基因芯片技术检测脑出血大鼠血肿周围组织早期基因表达

背景:任何生理状况的改变和表型的变化,最初的基础激发点是基因表达的改变,基因芯片技术是利用碱基配对的原理来快速、大量筛选目标基因的新方法,能整体宏观地研究生物体基因的表达及功能。目的:应用基因芯片技术研究大鼠脑出血早期差异表达基因,为探讨脑出血的病理机制奠定理论基础。设计:随机对照实验。单位:川北医学院附属医院神经内科。材料:实验于2002-10/2003-12在川北医学院附属医院完成。选取20只无特殊病原体级Wistar大鼠,雌雄各半,体质量220～260g,由重庆医科大学实验动物中心提供,将全部大鼠随机分成对照组和脑出血组,每组10只。方法:采用Ⅶ型胶原酶立体定位法制备大鼠急性脑出血动物模型,模型建立后4h取血肿周围组织和相同部位的正常脑组织进行基因芯片对照检测,用扫描仪扫描芯片荧光信号并进行计算机分析,用反转录-聚合酶链反应来考证基因表达谱的结果。主要观察指标:大鼠脑组织基因芯片检测结果及基因反转录-聚合酶链反应的计算结果。结果:大鼠急性脑出血后4h有差异表达基因129个,上调基因114个,下调基因15个,这些基因主要涉及到应激和免疫应答、细胞凋亡、能量代谢及信号转导。有关炎性损害的基因上调最为明显。反转录-聚合酶链反应计算结果显示,基因表示水平与芯片检测结果相符,说明基因芯片所建立的基因表达谱具有相当的可靠性。结论:脑出血早期存在多个差异表达基因,这些差异表达的基因可能在出血性脑损伤中发挥重要作用。     

Development of gene probes and evolutionary relationships of the PSE-4 bla gene to plasmid-mediated beta-lactamases of gram-negative bacteria

Six types of plasmid-mediated carbenicillinases can be distinguished on the basis of their substrate profiles, molecular mass isoelectric values and immunological properties. As yet, no structural classification has been attempted for these enzymes at the molecular level. We have isolated the PSE-4 structural gene responsible for carbenicillinase production in Pseudomonas aeruginosa strain Dalgleish and studied its expression in E. coli. A detailed physical map of the cloned fragment and the construction of deletion mutants permitted the precise localization of the PSE-4 structural gene. Various restriction endonuclease fragments known to be flanking or internal to the PSE-4 bla gene were used as DNA probes and tested for homologous sequences in other beta-lactamase genes. A collection of three restriction fragment probes internal or delimiting the PSE-4 structural gene were hybridized with purified plasmid DNA coding for 18 other beta-lactamases. Under high stringency conditions, only the PSE-1, CARB-3 and CARB-4 genes cross-hybridized with PSE-4; while one of the probes tested hybridized solely with CARB-3. Further analysis indicated that the PSE-1, PSE-4, CARB-3 and CARB-4 bla genes are related and could presumably have evolved from a common progenitor.     

Enhanced nuclear import and transfection efficiency of plasmid DNA using streptavidin-fused importin-beta.

In order to enhance the nuclear import of exogenous genes, novel plasmid DNA/importin-beta conjugates, which consist of a biotinylated plasmid DNA and a recombinant streptavidin-fused importin-beta, were prepared. The spacer length between plasmid DNA and biotin and the number of introduced biotin were adjusted. The microinjection of plasmid DNA/importin-beta conjugates into the cytoplasm of NIH3T3 cells resulted in the nuclear localization of conjugates and the higher expression efficiency, compared to intact plasmid DNA alone. These results indicate that plasmid DNA/importin-beta conjugates would be an important tool to enhance the nuclear localization of exogenous DNA in non-viral gene delivery system.     

Wt1基因与Ph~+白血病细胞系K562的生物学特性

Wt1基因是与造血调控、白血病发生及治疗预后密切相关的双功能基因,在急性髓系白血病及慢性粒细胞白血病进展期呈高表达,且与预后呈负相关,已用于临床微量残留病的监测。WT1蛋白不同亚细胞定位发挥不同生物学功能。本研究旨在探讨Ph+白血病细胞系K562中wt1 mRNA及蛋白的表达与定位,wt1抑制剂——姜黄素(curcumin)对K562细胞的增殖及细胞周期的影响及wt1在白血病发生发展中的相关作用机制。采用MTT法检测细胞增殖情况;流式细胞术分析细胞周期改变;免疫荧光技术及Western blot观察WT1蛋白的亚细胞定位及药物处理后表达水平的变化;实时定量PCR法观察药物处理后wt1及bcr/ablp210转录本水平的变化。结果表明,wt1 mRNA及蛋白在K562细胞高表达,姜黄素及格列卫均能降低wt1 mRNA及蛋白的表达水平,抑制细胞增殖;姜黄素使K562细胞停滞于G2/M期,而格列卫使其停滞于G0/G1期。结论:wt1基因表达改变可以影响Ph阳性细胞—K562的生长、增殖,调控wt1表达有可能成为Ph阳性白血病的新的治疗策略。     

背根神经节钠通道与疼痛

慢性疼痛是周围神经、组织损伤或炎性刺激所诱导的周围神经性病变的一种主要症状。最近一些研究表明背根神经节(dorsalrootganglion,DRG)钠通道表达及位置的改变与某些病理性疼痛有关,钠通道基因表达的可塑性及电生理的改变导致DRG细胞呈高兴奋性、产生自发动作电位及异常高频电活性,DRG钠通道在疼痛的病理生理中起做重要的作用。通过选择性地影响伤害性神经元产生动作电位及阻滞特异性钠通道可缓解神经性及炎性疼痛,有希望成为疼痛治疗的又一新领域。     

β-连环素在白血病细胞株的异常定位研究

β-连环素是Wnt信号通路关键的信号传递子，其在细胞的异常定位引起下游靶基因的转录是多种实体肿瘤发生的原因之一。由于造血细胞缺少典型的黏着连接（adherens junction，AJ），因此对β-连环素在血液肿瘤的表达及定位并无较多的研究。本研究探讨4种常见的白血病细胞株（Daudi，Jurkat，K562和Thp-1）中β-连环素蛋白的定位及β-连环素基因mRNA的表达水平，了解血液肿瘤中是否有β-连环素蛋白的定位异常，以期发现白血病新的发生机制，为寻找新的特异治疗靶点提供线索。用免疫细胞化学法检测β-连环素在白血病细胞系中的定位，用实时定量RT-PCR法测定β-连环素mRNA表达水平。结果表明：4种白血病细胞株有两种（Jurkat、Thp-1）存在β-连环素的异常定位，且β-连环素mRNA表达增高，但在Jurkat和Thp-1细胞中的β-连环素mRNA低于Daudi和K562细胞。这4种白血病细胞株中β-连环素基因的mRNA表达水平与其异常定位无相关性。结论：β-连环素定位异常可能参与部分血液肿瘤的发生，引起β-连环素定位异常的机制并不是发生在转录水平。     

脓毒症大鼠肝组织基因表达的研究

目的筛选脓毒症大鼠肝组织中与正常组织差异表达的基因并进行初步功能分析。方法雄性Wistar大鼠30只，随机分为模型组和空白对照组，每组15只。参照盲肠结扎穿孔术（CLP）制备大鼠脓毒症模型，采用含有4096个大鼠基因cDNA克隆的表达谱基因芯片，检测并分析脓毒症大鼠肝组织在CLP后24h的基因表达变化，并以计算机软件筛选出差异表达的基因。结果CLP后24h共筛选出522条与空白对照组相比出现差异的基因，占基因芯片总点数的12．7％，其中244条基因表达下调，278条基因表达上调。结论脓毒症导致的多器官功能障碍综合征（MODS），涉及到一系列与细胞周期、调控、细胞凋亡、免疫相关基因、各种基本生物化学物质代谢酶类基因和能量代谢相关基因、血液相关基因、癌基因相关基因、生长因子类基因、应激反应类基因、细胞信号转导相关基因、DNA结合转录和转录调节因子相关基因、DNA复制与修复相关基因、蛋白质翻译与修饰、加工、降解相关基因等相关的基因表达异常；采用基因芯片检测技术有利于全面揭示脓毒症中的基因表达模式，快速高效地发现新的研究目标和基因治疗途径。     

延边地区膝骨性关节炎患者滑膜组织基因谱的表达

背景：原发性骨性关节炎的发展是多种相关基因表达失常的缘故。采用mRNA基因表达谱芯片技术,检测膝关节骨性关节炎滑膜差异表达基因,有助于阐明骨性关节炎病理过程的相关机制。目的：比较延边地区膝关节骨性关节炎和正常关节滑膜的基因表达谱变化,筛选出与膝关节骨性关节炎相关的差异表达基因,并探讨其在骨性关节炎发病机制中的意义。方法：选择延边地区原发性膝关节骨性关节炎患者9例及正常对照者3例,应用mRNA基因表达谱芯片技术检测所获得的滑膜组织中表达差异的基因,筛选出表达差异基因并对其进行GO分析。结果与结论：膝关节骨性关节炎及正常对照者滑膜的差异表达基因分析结果显示,延边地区膝关节骨关节炎滑膜中的差异表达基因共有1261个,其中上调的基因有451个,下调的基因有810个。差异表达基因GO统计分析显示,信号转导、翻译调节、趋化作用、炎症应答、细胞增殖等多种基因的表达有差异。基因芯片技术能有效地筛选出骨性关节炎差异表达的基因,并进一步证实了骨性关节炎的病理过程是多种功能基因参与的复杂的病理过程。     

Improved herpes simplex virus type 1 amplicon vectors for proportional coexpression of positron emission tomography marker and therapeutic genes

For the development of efficient and safe gene therapy protocols for clinical application it is desirable to determine the tissue dose of vector-mediated therapeutic gene expression noninvasively in vivo. The herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET). Using bicistronic or multicistronic gene-expressing cassettes with tk as the PET marker gene, the quantitative analysis of tk gene expression may indirectly indicate the distribution and the level of expression of linked and proportionally coexpressed genes. Here, we describe the construction and functional evaluation of HSV-1 amplicon vectors mediating proportional coexpression of HSV-1-tk as PET marker gene and the enhanced green fluorescent protein gene (gfp) as proof of principle and cell culture marker gene and the Escherichia coli cytosine deaminase (cd) as therapeutic gene. Several double-/triple-gene constructs expressing HSV-1-tk, gfp, and E. coli cd were engineered based on gene fusion or the use of an internal ribosome entry site (IRES). Functional analysis in cell culture (green fluorescent protein [GFP] fluorescence and sensitivity to the prodrugs ganciclovir [GCV] and 5-fluorocytosine [5-FC]) and Western blots were carried out after infection of proliferating rat 9L gliosarcoma and human Gli36 glioma cells with helper virus-free packaged HSV-1 amplicon vectors. To study the ability of PET to differentiate various levels of tk expression noninvasively in vivo, retrovirally transduced and selected populations of rat F98 and human Gli36dEGFR glioma cells with defined levels of proportionally coexpressed tk and gfp genes were grown as subcutaneous tumors in nude rats and nude mice, and tk imaging by PET was performed. To study HSV-1 amplicon vector-mediated gene coexpression in vivo, HSV-1 amplicon vectors bearing coexpression constructs were injected (4 x 10(7) to 1 x 10(8) transducing units) into subcutaneously growing Gli36dEGFR gliomas in nude animals, and tk imaging was performed 24 hr later. All vector constructs mediated GFP expression and sensitized 9L and Gli36 cells toward GCV- and 5-FC-mediated cell killing in a drug dose-dependent manner, respectively. The levels of gene expression varied depending on the location of the genes within the constructs indicating the influence of the IRES on the level of expression of the second gene. Moreover, functional proportional coexpression of the PET marker gene HSV-1-tk and the linked therapeutic E. coli cd gene was observed. In selected tumor cell populations, subtle IRES-dependent differences of tk gene expression could be noninvasively distinguished by PET with good correlation between quantitative assays for IRES-dependent attenuated GFP and TK expression in culture and in vivo. After infection of subcutaneously growing gliomas with HSV-1 amplicon vectors, various levels of TK expression were found ranging from 0.011-0.062 percentage injected dose per gram (%ID/g). These values were 4.0- to 5.7-fold lower than positive control tumor cells. TK expression could be imaged by PET in vivo even with the tk gene located at the weak position downstream from the IRES. In conclusion, these HSV-1 amplicon vectors carrying HSV-1-tk as PET marker gene and any linked therapeutic gene will serve an indirect noninvasive assessment of the distribution of therapeutic gene expression by PET. Monitoring the correlation between primary transduction and therapeutic efficiency of a given vector is highly desirable for the development of safe and efficient gene therapy and vector application protocols in clinical applications.     

苦参碱诱导K562细胞分化早期的基因表达分析

目的　比较苦参碱诱导K5 6 2细胞前后的基因表达谱 ,寻找与肿瘤细胞分化相关的基因。方法　 0 .1mg/ml苦参碱作用K5 6 2细胞 3h后 ,采用基因芯片技术测定并分析对照组与苦参碱处理组间的基因表达谱差异。结果　在 84 6 5个候选基因中 ,筛选出 30个差异表达基因 ,苦参碱处理组与对照组比较 ,表达上调的基因有 12个 ,主要涉及代谢相关蛋白基因、细胞受体等 ;表达下调的有18个 ,包括抑癌基因、原癌基因、DNA结合与转录因子、代谢相关蛋白基因、细胞信号转导基因等。结论　肿瘤细胞诱导分化是多基因作用的综合结果 ,筛选的基因对了解肿瘤发生、发展与逆转分化 ,以及寻找潜在的药物作用靶点可能有意义。     

失血性休克大鼠肝脏基因表达谱的变化

目的 应用基因芯片技术分析失血性休克组(hemorrhage shock,HS)及对照组(sham hemorrhage shock,SHAM)大鼠肝脏差异基因表达谱,从分子水平探讨HS可能的病理生理机制.方法 20只雄性Wistar大鼠随机分成SHAM组和HS模型组,每组10只.采用5705点的大鼠寡核苷酸芯片分别检测HS大鼠及SHAM大鼠肝脏基因表达谱,重复三次并计算Ratio(R)均数,R≥2时表示基因上调;R≤0.5时表示基因下调.对差异表达基因的功能进行分析并用RT-PCR对其中9条基因的表达水平进行验证.结果 在大鼠5705条靶基因中,初步筛选出86条差异表达基因,其中上调基因72条,下调基因14条.根据基因的生物学功能,差异表达基因主要为:物质转运相关基因、转录调节相关基因、信号转导相关基因、应激反应相关基因、代谢相关基因、发育相关基因、细胞黏附相关基因等.结论 HS的发生是由多基因参与的复杂调控过程;芯片试验结果对进一步探讨HS可能的病理生理机制以及为探求HS治疗的新靶点提供了依据.     

霍乱弧菌O1血清群产毒株重复基因分析

目的对霍乱弧菌O1血清群产毒株基因组中重复基因数量分布、多样性及两种生物型中重复基因的差异进行分析。方法选择300株引起第6次和第7次大流行的O1群古典型和埃尔托型生物型菌株，使用BLAST和MCL等软件分析重复基因数量分布及两种生物型差异的重复基因；使用序列两两平均距离作为多样性的度量方法分析重复基因的遗传多样性以及在两种生物型之间存在遗传多样性差异的重复基因。结果每株菌平均包含242.63个同源基因簇，其中每株埃尔托型和古典型菌株分别平均包含244.85和209.74个。 筛选到13个在埃尔托生物型与古典生物型之间拷贝数存在显著差异同源基因簇，功能主要包括阴离子、氨基酸等物质的转运、定位、跨膜转运等。 对存在重复基因的同源基因簇的遗传多样性分析，发现在两种生物型之间多样性存在差异的同源基因簇50个，这些基因簇的功能主要包括阴离子、氨基酸、有机底物等转运、定植、底物特异的跨膜转运活性等。结论霍乱弧菌重复基因的进化有一定的功能倾向性，其特征解析对于理解其基因组适应性进化、菌株多样性及两种生物型基因组差异进化提供了新依据。     

Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes

Intravenous administration could become a delivery route of choice for prophylactic and curative gene therapies on condition that genes cross the capillary barrier and reach target tissues without being degraded. We investigated the kinetics and process of transgene delivery through mouse lung capillaries following DNA complexation with linear polyethylenimine (L-PEI) and intravenous injection. Using digoxin-labeled DNA we followed the cellular localization of DNA at different times after injection and correlated these findings with cell markers and transgene expression. At 2 h after injection some DNA was still localized on the interior of the capillary lumen, but other complexes had already crossed the barrier and resulted in gene expression. At 24 h after injection most labeled DNA was localised in pulmonary cells, as was transgene expression. Only rarely was transgene expression found in endothelial cells, suggesting that the complexes cross the capillary barrier rapidly. Levels of caspase-1-like activity did not increase following transfection implying that L-PEI/DNA complexes are transported across cellular barriers by a non-damaging, physiological process, without causing inflammation. The high levels of expression of different transgenes in pneumocytes indicates that transport of L-PEI/DNA complexes through the endothelial barrier does not affect their transfection capacity. These findings open up new possibilities for gene delivery and its application to the lung.     

应用基因芯片技术检测人脐带血CD34+细胞体外扩增的巨核细胞基因表达

目的 研究人脐带血CD34+细胞体外扩增巨核细胞的基因表达,从分子水平探讨巨核细胞的表达机制.方法 采用密度梯度离心法和免疫磁珠分选系统获取人脐带血CD34+细胞.100 ng/ml TPO诱导培养12 d后,应用抗CD+41单克隆抗体免疫磁珠法分选巨核细胞.应用基因芯片技术检测巨核细胞、非巨核细胞和meg-01细胞株...     

Evolutionary variation in gene expression is associated with dimorphism in eusocial vespid wasps

Phenotypic diversity is frequently generated by differences in gene expression. In this study, we addressed the relationship between homology in gene expression and phenotype among four species of eusocial wasps. Specifically, we investigated the evolution of caste‐specific and sex‐specific gene expression patterns associated with caste polyphenisms and sexual dimorphisms. We also identified several genes with functions relevant to their phenotype‐specific roles. Our results suggest that gene expression profiles associated with caste polyphenisms may evolve rapidly relative to those associated with sexes. Thus, caste‐biased genes may undergo less regulatory constraint or be subject to greater neutral variation in expression than sex‐biased genes.     

Housekeeping gene variability in normal and cancerous colorectal, pancreatic, esophageal, gastric and hepatic tissues

Careful normalization is essential for the accurate quantitation of mRNA levels in biopsy-sized tissue samples. Commonly, normalization of the target gene with an endogenous standard, mainly housekeeping genes (HKGs), is applied. However, differences in the expression levels of endogenous reference genes have been reported between different tissues and pathological states. Therefore, we were challenged to identify a set of endogenous reference genes whose mRNA expression levels would not change significantly between normal and cancerous tissues. Quantitative real-time PCR (Q-RT-PCR) analysis was applied to evaluate the variability in gene expression among 21 classical housekeeping genes in colorectal, pancreatic, esophageal and gastric cancer as well as in liver metastases in comparison to the corresponding normal tissue. Our results indicated that some housekeeping genes were candidates with relatively stable gene expression in several of the investigated tissues but for most of the HKGs under investigation our data have revealed distinct differences in the extent of variability in gene expression between the different tissues and pathological states. However, for each of the five tissues investigated we found a group of genes that were expressed at a constant level thus representing a panel of candidates that we can recommend as housekeeping genes in the respective tissue types. In summary, our results can be used as guidance for other scientists studying various carcinomas for tissue-specific selection of the optimal housekeeping gene (HKG) to be used in normalizing target gene expression.     

干扰素-α对K562细胞基因表达谱的调控研究

本研究查明干扰素-α对K562细胞基因表达谱的调控作用,为阐明IFN-α治疗慢性髓性白血病的作用机制提供依据.应用DNA芯片技术对IFN-α作用前后K562细胞基因表达谱的变化进行检测.结果表明：200 U/ml的IFN-α作用K562细胞1天后,没有1个基因表达差异在2.5倍以上,随后逐渐增多,到4天时达到高峰,在检测的基因中共有97个表达差异显著的基因,其中84个（86.60%）上调,13个（13.4%）下调.在97个表达差异显著的基因中,细胞调节蛋白类占23.71%,细胞受体类占14.43%,癌基因与抑癌基因占11.34%,细胞信号传导蛋白类占9.28%,细胞黏附分子占8.25%,其它基因占32.99%.第5天开始下降,但到第21天时仍有9个表达差异显著的基因.在细胞信号传导蛋白类中,参与JAK-STAT途径的JAK1基因经IFN-α刺激4天上调到3.78倍,JAK2上调到15.43倍.同时还发现信号转导子及转录活化子STAT1及STAT2分别上调到11.98和8.11倍.结论：IFN-α在浓度200 U/ml时,对K562细胞作用4天其基因表达变化最显著;IFN-α对K562细胞基因表达的调控是通过调节JAK-STAT途径实现的,此途径可能是FN-α治疗CML的机制之一.