DNA损伤诱导胃癌细胞端粒酶活性和TRF2表达增高

目的:检测在化疗药引起胃癌细胞DNA损伤过程中端粒酶、TRF1和TRF2的表达.方法:用不同浓度足叶乙甙处理胃癌细胞SGC7901和MKN28,分别在3,6,12,24和36h进行检测.采用实时定量TRAP分析检测端粒酶活性;用实时RT-PCR检测hTERTmRNA表达;用Westernblot和实时RT-PCR检测TRF1和TRF2表达.结果:两种细胞端粒酶活性及TRF2mRNA表达均在药物处理的早期明显增高(P<0.05),且显示明显的药物浓度依赖性(P<0.05);TRF1表达稍增高,但无统计学意义(P>0.05).hTERTmRNA表达无明显改变(P>0.05).TRF2表达在蛋白和mRNA水平均增高(P<0.05).结论:端粒酶和TRF2参与化疗药引起的胃癌细胞DNA损伤反应.     

TRF2小干扰RNA载体的构建及表达

目的:构建TRF2小干扰RNA载体,观察其在胃癌细胞中的表达．方法:根据pSilencer3．1-H1载体要求设计两对小干扰RNA,退火后连接入载体相应位点．经双酶切及测序鉴定后分别转染多药耐药衍生胃癌细胞SGC7901／ADR和SGC7901/VCR． G418选择培养液培养2 mo选取转染组及对照组细胞克隆．MTT法检测转染对细胞生长增殖速度的影响．细胞用阿霉素和足叶乙甙处理 6h后Western blot法检测TRF2表达．结果:成功构建TRF2小干扰RNA载体,建立稳定转染细胞株,转染后TRF2表达明显降低,对细胞的生长增殖速度无明显影响(P>0．05)．结论:成功构建TRF2小干扰RNA载体及稳定转染细胞株．     

酒精性肝病大鼠肝组织解偶联蛋白2的表达

解偶联蛋白(UCP)是线粒体内膜上可以调节质子跨膜转运的载体蛋白,具有解偶联活性,目前共发现5个亚型[1-2].其中U CP2通过调节质子跨膜转运参与能量消耗和脂质代谢,可能参与酒精性肝病(ALD)发病过程中的氧化应激与脂质过氧化.本研究旨在应用酒精灌胃建立大鼠ALD模型,动态观察UCP2蛋白及mRNA的表达情况,以探讨UCP2在ALD发病过程中的作用及其机制,为有效防治ALD提供新的理论依据.     

红霉素对香烟烟雾刺激的人巨噬细胞组蛋白去乙酰化酶2表达降低的抑制作用

目的 探讨红霉素对香烟烟雾刺激的人巨噬细胞组蛋白去乙酰化酶2（H DAC2）表达的影响及其机制研究.方法 体外培养人类单核细胞系U937细胞,用佛波脂将其诱导分化为人巨噬细胞.按传统方法制备好香烟烟雾提取物（CSE）用于实验.将传代后的细胞分组：对照组、CSE组、红霉素＋CSE组、HDAC的特异性抑制剂曲古霉素A（TSA）组.应用流式细胞仪检测人巨噬细胞活性氧（ROS）的含量;应用蛋白印迹实验（Western blot）检测HDAC2和核因子κB（NF-κB）蛋白表达;通过酶联免疫吸附试验检测细胞上清液中肿瘤坏死因子α（TNF-α）的浓度.结果 CSE可刺激人巨噬细胞释放ROS,具有浓度依赖性和时间依赖性;红霉素（1 mg/L）预孵育24 h可以增强1％ CSE抑制的人巨噬细胞HDAC2蛋白表达;红霉素（1 mg/L）预孵育24 h可以下调1％ CSE诱导的NF κB的活性;红霉素（1 mg/L）预孵育24 h可以抑制1％CSE诱导的TNF-α的合成和释放.结论 红霉素通过提高HDAC2蛋白表达进而抑制香烟烟雾诱导氧化应激导致的NF-κB活性增强和炎症介质TNF-α的合成和释放.     

解耦联蛋白2在急性肝功能衰竭大鼠肝脏中的动态表达和意义

目的 探讨急性肝功能衰竭(ALF)大鼠肝脏线粒体解耦联蛋白(UCP)2的表达趋势及意义.方法 健康雄性SD大鼠36只,分为对照组和模型组,模型组冉分为6、12、24、36和48 h 5个亚组,每组6只.模型组腹腔内注射D-氨基半乳糖(D-Gal)和脂多糖(LPS)诱导大鼠ALF模型.采用HE染色,光学显微镜下观察肝组织损伤情况,采用RT-PCR和免疫组织化学检测不同时间点肝脏UCP2 mRNA转录及其蛋白表达,同时测定各时间点血清ALT、AST和肝组织丙二醛(MDA)的变化.各实验组问数值比较采用SNK检验.结果 模型组肝组织呈炎性细胞浸润和明显坏死的ALF特征;模型组ALT、AST、MDA值均明显高于对照组[(24.0±2.0)U/L,(82.3士16.9)U/L,(2.55±0.22)μmol/g],且造模24 h达高峰[(8346.7±1363.1)U/L,(9766.7±1274.1)U/L,(8.34±1.13)μmol/g;均P<0.05];UCP2蛋白和UCP2 mRNA在正常肝组织中几乎不表达,D-Gal和LPS处理后6 h表达均硅著增加(P<0.05),24 h表达最强,且模型组相邻时间点之间差异有统计学意义(P<0.05).结论 成功构建大鼠ALF模型,大鼠ALF时UCP2蛋白和UCP2mRNA的表达水平与肝损伤程度及氧化应激水平有关.     

Decreased expression of Mac-2 (carbohydrate binding protein 35) and loss of its nuclear localization are associated with the neoplastic progression of colon carcinoma.

The Mac-2 lectin (carbohydrate binding protein 35) is a soluble, 32- to 35-kDa phosphoprotein that binds galactose-containing glycoconjugates. We report here that the colonic epithelium is a major site of Mac-2 expression in vivo based on immunohistochemistry of human tissue specimens. In this epithelium, proliferating cells at the base of the crypts do not express Mac-2 but its expression increases with differentiation along the crypt-to-surface axis. Mac-2 expression is concentrated in the nuclei of these differentiated epithelial cells. The progression from normal mucosa to adenoma to carcinoma is associated with significant changes in Mac-2 nuclear localization and expression. In all adenomas (9/9) and carcinomas (13/13) examined, Mac-2 was not present in the nucleus but was localized in the cytoplasm. Sequencing of Mac-2 cDNAs from normal mucosa and carcinoma revealed no specific mutations that could account for this loss of nuclear localization. We also observed a 5- to 10-fold decrease in Mac-2 mRNA levels in cancer compared to normal mucosa as well as a significant reduction in the amount of Mac-2 protein expressed. These observations suggest that Mac-2 exclusion from the nucleus and its decreased expression may be related to the neoplastic progression of colon cancer.     

Factors associated with longitudinal changes in serum concentrations of Mac-2 binding protein: A prospective 3-year observational study

Background and aimsMac-2 binding protein (M2BP) plays an important role in cell adhesion. In a recent cross-sectional study we reported that serum M2BP concentrations may reflect silent atherosclerosis. The aim of the present prospective follow-up study was to investigate possible relationships between changes in concentrations of M2BP and other factors over a >3-year period.Methods and resultsThe present study enrolled subjects who visited Enshu hospital from 2014 to 2015 for a periodic physical check-up and then attended for another physical check-up after >3 years (n = 174). Factors affecting changes in M2BP concentrations were investigated at both baseline and follow-up. Subjects with liver dysfunction, a history of hepatic disease, malignant neoplasm, or cardiovascular events at baseline were excluded. Univariate and multivariate regression analyses showed that changes in serum M2BP concentrations during the follow-up period were significantly associated with changes in low-density lipoprotein cholesterol (LDL-C), triglyceride, and oxidative stress marker derivatives of reactive oxygen metabolites (d-ROM) concentrations. Moreover, the increase in LDL-C was significantly greater in subjects in whom M2BP concentrations increased during the follow-up period. Logistic regression analysis with an endpoint of increased M2BP revealed that increased LDL-C was an independent determinant of an increase in M2BP during the follow-up period.ConclusionDuring the observation period of >3 years, serum M2BP concentrations were increased in subjects who also exhibited increases in levels of metabolic parameters, especially LDL-C, and the oxidative stress marker d-ROM. These results support that serum M2BP reflects one of the contributors to the progression of silent atherosclerosis.