姜黄素对胃癌SGC-7901细胞Nucleostemin基因表达的影响及意义

目的：研究姜黄素对胃癌SGC-7901细胞株NS基因表达的影响及凋亡诱导作用,探讨姜黄素抗肿瘤机制。方法：不同浓度姜黄素（0,10,20,40μmol·L^-1）和不同时间（12,24,48h）点处理细胞,利用CCK-8法检测细胞增殖抑制率,RT—PCR法检测姜黄素作用前后NS基因表达量的变化,TUNEL试剂盒法、流式细胞仪法检测细胞凋亡情况。结果：20μmol·L^-1以上浓度的姜黄素对体外培养的胃癌SGC-7901细胞株生长具有抑制作用并呈量效和时效关系;与对照组相比,姜黄素处理组NS基因表达量明显下降,细胞凋亡明显增加（凋亡指数〉26．61％,P〈0．05）。结论：姜黄素使胃癌SGC-7901细胞增殖减慢,凋亡增加,NS基因表达下降,可能是姜黄素抗肿瘤机制之一。     

鲍曼不动杆菌耐药表型与外排泵基因表达水平的研究

目的 探讨鲍曼不动杆菌临床分离株对常用抗菌药物的耐药性及与外排泵adeA基因表达水平之间的关系.方法 琼脂二倍稀释法检测鲍曼不动杆菌临床分离株对17种常用抗菌药物的最低抑菌浓度(MIC);PCR法扩增外排泵编码基因adeA:实时荧光定量RT-PCR(Real Time Fluorescent Quantitative RT-PCR)法检测adeA基因的mRNA表达水平.结果 药敏结果显示86株鲍曼不动杆菌对多粘菌素B全部敏感(100%),其次是美罗培南(73.2%)、亚胺培南(70.9%).耐药率最高的是庆大霉素(84.9%),其次为美罗西林(83.7%)和环丙沙星(79.1%).55株菌为多重耐药株(64%),其中5株(5/86)对多粘菌素B外的所有抗菌药物耐药(5.8%).adeA的检出阳性率为84.9%(73/86),Real Time RT-PCR结果显示多重耐药菌株adeA基因的mRNA相对表达量均高于敏感菌株,其中3株相对表达量为敏感菌株平均表达水平的30倍以上.结论 鲍曼不动杆菌临床分离株耐药情况严重,其主动外排系统adeA基因表达增强在多重耐药性形成中起重要作用.     

生物膜铜绿假单胞菌AmpC基因诱导表达的研究

目的研究铜绿假单胞菌的产酶基因AmpC在浮游和生物膜状态下的表达差异。方法改良的平板法建立铜绿假单胞菌生物膜模型,抗生素诱导浮游菌和生物膜菌AmpC基因表达,实时荧光定量聚合酶链反应测定PA01的AmpC基因表达水平。结果抗生素诱导前,PAO1浮游菌和生物膜菌的AmpC基因表达均较低,诱导后AmpC基因表达均明显上调。运用产最大AmpC酶活性浓度的抗生素诱导后,亚胺培南和头孢他啶诱导的生物膜菌的AmpC基因表达量高于其诱导的浮游菌。在浮游和生物膜状态下,亚胺培南诱导PAO1的AmpC基因表达量均高于头孢他啶。结论生物膜PA较浮游茵更易被诱导产生AmpC酶,亚胺培南的诱导能力高于头孢他啶。     

介导对氟喹诺酮类药物耐药的金黄色葡萄球菌norA基因的研究

目的研究导致对氟喹诺酮类药物耐药的金葡菌norA基因的介导机制。方法 K-B纸片扩散法测定细菌敏感性(Kirby-Bauer法),研究两种FQNS在菌体内的蓄积量,通过Real-time PCR方法检测金黄色葡萄球菌norA基因的表达。结果两种药物在敏感菌菌体内的稳态蓄积浓度明显高于耐药菌,所有实验菌株中均检测到norA基因的存在,经RealTime PCR扩增后,检测到高度耐药菌株的norA基因表达量较敏感菌菌株平均norA基因表达量高0～8倍;中度耐药菌株的norA基因表达量较敏感菌株高5～17倍。结论推测norA基因表达增加是菌体内药物蓄积量减少的主要原因之一;部分菌株的耐药可能没有norA基因参与,即使有norA基因参与,起作用也不相同;可能有其他外排泵共同参与金葡菌的耐药。     

芪参益气滴丸对大鼠心肌缺血Ppp3r1及Atp5d基因表达变化的影响

目的：通过观察大鼠心肌缺血后,芪参益气滴丸对其缺血区组织中钙调神经磷酸酶B亚基（Ppp3R1）和ATP合成酶亚基δ（Atp5d）基因表达的影响,探讨芪参益气滴丸对急性心肌缺血的保护作用机制。方法：24只大鼠随机分为正常对照组、模型组、芪参益气滴丸组,结扎左冠状动脉前降支主干建立大鼠心肌缺血模型。采用实时荧光定量PCR（real-timePCR）法检测不同组间Ppp3r1和Atp5d基因表达变化。结果：与空白对照组比较,模型组Ppp3r1基因表达量明显升高（P〈0.01）,Atp5d基因表达量明显降低（P〈0.05）。与模型组比较,芪参益气滴丸组Ppp3r1基因表达量降低（P〈0.05）,Atp5d基因表达量明显升高（P〈0.01）比较差异均具有统计学意义。结论：芪参益气滴丸对急性心肌缺血的作用保护机制可能与其能够降低Ppp3r1基因表达量,同时调控Atp5d基因升高有关。     

水杨酸毒扁豆碱滴眼剂定量分析的研究

0.25～0.5%水杨酸毒扁豆碱滴眼剂是医院常用制剂中的毒剧药,据上海、南京、无锡等医院制剂手册及药物制剂注解所载其处方组成为:水杨酸毒扁豆碱0.5g焦亚硫酸钠0.25g硼酸1.8g蒸馏水加至100ml其定量方法有旋光法、折光法和碱量法等但旋光法因受浓度的限制(药典规定最低浓度为1%),折光法受附加剂对析光率的影响,碱量法     

睾酮对人单核细胞中雄激素受体基因表达的影响

目的:研究不同浓度的睾酮对人单核细胞中雄激素受体(AR)基因表达的影响.方法:对培养的人单核细胞株THP-I予不同浓度的睾酮刺激,采用RT-PCR法检测细胞中AR基因表达量,进行半定量比较.结果:人的单核细胞中存在AR.刺激3 h后,睾酮浓度为10-6mol/L、10-5mol/L组和刺激6 h后10-9～10-5 mol/L的各组AR基因表达较对照组表达量明显减少,差异有统计学意义(P<0.05),给氟他胺(10-5mol/L)组和同时给予10-7mol/L睾酮组与对照组比较,AR的表达量差异无统计学意义(P>0.05).结论:在一定范围内,睾酮可减少人单核细胞中AR的表达,选择性受体阻滞剂氟他胺可使这种作用完全阻滞.     

耐多黏菌素肺炎克雷伯菌的耐药机制分析#br#

目的 诱导多黏菌素耐药的肺炎克雷伯菌，探究其对多黏菌素的耐药机制。方法 药物浓度倍增法诱导多黏菌素耐药株，微量肉汤稀释法测定最低抑菌浓度(minimum inhibitory concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC)；PCR扩增并测序确定突变部分；终点显色法检测内毒素含量；实时荧光定量PCR(qRT-PCR)检测相关基因表达量的变化。比浊法和活菌计数法分别测定生长曲线；结晶紫半定量法分析生物被膜形成能力。结果 诱导得到4株耐药株，菌株MIC和MBC均大幅提升且有一株mgrB上游基因突变，内毒素含量升高，mgrB表达量下降，PhoPQ和pmrHFIJKLM相关基因表达均上升。突变株生长状况与野生株基本一致，生物被膜形成能力增强，waaA基因表达上升。结论 肺炎克雷伯菌mgrB上游基因突变是多黏菌素耐药原因之一，并可引起内毒素含量升高和生物被膜形成能力增强。     

油脂性基质软膏制备经验点滴

常用的油脂性软膏剂基质材料有凡士林、液状石蜡、羊毛脂、植物油等,以凡士林最为常用,在工作实践中,我们对如何缩短软膏剂制备时间、提高工作效率、保证药品质量,有如下一点体会。 油脂性软膏的制备一般采用研和法、熔和法。医院制备量在５ｋｇ以上时多采用熔和法,但对不需要除去水分和灭菌者,基质熔化与软膏冷凝温度在《医院制剂规范》、《中国人民解放军医疗单位制剂规范》及常见教科书上均没指明,实际工作中往往只能凭工作经验或习惯。我们医院的软膏制备量通常在１０～４０ｋｇ。制备时先将凡士林５００９包装置水浴锅中加热,…     

复方间苯二酚洗剂中间苯二酚及苯酚的含量测定

<正> 复方间苯二酚洗剂的主要成分有间苯二酚、苯酚及硼酸等,其含量测定常用溴量法,由于苯酚及间苯二酚两者均有卤代反应,无法测定各组分的准确含量,据文献介绍间苯二酚—酚—硼酸溶液,在用溴量法测出总酚量后,再以核磁共振法测定两者的     

The optimal compound reference genes for qRT-PCR analysis in the developing rat long bones under physiological conditions and prenatal dexamethasone exposure model

Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool for evaluating gene expression, but its accuracy is affected by the stability of the reference genes used for normalization. The Minimum Information for Publication of Quantitative Real-time PCR Experiments (MIQE) guidelines indicated that it was important to use multiple stable reference genes as compound reference genes for acquiring optimal experimental results. In this study, the expression levels of eight candidate reference genes (SDHA, TBP, GAPDH, etc.) were detected by qRT-PCR in rat long bones at different developmental stages [gestation day (GD) 20, postnatal week (PW) 6 and PW12] under physiological conditions. Software geNorm, NormFinder, and BestKeeper were used to comprehensively evaluate the stability of the eight reference genes for screening out the most stable compound reference genes in each period. Additionally, the pathological model of prenatal dexamethasone exposure (PDE) was used to verify the stability and reliability of the selected compound reference genes. The result showed that two reference genes as compound reference genes for normalization were optimal. In the intrauterine period, SDHA and TBP could be selected as the compound reference genes, while YWHAZ and GAPDH could be selected at PW6 and PW12, and there was no significant gender difference in the selection of reference genes. The above compound reference genes remained stable in the PDE model and could make the statistical significance of the experimental results more remarkable. In conclusion, this study screened out the optimal compound reference genes in rat long bones before and after birth.     

mRNA expression profiles for the response of human tumor cell lines to the antimalarial drugs artesunate,arteether, and artemether

The antimalarial artemisinin derivatives artesunate (ART), arteether (ARE), and artemether (ARM) reveal remarkable antineoplastic activity. In the present investigation, we identified mRNA expression profiles associated with the response of tumor cells to ART, ARE, and ARM. We performed correlation and hierarchical cluster analyses of inhibition concentration 50% (IC(50)) values and basal mRNA expression levels of 464 genes deposited in the database of the National Cancer Institute, USA. Correlating IC(50) values of ART, ARE, and ARM and of 16 established antineoplastic drugs revealed that the artemisinin derivatives could not be assigned with a known class of drugs with defined mode(s) of action. The basal mRNA expression of 208 out of 464 genes (45%) correlated significantly with IC(50) values of at least one artemisinin derivative. These genes were from different classes (drug resistance genes, DNA damage and repair genes, apoptosis-regulating genes, proliferation-associated genes, oncogenes, tumor suppressor genes and cytokines). We identified two different gene clusters by hierarchical cluster analysis. One cluster contained predominately genes significantly correlated to all three artemisinin derivatives. This overlapping set of genes points to common molecular mechanisms of tumor inhibition by all three drugs in which genes affecting cellular proliferation may play an important role. The second cluster contained genes differentially associated with the response of artemisinin derivatives to cancer cells. The number of correlating drug resistance genes in this cluster increased in the order ART相似文献   

Genetic organization of Bungarus multicinctus protease inhibitor-like proteins

The structural organization of the genes encoding Bungarus multicinctus protease inhibitor-like proteins (PILPs), PILP-1, PILP-2 and PILP-3, are reported in this study. Unlike PILP-2 and PILP-3, recombinant PILP-1 exhibited inhibitory activity on trypsin. PILP genes and B chain genes shared identical organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 chain and B2 chain genes were absent in that of PILP genes. Noticeably, intronic insertion in the second intron of B chain genes appeared in the promoter region of PILP-1 gene, but not in that of PILP-2 and PILP-3 genes. Comparative analyses of PILP genes and B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that PILP genes and B chain genes originate from a common ancestor, and that accelerated evolution may diversify PILP and B chain genes as that proposed for snake venom phospholipase A(2), neurotoxin and cardiotoxin genes.     

Gene expression analysis of the lung following paraquat administration in rats using DNA microarray

Gene expression changes in the lungs induced by paraquat (PQ) administration were studied in rats using DNA microarrays that were detectable for 1,090 genes per DNA microarray. The rats were subjected to subacute PQ exposure (7 mg/kg, s.c., daily for eight administrations). Two days after the final administration, the rats were divided into two groups. Group 1 experienced significant body weight loss and displayed signs of subacute PQ toxicity, but Group 2 showed no significant effects due to the PQ treatment. A control group, Group 3, was also included. In the comparison of the gene expression levels in the animals from Group 1 or Group 2 to the control animals treated by vehicle, 48 genes in Group 1 and 29 genes from Group 2 were differentially expressed. The twenty-eight genes were common to these two groups. These differentially expressed genes following paraquat treatment were classified as follows: 5 neurotransmitter receptor genes; 4 transporter genes; 4 voltage-gated ion channel genes; 2 lipid metabolism enzyme genes; 2 G-proteins involved in endocytosis and exocytosis genes; 7 cytokine genes; 4 ADP ribosylation genes involved in cell death and regeneration; CFTR gene, which is the causal gene for cystic fibrosis; neurofibromatosis type 1 gene, which is the causal gene for the neurofibromatosis type 1 that is known to accompany pulmonary fibrosis; and the causal gene for spinocerebellar ataxia. These genes may prove to be the keys for the elucidation of the mechanism of PQ toxicity, e.g. PQ-induced pulmonary fibrosis.     

基于GEO芯片数据的肝癌特征基因生物信息学及预后关联性分析

目的 通过生物信息学方法利用GEO芯片数据分析肝癌组织中的特征基因及预后的相关性。方法 在GEO数据库中获取肝癌芯片数据,并利用GEO2R工具分析肝癌组织与正常肝组织之间的差异表达基因;利用GO数据库和KEGG数据库对差异表达基因进行富集和功能注释;基于STRING数据库和Cytoscape软件构建蛋白质互相作用网络（protein-protein interaction,PPI）,分析其关键基因;用在线工具Kaplan Meier-Plotter对这些关键基因进行生存分析;用The Human Protein Atlas数据库对这些关键基因在肝癌组织中的蛋白质表达进行免疫组化分析。结果 共鉴定出338个差异表达基因,其中97个为上调基因,241个为下调基因。其中上调的差异表达基因显著富集在细胞核有丝分裂、细胞分裂、有丝分裂成对染色单体分离、成对染色单体凝聚等生物过程;下调的差异表达基因显著富集在环氧化酶P450途径、氧化还原过程、外源性药物分解代谢过程等生物过程。根据PPI网络,对关键模块的24个关键基因进行鉴定,发现这些关键基因的高表达与肝癌患者的生存率低有相关性,并截取关键差异表达基因免疫染色代表性图像。结论 本研究发现的关键差异基因有助于更全面地了解肝癌发生的分子机制,可作为肝癌预后的生物标志物以及潜在的肝癌治疗分子靶点。     

基于TCGA数据库的胆囊癌关键基因及预后基因的挖掘与分析

目的 通过TCGA数据库深入挖掘胆囊癌发生的关键基因,寻找胆囊癌的预后基因。方法 从癌症基因组图谱（TCGA）数据库中下载胆囊癌及癌旁正常组织转录组数据,采用R软件中的edgeR包对数据进行差异表达分析,将获取的差异表达基因进行GO和KEGG富集分析,并通过STRING在线生物信息学工具构建蛋白质-蛋白质相互作用（PPI）网络,通过Cytoscape软件进行关键基因筛选,利用R软件中的survival包对关键基因进行生存预后分析。结果 共获取胆囊癌差异表达基因1 766个,其中上调基因1 172个,下调基因594个。差异基因主要富集于环氧酶P450途径、细胞器膜、四烯酸环氧酶活性和代谢途径。构建PPI网络,获取10个关键基因,分别是BUB1、BUB1B、CDK1、UBE2C、KIF2C、AURKB、CDC20、KIF23、CCNB2和KIF20A。生存分析显示,KIF23与胆囊癌的预后显著相关。结论 基于TCGA数据库挖掘出10个胆囊癌关键基因,有助于深入了解胆囊癌的发生发展过程,KIF23有可能成为胆囊癌潜在的治疗靶点及预后标志物。     

应用基因芯片技术研究非毒性结节性甲状腺肿基因表达的初步研究

目的筛查非毒性结节性甲状腺肿（NTNG）相关基因,探讨NTNG发生机制。方法分别抽提NTNG患者甲状腺组织及正常甲状腺组织总RNA,逆转录合成相应以Cy5和Cy3标记的探针与基因表达谱芯片进行杂交,扫描芯片荧光信号图像,用对扫描图像进行数字化处理和分析。利用实时荧光定量PCR技术对筛选出的部分表达差异明显的基因进行验证。结果筛选出表达差异明显的基因48个,其中NTNG组中表达上调的基因36条,表达下调的基因12条。荧光定量PCR结果与基因芯片一致。结论NTNG患者甲状腺组织与正常甲状腺组织之间存在明显的基因表达差异,为NTNG患者提供相当数量的与细胞外基质、细胞因子、受体信号转细胞信号传递等相关的差异表达基因,为NTNG早期诊断和治疗提供了线索。     

Screening for control genes in mouse hippocampus after transient forebrain ischemia using high-density oligonucleotide array

In conventional relative gene expression analysis (Northern blotting, RT-PCR, and in situ hybridization), housekeeping genes such as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin genes, whose expression levels are considered stable, have been used as control genes for normalization of RNA quantitation. However, it has been reported that the expression levels of these two control genes are affected by ischemia. Therefore, we have been searching for novel control genes whose expression levels are stable in a mouse model of transient forebrain ischemia. Using the GeneChip Mu6500 array set, we monitored the expression levels of approximately 6000 murine genes in the mouse hippocampus during 24 h of ischemia-reperfusion. To select stable genes, we applied the restricted criterion of a 1.5-fold change in expression level as the threshold. By adding statistical analysis with this criterion, we identified 10 genes as candidates for control genes from the GeneChip data. In this criterion, GAPDH and beta-actin genes were not included in the 10 genes as candidates for control genes. The present findings might be relevant to the use of control genes in quantitation of RNA, particularly in the study of mouse transient forebrain ischemia.     

Gene interaction network analysis suggests differences between high and low doses of acetaminophen

Bayesian networks for quantifying linkages between genes were applied to detect differences in gene expression interaction networks between multiple doses of acetaminophen at multiple time points. Seventeen (17) genes were selected from the gene expression profiles from livers of rats orally exposed to 50, 150 and 1500 mg/kg acetaminophen (APAP) at 6, 24 and 48 h after exposure using a variety of statistical and bioinformatics approaches. The selected genes are related to three biological categories: apoptosis, oxidative stress and other. Gene interaction networks between all 17 genes were identified for the nine dose-time observation points by the TAO-Gen algorithm. Using k-means clustering analysis, the estimated nine networks could be clustered into two consensus networks, the first consisting of the low and middle dose groups, and the second consisting of the high dose. The analysis suggests that the networks could be segregated by doses and were consistent in structure over time of observation within grouped doses. The consensus networks were quantified to calculate the probability distribution for the strength of the linkage between genes connected in the networks. The quantifying analysis showed that, at lower doses, the genes related to the oxidative stress signaling pathway did not interact with the apoptosis-related genes. In contrast, the high-dose network demonstrated significant interactions between the oxidative stress genes and the apoptosis genes and also demonstrated a different network between genes in the oxidative stress pathway. The approaches shown here could provide predictive information to understand high- versus low-dose mechanisms of toxicity.     

整合生物信息学法与加权基因共表达网络分析联合分析脑胶质瘤特征基因

目的 利用脑胶质瘤(Glioblastoma, GBM)芯片数据,采用整合生物信息学法和加权基因共表达网络分析法(weighted gene correlation network analysis, WGCNA),寻找肿瘤发病特征基因、关键通路以及转录调控机制。方法 利用GEO数据库中高通量基因芯片数据,通过整合生物信息学法筛选出差异基因;利用WGCNA分析脑胶质瘤关键基因（Hub基因）;采用维恩分析法,整合这些差异基因与hub基因,筛选出脑胶质瘤特征基因。采用GO基因功能注释和KEGG通路富集,分析脑胶质瘤特征基因所富集的功能和通路。利用Kaplan-Meier分析特征基因与脑胶质瘤生成率的关系。利用GCBI数据分析平台,分析调控这些特征基因的转录因子。结果 经分析,发现273个特征基因。这些特征基因主要可影响离子通道、蛋白激酶、γ-氨基丁酸受体功能。CHD5,SYP,PHYHIP表达水平与脑胶质瘤生存率显著相关;转录因子Sp1、Sp3、REST可能是调控这些特征基因的关键因子。结论 本研究利用生物信息学的方法,从多种角度定义了脑胶质瘤的特征基因及调控机制,为其精准治疗提供了依据。