氯霉素处理对大鼠脑皮质细胞色素氧化酶亚基表达协调性的影响

目的 通过应用线粒体蛋白合成特异抑制剂氯霉素(CAP)处理后 ,观察大鼠脑皮质线粒体COX亚基Ⅰ和Ⅳ以及线粒体转录因子 (mtTFA)、核呼吸因子 1(NRF 1)等相关转录调控因子的基因表达改变 ,以了解在特异性阻断线粒体蛋白翻译后细胞核与线粒体基因组在转录与翻译水平上的变化 ,探讨mtDNA与nDNA组之间的相互关系。方法 成年♂Wistar系大鼠分为对照组和药物处理组 ,药物处理连续7d腹腔注射 0 5 %CAP ,10 0mg·kg-1,1次 / 12h。RT PCR分析脑组织COXI、COXIV、NRF 1及mtTFAmRNA水平 ,Westernblot分析脑组织线粒体COXI、COXIV及组织NRF 1蛋白。结果 CAP组脑线粒体COX亚基Ⅰ蛋白含量降低(P <0 0 5 ) ,COXⅣ /COXⅠ比值增高 ,COXImRNA水平以及COXIVmRNA与蛋白水平均无改变 (P >0 0 5 )。同时 ,mtTFA的mRNA以及NRF 1的mRNA及蛋白含量与对照组比较均无差异。结论 线粒体内COXⅠ蛋白水平的变化对mtDNA的转录不存在反馈调节机制 ,而且线粒体基因组对细胞核基因组也没有可观察到的反馈效应。

鼠白细胞介素-12双亚基共表达质粒的构建及在体内外的表达

目的 :构建双亚基共表达鼠白细胞介素 - 12 (m IL- 12 )真核表达质粒 ,并观察其在体内外的表达。方法 :将m IL - 12 p35和 p4 0全长编码 c DNA构建在 pc DN A 3.1载体上 ,然后把 p35表达单元 (CMV- p35 - BGH PA)插入pc DNA 3.1/ p4 0载体 ,使两个目的基因均受各自的启动子 CMV控制 ,构建成 m IL - 12双亚基共表达质粒 p Cm IL -12 ,并进行体内外表达。结果 :p Cm IL - 12在体外转染 COS- 7细胞后 ,经 EL ISA证实有 m IL- 12表达 ,其表达上清能在体外明显增强小鼠 NK细胞活性。小鼠皮内注射 p Cm IL - 12亦能增强小鼠 NK细胞活性。结论 :所构建的质粒在体内外均能表达有生物学活性的 m IL-

听源性惊厥大鼠惊厥后NMDA受体NR1亚基蛋白表达和功能研究

目的 :探讨惊厥后大脑皮质N 甲基 D 门冬氨酸 (NMDA)受体亚基NR1蛋白表达及其功能改变与癫痫发病机制的关系。方法 :P77PMC大鼠按惊厥后不同时间随机分为 6组 ,免疫组化每组 6个样本 ,配基结合实验每组 5个样本 ,制备大脑冰冻切片和大脑皮质突触膜。利用免疫组化技术 ,观察听源性癫痫易感大鼠P77PMC惊厥后不同时间NR1亚基蛋白的表达 ,应用配基结合实验观察听源性惊厥大鼠P77PMC惊厥后大脑皮质突触膜对3H MK80 1的结合改变 ,以及NMDA受体激动剂对其结合的影响。结果 :大脑皮质在惊厥后 4hNR1蛋白表达即开始增加 ,2 4～48h达高峰。反应体系中无任何NMDA受体激动剂时 ,惊厥后大脑皮质突触膜对 3H MK80 1的结合有增加趋势 ,但差异无显著性 (P >0 .0 5 ) ;当反应液中加入NMDA受体激动剂时 ,惊厥后大脑皮质突触膜对 3H MK80 1的结合明显增加 ,除惊厥后 4h与正常对照组比较差异无显著性外 ,其他惊厥后各时间点与正常对照组比较差异均有显著性(P <0 0 1)。结论 :听源性惊厥大鼠P77PMC惊厥后 ,大脑皮质NMDA受体NR1亚基蛋白表达增加 ,NMDA受体对其激动剂的敏感性增加 ,提示NR1亚基参与了P77PMC大鼠惊厥的发生与发展 ;

基于TCGA数据库探索POLA2基因在肝癌中的表达及其与预后的相关性

目的探索DNA聚合酶α亚基B(POLA2)在肝癌中的表达,分析POLA2表达与肝癌患者临床病理特征和预后的关系。方法利用肿瘤基因组图谱(TCGA)数据库和qRT-PCR技术分析POLA2在肝癌组织及肝癌细胞中的表达水平,利用人类蛋白表达图谱(HPA)数据库评估POLA2蛋白在肝癌组织中的表达水平;应用ROC曲线评估POLA2对肝癌诊断的敏感性,采用Logistic回归分析POLA2表达与肝癌临床病理特征的相关性,通过Kaplan-Meier法、单及多因素Cox回归模型分析POLA2的表达与肝癌患者预后的关系。并进一步寻找TCGA数据库中POLA2共表达基因,进行基因本体(GO)生物功能及京都基因与基因组百科全书(KEGG)通路富集分析。同时通过肿瘤免疫评估资源(TIMER)数据库分析POLA2与肝癌免疫浸润的相关性。结果POLA2在肝癌组织和肝癌细胞中呈显著高表达(均P<0.05),ROC曲线下面积为0.954。POLA2表达与肝癌TNM分期、组织学分级和临床分期均密切相关(均P<0.01),单及多变量Cox分析表明高表达的POLA2可作为肝细胞癌(HCC)的独立生物学标志物。GO生物功能及KEGG通路富集分析提示,POLA2基因可能参与保持解旋酶活性和DNA依赖的ATP酶活性,主要富集于细胞周期、DNA复制和范可尼贫血通路。TIMER数据库分析显示POLA2与免疫浸润细胞(Th2细胞、B细胞、CD4+T细胞、巨噬细胞、中性粒细胞)具有显著正相关(均P<0.01)。结论POLA2表达与肝癌患者预后及免疫浸润具有相关性,可作为肝癌诊断及预后评估的重要生物学靶标。

向日葵V-ATPasea3亚基基因的克隆及表达分析

采用RACE技术,从向日葵P50中克隆V-ATPase a3亚基基因c DNA全长,并进行生物信息学分析;利用实时荧光定量PCR分析不同浓度、不同时间的Na Cl、ABA和PEG模拟干旱胁迫条件下V-ATPase a3亚基基因的表达特征,以及相同胁迫条件下该基因在向日葵不同器官的表达特征。序列分析表明,该基因c DNA全长2 873bp,含5'-UTR 109bp、3'-UTR 295bp及编码区2 469bp,编码822个氨基酸,其编码蛋白质的理论分子质量为204.55k Da,等电点为6.29,Gen Bank登录号为KU315054。该基因编码的蛋白质为疏水性的跨膜蛋白,亚细胞定位预测其在质膜上。向日葵V-ATPase a3亚基与已报道的10种植物的V-ATPase a3亚基的同源蛋白有高度相似的保守区域,在进化上与朝鲜蓟的亲缘关系最近。实时荧光定量PCR结果表明,向日葵受到Na Cl、ABA和PEG模拟干旱三种非生物胁迫后,V-ATPase a3亚基基因均上调表达,但表达模式不同,不同器官存在特异性表达差异。研究认为,V-ATPase a3亚基基因响应了向日葵非生物胁迫的应答,为加强对V-ATPase基因的利用奠定基础。

Gene Cloning and Expression of PKA Catalytic Subunit and Its Activity in Arabidopsis thaliana

Protein phosphorylation and dephosphorylation are the general means of regulation metabolism within a cell. A PKA catalytic subunit was found in Arabidopsis genome using Blast software. The cDNA was cloned by RT-PCR and sequencing result indicated a high degree of homology at protein level. The cDNA was subcloned into pET30a (+) and expressed in E. coli at different temperatures. The target protein was insoluble when induced at 37degreesC, while dissolvable if induced at 22degreesC with 0.01 mmol/L IPTG. Ni2+-NTA affinity chromatography was used to purify the target protein, which was shown to have cAMP-dependent protein kinase activity. Western blotting analysis indicated that stress treatments affected the expression of PKA catalytic subunit at protein level just to a small extent.

Expression Level of u-PAR in Different Invasive PC-3M Cell Subclones

Objective: To select a target molecule associated with invasive potential in PC-3M cell. Methods: Cell subclones were isolated from PC-3M cell line with the method of limited dilution and their invasive ability characterized by monolayer invasion assay. The expression of u-PAR in the cell subclones at mRNA and protein levels was assayed respectively by non-competitive quantitative RT-PCR and immunohistochemical assay. Results: The expression level of u-PAR in highly invasive cell subclones was higher than that of lower invasive subclones. Conclusion: The higher expression level of u-PAR is associated with the relative strong invasive ability of PC-3M subclones. It is indicated that the u-PAR might be a promising target molecule for inhibiting invasion of highly invasive PC-3M cell subclones.

Value of endoscopic methylene blue and Lugol's iodine double staining and detection of GST-n and telomerase in the early diagnosis of esophageal carcinoma

AIM：To explore the expressions of GST-π and telomerase activity in esophageal carcinoma and premalignant lesions and to investigate the value of endoscopic methylene blue （MB） and Lugol＇s iodine double staining. METHODS： Seventy-two patients with esophagopathy were sprayed endoscopically with MB and Lugol＇s iodine in proper order and the areas stained blue and brown, and the area between the blue and brown stains were obtained. Depending on the pattern of mucosal staining, biopsy specimen was obtained. GST-π and telomerase activity in specimens were examined by immunohistochemistry and PCR-based silver staining telomeric repeat amplification protocol, respectively. RESULTS： After MB and Lugol＇s iodine staining, the area between both the colors was obtained in 64 of the 72 patients and the areas were stained blue and brown in all of the 72 patients. Association test of two simultaneous ordinal categorical data showed a correlation between the esophageal mucosal staining and the esophageal histology （P〈0.005）. The expression of GST-π and telomerase activity in esophageal carcinoma and premalignant lesions increased. The expression of GST-π and telomerase activity in dysplasia and carcinoma was significantly higher than that in normal epithelium （P〈0.005）. The expression in hyperplasia was slightly higher than that in normal epithelium. With the lesions progressing from low- to moderate- to high-grade dysplasia, the positive rate increased （P〈0.025）. Expression of GST-π was correlated with that of telomerase activity in dysplasia and carcinoma （φ= 0.4831, P〈0.005;φ= 0.3031, P〈0.025, respectively）; but there was no correlation between them in normal epithelium and hyperplasia. CONCLUSION： The expression of GST-π and telomerase may be an early event in the carcinogenesis of esophagus. They may play an induced and synergistic role with each other in the carcinogenesis of esophagus. Endoscopic MB and Lugol＇s iodine double staining and detection of GST-π and telomerase activity may contribute to the early diagnosis of esophageal carcinoma.

Gastric cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine in rat with ulcers and expressions of ras and c-erbB2 genes

Objective: To observe the series of pathological changes during the development of gastric adenocarcinoma in ulcerative rats induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and the expression profile of related oncogenic protein.Methods: MNNG was administered in rats with ulcers due to acetic acid treatment to induce gastric cancer, and the protein expressions of ras and c-erbB2 genes in the ulcer were examined immunohistochemically along with pathological examination.Results: The incidence of gastric adenocarcinoma in the model group reaches 40% (6/15), while none of the rats developed cancer in the control group with ulcers.Positive expressions of the proteins of p21ras and c-erbB2 were observed in the tissues undergoing canceration in the 6 rats of model group, but were not observed in the 5 control rats; p53 protein expression, however, failed to be detected in both groups.Conclusion: A new animal model of gastric cancer has been established in rats with gastric ulcer after MNNG treatment, which may facilitate the pharmacological research of gastric cancer.

EXPRESSION AND SUBCELLULAR LOCALIZATION OF P9-ZFD PROTEIN IN PATIENTS WITH MYASTHENIA GRAVIS

To express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-re-lated gene P9 ( P9-ZFD ) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG. Methods The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET-24a, and the P9-ZFD recombinant protein was induced via E.coli. BL21 (DE3) and purified by histidine affinity chromato-graphy. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expres-sion and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied. Results The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control. Conclusion P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

GABA stimulates human hepatocellular carcinoma growth through overexpressed GABAA receptor theta subunit

AIM: To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC). METHODS: Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang's liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed. RESULTS: We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo , and found that GABA increased HCC growth in a dosedependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ. CONCLUSION: GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC.

Trans-10,cis-12, not cis-9,trans-11, conjugated linoleic acid decreases ErbB3 expression in HT-29 human colon cancer cells

AIM： To examine whether trans-10,cis-12 CLA （t10C12） or cis-9,trans-11 CLA （c9t11） inhibits heregulin （HRG）-β- stimulated cell growth and HRG-β-ErbB3 signaling in HT-29 cells. METHODS： We cultured HT-29 cells in the absence or presence of the CLA isomers and/or the ErbB3 ligand HRG-β. MTT assay, [^3H]thymidine incorporation, Annexin V staining, RT-PCR, Western blotting, immunoprecipitation, and in vitro kinase assay were performed. RESULTS： HRG-β increased cell growth, but did not prevent DNA t10c12-induced growth inhibition. T10C12 inhibited DNA synthesis and induced apoptosis of HT-29 cells, whereas c9t11 had no effect. T10c12 decreased the levels of ErbB1, ErbB2, and ErbB3 proteins and transcripts in a dose-dependent manner, whereas c9t11 had no effect. Immunoprecipitation/ Western blot studies revealed that t10c12 inhibited HRG- β-stimulated phosphorylation of ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase （PI3K） to ErbB3, ErbB3-associated PI3K activities, and phosphorylation of Akt. However, c9t11 had no effect on phospho Aid： levels. Neither t10c12 nor c9t11 had any effect on HRG-β-induced phosphorylation of ERK-1/2. CONCLUSION： These results indicate that the inhibition of HT-29 cell growth by t10c12 may be induced via its modulation of ErbB3 signaling leading to inhibition of Akt activation.

AMPK Subunit Expression Regulates Intramuscular Fat Content and Muscle Fiber Type in Chickens

The objective of this study was to assess the role of AMPK in intramuscular fat(IMF) and fiber type in chicken muscle. The chickens were slaughtered and their muscles were collected at the ages of 4, 8, and 16 weeks so as to determine the IMF contents, as well as the expression levels of AMPK subunits, regulators of adipogenesis. In addition, the myosin heavy chains(My HCs) in thigh muscle tissues were also measured. The results showed that the IMF contents in 16-week old chickens were higher than those in 4 and 8-week-old chickens(P<0.05).The expression levels of fatty acid synthase(FAS) and fatty aicd translocase CD36(FAT/CD36) m RNA were increased significantly in samples collected at the ages of4 and 16 weeks(P<0.05). The expression levels of My HC IIa and IIb differed significantly among all the developmental stages(P <0.05). The AMPKα2, AMPKγ1,and AMPKγ3 m RNA levels were dramatically decreased with the increase of age(P <0.05). To examine the role of AMPK in adipogenesis regulation, the SV cells were cultured in an adipogenesis medium and treated with AICAR and Compound C respectively, the specific activator and inhibit of AMPK. The Compound C induced dramatically a greater expression of C/EBPβ, SREBP1 and PPARγ(P <0.05). In conclusion, the expression of AMPKα2, AMPKγ1, and AMPKγ3 m RNA is significantly correlated with the adipogenesis in skeletal muscle of chickens.

Transgenic mice overexpressingγ-aminobutyric acid transporter subtypeⅠ develop obesity

Transgenic mice ubiquitously overexpressing murine γ aminobutyric acid transporter subtype Ⅰ were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgeinc mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. Tills preliminary finding indicates that the inappropriate level of γ-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.

MOLECULAR CLONING, EXPRESSION AND SUBCELLULAR LOCALIZATION OF HUMAN OCT-4 PROTEIN

Objective To clone, express, purify human Oct-4 and detect its subcellular localization. Methods Human Oct-4 cDNA was amplified by RT-PCR strategy. Oct-4 protein was induced and expressed in BL21(DE3) strain. Furthermore, the protein was purified with Ni-NTA resin. Subsequently, site-directed mutagen-esis of Oct-4 (aa 236 - 240) was introduced. Finally, the subcellular localization of wide type Oct-4 and mutant Oct-4 was examined by immunofluorescent cytochemistry staining and confocal laser scanning microscope analysis. Results The full length cDNA of human Oct-4 was 1083 bp. Human Oct-4 encoded a 55 kd protein by prokaryotic vector in E coli. Compared with pure nuclear localization of wide type Oct-4, mutant Oct-4 was mostly enriched in the cytoplasm. Conclusion The cloning, expression and investigation of subcellular localization of human Oct-4 are basis of studying its biological function.

Molecular subtypes of colorectal cancer:Evaluation of outcomes and treatment

Colorectal cancer(CRC) is a biologically heterogeneous disease with diverse clinical outcomes and responses to treatment. In the past two to three decades, a major effort has focused on classifying colorectal cancer subtypes based on causation, etiology, gene expression profiles, different pathways, and translational data from clinical trials. The goal is to uncover prognostic and predictive factors for outcomes in patients with colorectal cancer and to guide therapeutic approaches and management for the improvement of overall survival. Significant advances have been achieved in this area. However, tremendous work is still needed to accomplish the goal of better understanding intratumoral heterogeneity and the influence of the colonic environment, among other facets of colorectal cancer.