槲皮素对小鼠体内和体外NIH-3T3细胞的抗氧化作用及其机理

目的 比较槲皮素对小鼠体内和体外H2O2处理前/后的NIH-3T3细胞抗氧化效应的差异并探讨其作用机理。方法 将NIH-3T3细胞分为4组,槲皮素前保护组（Qb）：槲皮素后保护组(Qa)：H2O2组 (H2O2)和对照组(C)。 用试剂盒检测细胞内T-AOC、SOD、GSH-Px、GSH、MDA、NOS和NO2-/NO3-的水平;MTT和TUNEL法检测细胞凋亡率和生存率;免疫印迹和免疫组化技术检测细胞的cyclin D1、PTEN、NF-kB、HSP-70、Bcl-2、Bax、 及caspase-3的表达。另将20只Wistar大鼠等分为对照组和实验组,检测槲皮素灌胃前、后1、2及24h血浆内T-AOC、SOD、GSH-Px、GSH、MDA、NOS和NO2-/NO3-的水平。结果H2O2组细胞凋亡率增高,cyclin D1、PTEN及Bcl-2的表达下调;Bax、HSP-70、NF-kB及caspase-3的表达上调;T-AOC,SOD,GSH-Px及GSH水平降低,NOS、NO2-/NO3-及MDA增高。动物灌胃后1h/2h血浆中T-AOC、SOD、GSH-Px、GSH,NOS及NO2-/NO3-的水平增高,MDA降低;24h后基本恢复。结论 槲皮素在体内外皆可通过调节抗氧化酶体系发挥抗氧化作用,其效应依据H2O2处理与否和处理前/后等细胞的异质性而呈现一定差异。     

携带pcDNA3s与EGFPC3质粒的重组减毒沙门氏菌的免疫性与EGFP的表达

目的研究HBsAg DNA重组减毒鼠伤寒沙门氏菌的免疫性与增强型绿色荧光蛋白(EGFP)基因EGFPC3在小鼠体内的表达。方法将质粒pcDNA3s与pEGFPC3分别电转化减毒鼠伤寒沙门氏菌SL8786,构建重组减毒鼠伤寒沙门氏菌SL8786/EGFPC3与SL8786/pcDNA3s。利用时间分辨荧光免疫分析技术(TRFIA)检测小鼠的血清抗体的含量,以及重组菌引起的T细胞增殖和细胞毒性T淋巴细胞(CTL)应答。用荧光显微镜观察EGFPC3表达。结果口服SL8786/p cDNA3s免疫小鼠后,可诱导产生较强的特异性CTL应答。口服免疫小鼠后第11周抗-HBs含量最高。用流式细胞术检测贴壁培养的2只小鼠的脾细胞中SL8786/EGFPC3阳性率分别为19.20%和17.36%,而SL8786/pcDNA3口服的2只小鼠脾细胞中的EGFP阳性率分别仅为1.95%和1.63%。给小鼠口服SL8786/EGFPC33周后,在小鼠肝脏、脾脏、肾脏、胸腺、十二指肠及肌肉等组织中,均有EGFPC3的表达。结论口服SL8786/pcDNA3s在小鼠体内诱导了特异性细胞和体液免疫应答。携带EGFPC3的重组鼠伤寒沙门氏菌SL8786/EGFPC3在小鼠的部分组织中产生绿色荧光表达,该重组菌的构建为减毒沙门氏菌作为活载体基因工程疫苗的研制提供了一个优良模型。     

大肠癌组织中PIK3CA和PIK3CB的表达及意义

目的观察癌基因PIK3CA、PIK3CB在大肠癌组织中的表达及其相互关系,并分析PIK3CA和PIK3CB的表达对大肠癌患者预后的影响。方法采用免疫组化EnVision法染色并结合图像进行分析,探讨PIK3CA和PIK3CB在大肠癌组织及正常大肠黏膜组织中的表达;应用RT-PCR和Western blot技术分别检测PIK3CA mRNA和PIK3CB mRNA及其相应蛋白的表达;回访125例大肠癌术后患者,生存分析用Kaplan-Meier法,生存曲线用Log-Rank检验。结果在300例大肠癌蜡块组织及正常黏膜组织中,PIK3CA表达的平均光密度值分别为:10.087 9±2.148 7、6.937 6±2.065 3,PIK3CB表达的平均光密度值分别为:15.870 6±2.877 7、8.693 2±2.442 4。PIK3CA和PIK3CB在大肠癌组织中的表达具有相关性(r=0.68,P<0.05);RT-PCR检测结果显示,与正常大肠黏膜组织相比,大肠癌组织中PIK3CA mRNA和PIK3CB mRNA的表达水平均明显升高,且二者表达结果呈正相关(r=0.74,P<0.05);Western blot法检测发现PIK3CA和PIK3CB蛋白表达水平同样升高,二者表达结果呈正相关(r=0.71,P<0.05);生存分析显示PIK3CA和PIK3CB高表达与患者死亡风险具有明显相关性。结论 PIK3CA和PIK3CB在大肠癌组织中高表达,与正常黏膜组织中的表达相比差异较明显,且PIK3CA和PIK3CB在大肠癌的发生、发展中具有协同作用,PIK3CA、PIK3CB高表达亦是大肠癌患者预后相关的因素之一。检测大肠癌患者PIK3CA和PIK3CB的表达对于控制大肠癌的进展、确定大肠癌的靶点治疗及判断预后具有重要意义。     

pFlag-dlx3载体的构建及在iMDP3中的表达

目的构建真核表达载体p Flag-dlx3,并将其转染成牙本质样细胞株i MDP3,检测外源dlx3基因在i MDP3细胞内的表达。方法提取新生小鼠牙髓总RNA,RT-PCR扩增Dlx3目的基因片段,并将该片段克隆至PCR-TopoⅡ载体中;经鉴定正确后目的基因与表达载体p Flag-CMV连接;Lipofectamin 2000介导重组质粒p Flag-dlx3转染i MDP3;Western-blot鉴定外源性dlx3在细胞内的表达。结果重组p Flag-dlx3质粒经酶切、测序鉴定正确;i MDP3细胞内检测到外源dlx3蛋白的表达;转染p Flag-dlx3组dlx3蛋白的表达量显著高于正常组。结论成功构建真核表达载体p Flag-dlx3,且外源dlx3基因可在i MDP3细胞内过表达。     

Ezh2、Runx3和caspase-3在子宫内膜样腺癌中的表达及相关性

目的 探讨Ezh2、Runx3和caspase-3在正常子宫内膜、子宫内膜增殖症和子宫内膜样腺癌组织中的表达及相关性.方法 采用组织芯片技术和免疫组织化学SP法检测30例正常子宫内膜组织、30例单纯性增生内膜、30例复杂伴不典型增生内膜、72例子宫内膜样腺癌组织中Ezh2、Runx3和caspase-3蛋白的表达.结果 Ezh2、Runx3和caspase-3蛋白在子宫内膜样腺癌组织中的阳性表达率分别为83.3%(60/72)、26.4%(19/72)、33.3%(24/72);Ezh2在腺癌组织中的阳性率明显高于正常和各型增生内膜(16.7%、33.3%、63.3%;P<0.05),Runx3在腺癌组织中的阳性率明显低于正常和单纯性增生内膜(80.0%、56.7%;P<0.01),caspase-3在腺癌组织中的阳性率明显低于正常和各型增生内膜(86.7%、73.3%、63.3%;P<0.01),差异均有统计学意义.Ezh2和Runx3的阳性表达与组织分级、临床分期和肌层浸润深度均有关(均P<0.05),与淋巴结转移均无关(P>0.05);caspase-3的阳性表达与组织分级有关(P<0.05),而与临床分期、浸润深度及淋巴结转移均无关(P>0.05).Ezh2与Runx3的蛋白表达呈负相关(rs=-0.262,P<0.05).结论 Ezh2、Runx3和caspase-3可能在子宫内膜样腺癌的发生发展过程中发挥重要作用,联合检测其表达可为子宫内膜样腺癌的预后监测及新基因靶点的探寻提供依据.

Abstract：

Objective To investigate the role of the expression of Ezh2, Runx3 and caspase-3 proteins and their correlation in the pathogenesis of endometrial carcinoma. Methods Expression of Ezh2, Runx3 and caspase-3 proteins was examined by tissue microarray technique and immunohistochemistry (SP method) in 72 cases of endometrial adenocarcinomas, 60 endometrial hyperplasia and 30 normal endometrial tissues. Results The positive expression rates of Ezh2, Runx3 and caspase-3 proteins in endometrial adenocarcinomas were 83.3%(60/72), 26.4%(19/72) and 33.3%(24/72), respectively. The positive rate of Ezh2 protein in endometrial carcinomas was higher than that in normal endometrium and endometrial hyperplasia(16.7%, 33.3%, 63.3%;P<0.05). However, the positive rate of Runx3 in endometrial carcinomas was lower than that in normal endometrium and endometrial hyperplasia(80.0%,56.7%; P<0.01). The positive rate of caspase-3 protein in endometrial carcinomas was lower than that in normal endometrium and endometrial hyperplasia(86.7%, 73.3%, 63.3%;P<0.01). Positive expression of Ezh2 and Runx3 was related to the histological grade, FIGO stage, and depth of invasion of endometrial adenocarcinomas(P<0.05), but it was not related to the lymph node metastasis (P>0.05). Positive expression of caspase-3 protein was related to the histological grade(P<0.05), but it was not related to the FIGO stage, depth of invasion and the lymph node metastasis of endometrial adenocarcinomas (P>0.05). The expression of Ezh2 protein was negatively correlated to that of Runx3 (rs=-0.262, P<0.05). Conclusions Abnormal expression of Ezh2, Runx3 and caspase-3 proteins is associated with the development and progression of endometrioid adenocarcinoma. Combined analysis of Ezh2, Runx3 and caspase-3 may offer prognostic information for patients with endometrial cancer.     

Expression and localization of Aquaporin 3 (AQP3) in folliculogenesis of ewes

The mRNA expression and localization of Aquaporin 3 (AQP3) were investigated in the ovarian follicles of ewes at different stages of development (primordial, primary, secondary, small, and large antral). The gene expression was quantified by qPCR, while the protein identification and localization were determined by Western blot and immunohistochemistry, respectively. Analysis revealed that AQP3 mRNA was detected only in the antral follicles, whereas the protein expression was detected in the oocyte and granulosa cells in all stages of follicular development. The latter observation suggests that the presence of AQP3 in follicles of all categories, especially in the antral follicles, provides novel insights on the mechanisms that regulate the flow of water between cells during the formation of antral follicles in sheep.     

MMP-3和TIMP-3在卵巢癌中的表达及其意义

目的探讨MMP-3和TIMP-3在卵巢癌的表达及其意义。方法本文采用光镜、透射电镜和免疫组化方法对27例卵巢癌组织中的MMP-3及TIMP-3表达进行检测。结果结果表明,临床分期为晚期的卵巢癌组织中MMP-3阳性细胞的数密度和面密度明显高于早期,而TIMP-3则低于早期。电镜下,早期淋巴细胞和树突状细胞浸润较多,癌细胞没有穿过基底膜;晚期淋巴细胞和树突状细胞浸润较少,可见癌细胞穿基底膜。结论MMP-3和TIMP-3的表达程度及MMP-3/TIMP-3的比值,可作为判断卵巢癌病程的指标。     

Mapping the amplification of EIF3S3 in breast and prostate cancer

Gain of chromosome arm 8q is a frequent genetic alteration in breast and prostate cancer. Two amplified subregions, 8q21 and 8q23-24, have been identified with comparative genomic hybridization (CGH). We have recently demonstrated that the EIF3S3 (eIF3-p40) gene, located at 8q23, is often amplified and overexpressed in both breast and prostate cancer. Here, we used fluorescence in situ hybridization (FISH) to map the amplified region around EIF3S3 in primary breast cancers and cell lines. The size of the common highly amplified region was about 2.5 Mb between the markers D8S1668 and WI-7959. Next, we analyzed the expression of all expressed sequence tags (ESTs) located within and near this region by RNA slot blot hybridization. In addition to EIF3S3, three anonymous ESTs and EXT1 were found to be highly expressed in cancer cell lines with the amplification at 8q23-q24. However, the anonymous ESTs were located outside the minimal highly amplified region and EXT1 was overexpressed only in one of the cancer cell lines with 8q amplification. Since EIF3S3 was the only consistently overexpressed gene located in the minimal highly amplified region, it is the strongest candidate target gene for 8q23-q24 amplification.     

Autoradiographic localization of3H-GABA and3H-muscimol binding in rat cerebellar cultures

Summary Autoradiographic studies were conducted on the binding of³H-GABA and³H-muscimol in cultures of rat cerebellum. Binding sites for both substances were observed on many cerebellar neurones, such as Purkinje cells and interneurones, but not on glial cells. Binding of³H-GABA and³H-muscimol was inhibited by unlabelled GABA and by the GABA antagonist bicuculline.     

Degradation of P(3HB) and P(3HB-co-3HV) in biological media

The biodegradability of oriented fibers made of polyhydroxybutyrate (P(3HB)) and its co-polymer with β-hydroxyvalerate (P(3HB-co-3HV)) was investigated in buffer solutions and in biological media in vitro and in vivo. The fibers of both polymer types demonstrated resistance to hydrolytic degradation in buffer solutions at 38°C and pH from 4.5 to 7.0 (for up to 180 days). It has been found that the biodegradation of the fibers in vitro in blood and serum and in vivo is accompanied by weight losses and minor changes in the microstructure with no significant losses in the tensile strength over a long time (up to 180 days). The biodegradation rate of the less crystalline co-polymer P(3HB-co-3HV) fibers was 1.4–2.0-times higher than that of the homopolymer P(3HB). It has also been shown that the degradation of the fibers in vivo is influenced both by tissue fluid enzymes and cells (macrophages and foreign-body giant cells). The fibers were eroded on the surface only with no gross defects and no dramatic effects on their mechanical performance.     

Degradation of P(3HB) and P(3HB-co-3HV) in biological media

The biodegradability of oriented fibers made of polyhydroxybutyrate (P(3HB)) and its co-polymer with beta-hydroxyvalerate (P(3HB-co-3HV)) was investigated in buffer solutions and in biological media in vitro and in vivo. The fibers of both polymer types demonstrated resistance to hydrolytic degradation in buffer solutions at 38 degrees C and pH from 4.5 to 7.0 (for up to 180 days). It has been found that the biodegradation of the fibers in vitro in blood and serum and in vivo is accompanied by weight losses and minor changes in the microstructure with no significant losses in the tensile strength over a long time (up to 180 days). The biodegradation rate of the less crystalline co-polymer P(3HB-co-3HV) fibers was 1.4-2.0-times higher than that of the homopolymer P(3HB). It has also been shown that the degradation of the fibers in vivo is influenced both by tissue fluid enzymes and cells (macrophages and foreign-body giant cells). The fibers were eroded on the surface only with no gross defects and no dramatic effects on their mechanical performance.     

Electrochemical copolymerization of 3-dodecylthiophene and 3-methylthiophene

3-Dodecylthiophene ( 1a ) and 3-methylthiophene ( 1b ) were copolymerized electrochemically. The visible and IR spectra of the neutral copolymers prove the copolymers to be of random structure, which can be denoted as that of poly(3-alkyl-2,5-thienylene)s. 1b polymerizes with preference over 1a . With increasing fraction of monomeric units of 1b in the copolymers, the conductivity increases and the solubility of the neutral copolymers decreases. The copolymer containing 58 mol-% monomeric units of 1b has a high conductivity of 220 S/cm and a good solubility of 75 wt.-% in chloroform. ¹H NMR spectra of poly(3-dodecylthienylene) and copolymers show two chemical shifts for the α(1)-methylene protons of the monomeric units of 1a as well as of the methyl protons of the monomeric units of 1b . This fact may be explained by the presence of head-to-head and other constitutions.     

14-3-3 Protein isoforms and atypical patterns of the 14-3-3 assay in the diagnosis of Creutzfeldt-Jakob disease

A positive 14-3-3 assay is a criterion for probable Creutzfeldt-Jakob disease (CJD). Cerebrospinal fluid (CSF) 14-3-3 is usually detected by immunoblot using an antibody that recognizes all of the 14-3-3 isoforms. In a few cases, the antibody recognizes an inferior band and this pattern is associated with false positive results. We analyzed 43 CSF (26 CJD, 17 controls) samples using antibodies against specific isoforms (beta, epsilon, gamma, tau, xi) and compared the results with those obtained with the standard antibody. The anti-gamma and anti-beta antibody achieved similar results but the presence of atypical patterns made the standard antibody more accurate for the CJD diagnosis. To study the nature of the inferior band, CSF samples were probed with antibodies against light chain immunoglobulins, and immunoblots of human IgG with the standard antibody. The experiments suggested a cross-reaction of the anti-14-3-3 antibody with light chain immunoglobulins.