central focus of aging research is to determine how calorie restriction (CR) extends lifespan and delays diseases of aging. SIRT1, the mammalian ortholog of Sir2 in yeast, is a longevity factor which mediates dietary restriction in diverse species. In addition, SIRT1 plays a protective role in several models of neurodegenerative disease. We tested the role of SIRT1 in mediating the effects of CR in a mouse model of prion disease. Prion diseases are protein misfolding disorders of the central nervous system with many similarities to other neurodegenerative diseases, including deposition of aggregated protein, gliosis, and loss of synapses and neurons. We report that the onset of prion disease is delayed by CR and in the SIRT1 KO mice fed ad libitum. CR exerts no further effect on the SIRT1 KO strain, suggesting the effects of CR and SIRT1 deletion are mechanistically coupled. In conjunction, SIRT1 is downregulated in certain brain regions of CR mice. The expression of PrP mRNA and protein is reduced in the brains of CR mice and in SIRT1 knockout mice, suggesting a possible mechanism for the delayed onset of disease, as PrP levels are a critical determinant of how quickly mice succumb to prion disease. Surprisingly, CR greatly shortens the duration of clinical symptoms of prion disease and ultimately shortens lifespan of prion-inoculated mice in a manner that is independent of SIRT1. Taken together, our results suggest a more complex interplay between CR, SIRT1, and neurodegenerative diseases than previously appreciated.     

热卡限制对高脂饲养大鼠肝脏糖异生相关基因表达的影响

目的 观察热卡限制对高脂饲养大鼠肝脏Forkhead转录因子O1(FoxO1)、磷酸烯醇丙酮酸羧激酶(PEPCK)和葡萄糖6磷酸酶(G-6-P)mRNA表达的影响,探讨其可能机制.方法 24只雄性Wistar大鼠随机分为正常对照组(7只)、高脂组(9只)和热卡限制组(8只),分别给予正常饮食、高脂饮食和60%热卡限制饮食,共饲养12周.实验终点时取空腹血测血糖、胰岛素、甘油三酯和总胆固醇;称内脏脂肪重量及体重,计算内脏脂肪重量占体重百分比;逆转录聚合酶链法检测肝脏FoxO1、PEPCK和G-6-P mRNA表达变化;光镜观察肝脏组织学改变.结果 高脂饮食大鼠出现明显腹型肥胖,空腹血糖、空腹胰岛素、甘油三酯和总胆固醇均升高,FoxO1、PEPCK和G-6-P mRNA表达较正常组分别增加18.9%、33.8%和24.6%(P值均＜0.01),且光镜下出现肝脏脂肪变性;热卡限制后大鼠体重、内脏脂肪含量显著下降,空腹血糖、空腹胰岛素、甘油三酯、总胆固醇均有所下降,FoxO1、PEPCK和G-6-P mRNA表达较正常组分别减少26.6%、35.0%和34.3%(P值均＜0.01),同时镜下脂肪变性有所好转.结论 热卡限制能有效降低FoxO1、PEPCK和G-6-P基因表达,增强胰岛素信号传导,抑制肝脏糖异生,调节糖代谢.     

氯化钴对RNA干扰食管鳞癌细胞HIF-1α基因表达及功能的影响

目的研究氯化钴模拟缺氧对RNA干扰的缺氧诱导因子-1α（HIF-1α）基因表达及功能的影响，观察在人食管鳞癌细胞系Eca-109中RNA干扰抑制HIF-1α的效果。方法选择4组细胞分别为食管癌Eca-109细胞和3株稳定转染HIF-1αSiRNA的Eca-109细胞（本实验室编号分别为H2／14号、H3／15号、H2／20号细胞），分别用200、400、600、800μmol／L氯化钴模拟缺氧，缺氧培养时间分别为12、24．48、72h，采用Western印迹和逆转录聚合酶链反应（RT-PCR）检测HIF—1α蛋白及mRNA表达，同时Western印迹检测血红素加氧酶（HO-1）、基质金属蛋白酶-2（MMP-2）、葡萄糖转运体-1（Glut-1）、P53等相关基因的表达。结果4组细胞HIF-1α蛋白表达均随氯化钴浓度加大而增加，常氧与400μmol／L氯化钴处理24h后筛选出H3／15号细胞HIF-1α蛋白表达最少，与其它3组相比差异有统计学意义（P〈0．05），mRNA检测差异无统计学意义；干扰细胞常氧下HO-1、MMP-2、Glut-1、P53表达不同程度减少，缺氧后HO-1、P53表达增加，MMP-2、Glut-1表达无明显变化。结论RNA干扰能抑制Eca-109细胞的HIF-1α基因表达，从而不同程度减少HO-1、MMP-2、Glut-1、P53等相关基因表达；经氯化钴模拟缺氧筛选出干扰效果较好的一株细胞，为进一步研究HIF-1α的功能奠定了基础。     

The protective effect of erythropoietin on ischaemia/reperfusion injury of liver

BackgroundBesides its haematopoietic effect, erythropoietin (EPO) has multiple protective effects, i.e. antiapoptotic, antioxidant and angiogenic properties. The neuroprotective effects of EPO against ischaemia have all been demonstrated in cell culture and animal models. The aim of the study was to evaluate the effect of erythropoietin on ischaemia-reperfusion injury (I/R injury) of the liver.MethodsForty-eight adult male Sprague-Dawley rats weighing 250–300 g were divided into three groups: group I, hepatic ischaemia-reperfusion (Hepatic I/R); group II, hepatic ischaemia-reperfusion + EPO (Hepatic I/R+ EPO); group III, sham. Hepatic ischaemia was created by placing a microvascular clamp on the hepatic pedicle for 45 minutes. EPO was given to group II at a dose of 1000 U/kg 120 minutes before the onset of the ischaemia. Blood samples and liver tissues were obtained after 45 minutes of reperfusion from half of the rats in each group. The remaining rats were killed after a 24-hour observation period and blood and tissue samples were obtained. Blood alanine aminotransferase, tumour necrosis factor-α (TNF-α), interleukin-2 (IL-2) and liver tissue malondialdehyde (MDA) levels were determined. Liver tissue histopathology was also evaluated by light microscopy.ResultsIn rats with hepatic ischaemia, serum levels of ALT, TNF-α, IL-2 and liver tissue levels of MDA were reduced by the administration of erythropoietin and the histopathological score was also less severe.ConclusionThis study demonstrates that pre-ischaemic administration of EPO has protective effects on hepatic I/R injury.     

Effect of calorie restriction on liver and kidney glutathione in aging emory mice

Increases in antioxidant defense capacity have been associated with increases in the health and life span of calorie restricted animals. Emory mice develop late-life cataract, a lesion associated with oxidative damage and loss of lens glutathione (GSH). The effect of calorie restriction on GSH in liver and kidney in this model has not been explored. GSH and oxidized GSH (GSSG) were measured by HPLC in liver and kidney of Emory mice fed a control diet (C; 85% calories of ad-lib fed mice) or 60% calorie intake of C (R; 40% calorie restriction relative to C mice) for up to 22 mo age. Liver GSH concentration increased significantly in C and R mice from 4.5 to 12 mo old with no difference observed between the two groups. At 22 mo of age, liver GSH was lower than that of 12 mo old in both groups. As compared with GSH at 12 mo old, this decrease was almost twice as greater in C (70%, p=0.001) than in R mice (36%, p=0.02), so that R mice had a significantly higher concentration of GSH in liver than C mice at 22 mo of age (R = 32.8+5.1, C= 22.1+8.3 imol GSH/g protein, p<0.01). Liver GSSG was similar in C and R mice at 12 mo of age (4.45+1.35 vs. 4.75+1.83 imol GSSG/g protein), but increased in R mice at 22 mo (R=5:43±1.48; C=3.22±1.02, p<0.01). Therefore, at 22 mo old, total liver glutathione (GSH+GSSG) was higher in R than in C mice. There was no significant difference in GSH, GSSG and total GSH in kidney from C and R mice at these ages. Thus, calorie restriction reduces the age-related loss of GSH antioxidant capacity in liver but not kidney of Emory mice.     

Prognostic signif icance of HIF-2α/EPAS1 expression in hepatocellular carcinoma

AIM: To evaluate the prognostic signif icance of HIF- 2α/EPAS1 expression in hepatocellular carcinoma (HCC). METHODS: Surgical specimens from 315 patients with HCC as well as 196 adjacent noncancerous lesions and 22 cases of normal liver tissue were investigated by immunohistochemistry (IHC) for HIF-2α/EPAS1 using a standard detection system. Correlations with clinicopathological factors, VEGF, microvessel density (MVD), and prognosis were analyzed. RESULTS: Immunoreactivity of HIF-2α/EPAS1 was positive in 69.5% of HCC, 55.6% of adjacent noncancerous tissue, and 0% of normal liver tissue. And it was significantly correlated with tumor grade, venous invasion, intrahepatic metastasis, necrosis, and capsule infiltration. Correlation analysis of HIF-2α/EPAS1 with angiogenic factor VEGF (P < 0.001), and MVD (P = 0.016) was also noted. HIF-2α/EPAS1 protein was less frequently expressed in low MVD cases, whereas a high rate of expression was noted in cases with both medium and high MVD (P = 0.042). By Kaplan-Meier analysis, strong HIF-2α/EPAS1 staining (> 50% of tumor cells) in HCC correlated with a shortened survival in patients (Cox's regression, P < 0.001, r = 3.699). CONCLUSION: We conclude that HIF-2α/EPAS1 expression may play an important role in tumor progression and prognosis of HCC. Assessment of HIF-2α/EPAS1 expression in HCC may be used as a diagnostic tool and possibly a target in the treatment of HCC.     

CR1和CR3在慢性阻塞性肺疾病急性加重期的临床意义

目的初步探讨补体受体1和3（CR1和CR3）在COPD急性加重期的临床意义。方法采用流式细胞学技术测定CR1和CR3在慢性阻塞性肺疾病急性加重期,稳定期患者及健康对照组外周血中性粒细胞上的表达,并比较AECOPD患者细菌检出组与无细菌检出组中CR1和CR3的表达差异。结果 CR1和CR3的表达在细菌检出组和无细菌检出组,稳定期组,健康对照组之间存在显著的差异（P〈0.01）。结论 CR1和CR3在中性粒细胞表面的表达可以作为AECOPD患者细菌感染的一项免疫学指标。