基因芯片检测吴茱萸碱对小鼠DC细胞功能调控相关基因表达的影响

目的：利用基因芯片技术检测吴茱萸碱对小鼠骨髓来源DC功能调控相关基因表达的影响。方法：分离BALB／c小鼠骨髓细胞，经GM-CSF体外诱导培养10天的未成熟DC细胞（iDC）经不同因素处理并分为：对照组（Ⅰ）、吴茱萸碱组（EVO）（Ⅱ）、内毒素（LPS）组（Ⅲ）、EVO＋LPS组（Ⅳ），24小时后收集各组细胞，抽提总RNA，利用SuperArray公司小鼠DC与抗原提呈细胞基因芯片MM-604对细胞功能相关基因进行检测。结果：Ⅱ／Ⅰ上调≥2倍基因7个，下调≥2倍基因10个；Ⅲ／Ⅰ上调≥2倍基因37个，下调≥2倍基因12个；Ⅳ／Ⅱ上调≥2倍基因46个，下调≥2倍基因7个；Ⅳ／Ⅲ上调≥2倍基因24个，下调≥2基因2个，对这些基因功能进行检索分类，主要涉及细胞因子分泌及其受体表达、抗原摄取、抗原提呈、细胞表面受体、信号传导。结论：吴茱萸碱对DC作用涉及多个基因的表达调控，这些基因控制并影响着DC功能、分化和成熟，为进一步寻找药物靶点提供了线索。     

基因芯片筛选抗胸腺细胞血清性肾炎大鼠肾组织基因表达差异的初步研究

目的： 利用基因芯片检查大鼠系膜增生性肾炎，即抗胸腺细胞血清性肾炎(ATSN)模型大鼠发病 40 min 和 5 d 时肾组织相关基因的表达情况，探讨ATSN大鼠不同时相肾组织基因表达的差异并作功能分析。 方法： 用抗胸腺细胞抗血清（ATS）复制大鼠ATSN模型，抽提发病 40 min 和 5 d 时肾组织RNA，分别用逆转录合成荧光分子掺入的cDNA作探针进行基因芯片杂交。用Agilent 扫描仪扫描，Imagene软件读取数据，最后用Genespring进行normalize处理和差异表达基因的筛选。差异基因筛选标准为实验组/对照组比值（ratio）>或=2为上调基因，实验组/对照组ratio<或=0.5为下调基因。然后选取上调和下调基因登录GenBank查找其相关功能。 结果： 在 9 234 个大鼠基因中（去除质控基因），ATSN大鼠 40 min 时肾组织上调基因为341个，其中部分为凋亡相关的调控基因，部分为增生相关基因及一些与信号转导有关的基因；下调基因为392个，其中一些是酶类基因，部分为生长因子和细胞因子受体基因等，其余大多上调和下调基因功能不详。ATSN发病 5 d 时，肾组织上调基因是213个，包括增生相关基因、信号转导相关基因等；下调基因119个，其中部分为酶类和细胞因子相关基因。5 d 时上调和下调基因大多功能不清楚。 结论： 大鼠ATSN发病早期（40 min），致病相关基因已被激活（如凋亡和增生相关基因），其中相关信号转导分子基因也表达上调，而在ATSN大鼠发病 5 d 时，基因表达谱有所改变，其中涉及的基因多为增生相关基因和信号转导相关基因。     

颞叶癫痫大鼠海马差异表达基因和蛋白质的筛选研究

目的：寻找颞叶癫痫大鼠海马组织的差异表达基因和蛋白质，以期为进一步探讨颞叶癫痫的发病机制，寻找新的治疗靶点和研发新的治疗手段奠定基础。方法:运用cDNA微阵列、二维电泳和MALDI-TOF-MS技术，分析氯化锂-匹罗卡品（LiCl-PILO）致痫大鼠模型海马组织的基因表达谱和蛋白质表达谱，并对发现的差异表达基因和差异表达蛋白质进行分析和鉴定结果和。结论：发现LiCl-PILO致痫大鼠海马组织中192个基因差异表达，159条可在GenBank中登陆，其中表达上调的基因84条，表达下调的基因75条；筛选到78个差异表达蛋白质斑点，其中31个在癫痫组表达下调，47个在癫痫组表达上调。有5个蛋白质最终鉴定确认。本研究结果为运用蛋白质组学方法寻找癫痫治疗新靶点研究提供实验依据。     

雌激素对自发性高血压大鼠颅内动脉血管壁TNF-α和MMP-9表达的影响

目的: 探讨雌激素对颅内动脉血管壁炎症反应和血管重塑的可能作用。方法: 雌性自发性高血压大鼠(SHR)32只,随机分为4组:自发性高血压模型对照组、卵巢切除去势组、去势后雌激素替代组和卵巢切除去势对照组,每组8只。放射免疫法检测各组静脉血的雌激素水平,获取标本后光镜下观察血管壁的病理变化,运用Western blotting 检测颅内动脉Willis环血管壁组织TNF-α和MMP-9蛋白的含量。结果: 自发性高血压模型对照组雌激素水平明显高于卵巢切除去势组(P<0.01);卵巢切除去势后雌二醇替代组的雌激素水平较卵巢切除去势对照组明显增高(P<0.01)。所有大鼠均未见进展期动脉瘤。在自发性高血压模型对照组中未见动脉瘤样改变,在卵巢切除去势组、卵巢切除去势对照组和卵巢去势后雌激素替代组中分别发现2个、3个和1个早期动脉瘤样改变。自发性高血压模型对照组颅内动脉血管壁TNF-α和MMP-9的含量明显低于卵巢切除去势组,雌二醇替代治疗组颅内动脉血管壁的TNF-α和MMP-9表达明显低于卵巢去势对照组(P<0.01)。结论: 雌激素能通过抑制颅内动脉的炎性反应,降低血管壁MMP-9的表达,影响颅内动脉的血管重塑。     

心肌成纤维细胞在血管紧张素Ⅱ刺激下表达下调基因的分离和鉴定

目的：对成年大鼠心肌成纤维细胞（CF）受血管紧张素Ⅱ（AngⅡ）刺激后下调基因表达谱进行筛选及分析。 方法： 以受AngⅡ刺激CF为驱动方，未刺激CF为实验方，进行抑制消减杂交（SSH）建立消减cDNA文库。经斑点杂交筛选文库后将部分阳性克隆测序及进行同源性分析。 结果： 共获得17条下调表达基因，分别与胞内信号转导、转录抑制、纤维沉积和细胞骨架重构相关，并克隆到4条新基因表达序列标签（EST）。 结论： SSH有效地克隆了成年大鼠CF受AngⅡ刺激后下调表达基因，对这些基因的研究将有助于阐明心肌改建的分子机制。     

雌激素对卵巢摘除大鼠心内神经节细胞Bcl-2和Bax表达的影响

本实验建立了去卵巢和雌激素替代疗法动物模型,用免疫组织化学SABC法结合图象分析方法,观察大鼠心内神经节细胞中Bcl-2和Bax的变化,探讨雌激素对卵巢摘除大鼠心内神经节细胞中Bcl-2和Bax表达的影响。去卵巢(OVX)大鼠心内神经节细胞中Bcl-2的表达较正常对照组明显减弱(P<0.05),雌激素替代治疗组大鼠心内神经节中Bcl-2的表达与正常对照组相比无显著性差异(P>0.05),而较卵巢摘除组显著增高(P<0.05);卵巢摘除后同时给予特异性雌激素受体阻断剂(TAM)和17β-雌二醇(OVX+TAM+ERT)组大鼠心内神经节中Bcl-2的表达较正常对照组显著减弱(P<0.05),但与OVX组无显著性差异(P>.05)。各组中Bax的表达无显著性差异(P>0.05)。实验结果提示雌激素能上调大鼠心内神经节细胞中Bcl-2的表达,但对Bax的表达无影响。     

胃癌癌变相关基因表达的cDNA微阵列研究

目的 建立胃癌基因表达谱,筛选胃癌相关基因。方法 用含10000个已知基因和7000个ESTs(expressed sequence tags)的cDNA微阵列分析胃癌和癌旁正常胃黏膜基因表达谱的变化,半定量RT-PCR研究差异表达基因与胃癌的关系。结果 二倍以上的差异表达基因359个,其中在胃癌组织中表达上调271个,表达下调88个;二倍以上的差异表达ESTs 28个,其中在胃癌组织中表达上调24个,下调4个。RT-PCR进一步证实碳酸酐酶Ⅱ在胃癌组织中存在表达下调,胰岛素样生长因子结合蛋白4在胃癌组织中存在表达上调。结论 发现碳酸酐酶Ⅱ、胰岛素样生长因子结合蛋白4可能与胃癌发生有关,为进一步寻找和克隆胃癌相关基因提供了重要的研究线索。     

利用生物芯片技术检测人参皂苷Rg1对小鼠树突状细胞功能调控相关基因表达的影响

目的:利用基因芯片技术检测人参皂苷Rg1对小鼠骨髓来源DC细胞功能调控相关基因表达的影响.方法:分离C57小鼠骨髓细胞,经GM-CSF、IL-4体外诱导培养6天的未成熟DC细胞(iDC)经不同因素处理并分为:对照组(Ⅰ)、人参皂苷Rg1组(Ⅱ)、内毒素(LPS)组(Ⅲ)、Rg1+LPS组(Ⅳ),24小时后收集各组细胞,抽提总RNA,利用树突状和抗原呈递细胞基因芯片对细胞功能相关基因进行检测.结果:(Ⅱ/Ⅰ)上调≥2倍基因23个,下调≥2倍基因45个;(Ⅲ/Ⅰ)上调≥2倍基因15个,下调≥2倍基因47个;(Ⅳ/Ⅱ)上调≥2倍基因35个,下调≥2倍基因15个;(Ⅳ/Ⅲ)上调≥2倍基因37个,下调≥2基因10个.这些基因功能主要涉及细胞因子分泌及其受体表达、抗原摄取、抗原提呈、细胞表面受体、信号传导.结论:人参皂苷Rg1对DC作用涉及多个基因的表达调控,这些基因控制并影响着DC功能、分化和成熟,为进一步寻找药物靶点提供了线索.     

雌激素对卵巢摘除大鼠心内神经节细胞胆碱乙酰基转移酶和一氧化氮合酶表达的影响

目的：探讨雌激素对卵巢摘除大鼠心内神经节细胞中神经递质的影响。方法：建立去卵巢(OVX)和雌激素替代疗法动物模型，2个月后用免疫组化结合图象分析方法观察大鼠心内神经节细胞中胆碱乙酰基转移酶(ChAT)和一氧化氮合酶(NOS1)的变化。结果：在OVX大鼠心内神经节细胞中ChAT和NOS1较正常对照组明显减少，雌激素(E2)替代治疗组与正常对照组相比无明显变化，OVX 雌激素受体拮抗剂(TAM)组较正常对照组显著减少，但与OVX组无明显差异。结论：雌激素能上调大鼠(OVX)心内神经节细胞中ChAT和NOS1的表达，雌激素可能够通过对心内神经节细胞中神经递质的调节，间接发挥对心血管功能的调节作用。     

基于癌症基因组图谱筛查卵巢浆液性囊腺癌相关基因

目的:利用全基因组表达谱芯片筛查与卵巢浆液性囊腺癌发生相关的基因,对在卵巢浆液性囊腺癌发生过程中可能参与的基因间的信号转导通路进行分析。方法:选取癌症基因组图谱(TCGA)数据库中卵巢浆液性囊腺癌的Affymetrix Gene Chip Human Exon 1.0 ST Array数据共16张,分别为卵巢浆液性囊腺癌组8张和正常组8张,筛选出差异表达基因,并进行基因本体(gene ontology,GO)分析和信号通路分析,构建卵巢浆液性囊腺癌相关基因间的信号转导通路,分析网络中具有重要作用的基因。结果:共筛选出1 144个在卵巢癌中差异表达的基因,其中表达上调的基因有747个,表达下调的基因有397个。GO分析得到上调差异基因的显著性功能分析结果362项,下调差异基因的显著性功能分析结果 160项(P0.05)。其中包括与肿瘤发生相关的基因功能有细胞周期、DNA复制、细胞增殖、细胞凋亡、细胞黏附等。信号通路分析得到45个显著上调信号通路和14个显著下调信号通路(P0.05)。其中参与肿瘤发生相关的信号通路主要有细胞周期、P53信号通路、DNA复制、肿瘤中的信号通路、PI3K-Akt信号通路、ECM-receptor信号通路、细胞黏附因子、细胞凋亡等。挑选显著性基因功能和信号通路分析的交集基因229个,构建显著性GO与信号通路基因间信号转导网络。分析发现CDK1、PLK1、MCM3和PGK1这4个基因在卵巢癌的基因调控网络中具有重要作用。结论:卵巢浆液性囊腺癌中有大量差异表达基因,差异表达的基因在多个与肿瘤发生密切相关的信号通路中发挥重要的调控作用。     

Estrogen replacement reverses ovariectomy-induced vaginal hyperalgesia in the rat

OBJECTIVES: The loss of ovarian function in women through aging or oophorectomy is often associated with the development of vaginal hyperalgesia that can be alleviated with estrogen replacement. This study examined if ovariectomy in rats would similarly give rise to vaginal hyperalgesia, and, if so, whether estrogen replacement would alleviate it. METHODS: Female rats were trained to perform an operant response to escape vaginal distention delivered by inflating a balloon located in mid-vaginal canal. Percent escape responses to eight different volumes of distention measured in normally cycling rats were compared with measures made in the same rats following ovariectomy (OVX) or sham ovariectomy (shamOVX), and then, in the OVX group, estrogen replacement (OVX+E2). Pressures exerted by the eight volumes on the vaginal wall were also measured, thereby permitting assessment of vaginal tone. RESULTS: Whereas overall escape response percentages after OVX, but not shamOVX, were significantly higher to the largest six distention volumes compared with responses during cycling, there were individual differences in the amount of hyperalgesia. Following OVX+E2, escape response percentages decreased in all but one rat. Vaginal tone after OVX, shamOVX or OVX+E2 did not differ from overall vaginal tone in cycling rats. CONCLUSIONS: Ovariectomy in rats evokes a variable amount of vaginal hyperalgesia that can be alleviated by estrogen replacement in most cases. Thus, the ovariectomized rat appears to provide a useful model for the study of mechanisms underlying the dyspareunia that is associated with loss of ovarian function in women.     

力生长因子E肽与应力刺激对成骨细胞基因表达的影响

目的应用基因芯片技术分析在力生长因子E肽(MGF-Ct24E)和应力作用下成骨细胞基因表达的差异。方法体外培养原代成骨细胞,分别对细胞施加周期性拉伸刺激(应变12%,频率0.5 Hz)和MGF-Ct24E(50 mg/L)直接作用,基因芯片技术分析力学刺激和MGF-Ct24E作用对原代成骨细胞基因表达谱的影响,并用定量PCR验证基因芯片实验结果。结果与对照组相比,力学加载组共发现差异表达基因1 866个,其中上调基因1 113个,下调基因753个;MGF-Ct24E处理组共发现差异表达基因1 178个,其中上调基因796个,下调基因382个。GO分析发现两者的差异基因表达谱具有一致性,并且差异表达基因主要涉及细胞增殖与分化调节、细胞对应力刺激的响应和力学转导通路等。定量PCR实验结果验证的差异表达基因与芯片实验结果一致。结论基因表达谱分析显示应力刺激和MGF-Ct24E作用对成骨细胞的基因表达具有类似的调控效应,为后续使用MGF-Ct24E治疗骨修复以弥补应力刺激不足的研究提供了新的思路。     

Immunohistochemical analysis of the effects of estrogen on intraarticular neurogenic inflammation in a rat anterior cruciate ligament transection model of osteoarthritis

Synovitis is considered as one of the factors associated with the pathogenesis of osteoarthritis (OA). There is currently a significant amount of research linking estrogen deficiencies with the development of OA in estrogen-deficient women, including postmenopausal women; however, the exact etiology remains unclear. Various neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), have been shown to contribute to synovitis in OA joints, and the influence of estrogen on the expressions of SP and CGRP in the synovium of OA joints has been noted. After ovariectomy (OVX) followed by estradiol (E2) replacement, 24 female rats were divided into three groups: OVX group, OVX + E2 replacement group (E2 group), and a sham group. All rats underwent transection of the anterior cruciate ligament at the same time. After 30 days, the histological findings of knee joints by hematoxylin-eosin staining and immunofluorescence staining of protein gene product 9.5 (pan-neuronal marker), SP, and CGRP were compared among experimental groups. The degree of synovitis in the OVX group was higher than in the E2 and sham groups. No significant differences in the density of protein gene product 9.5-immunoreactive nerve fibers were observed among the three experimental groups, but the density of SP- or CGRP-immunoreactive nerve fibers in the OVX group was significantly higher than in the E2 and sham groups. These findings suggest that estrogen partly regulates intraarticular neurogenic inflammation in OA joints by modulating the expressions of neuropeptides in the synovium.     

应用基因表达谱芯片观察小鼠心肌缺血后基因表达的变化以及四逆汤对其影响

目的:应用基因表达谱芯片观察小鼠心肌缺血后基因表达的变化以及四逆汤对其影响。方法:昆明种小鼠,随机分为对照组、缺血组、缺血加四逆汤组。提取各组心肌组织总RNA,纯化mRNA,与含有2304条小鼠基因的cDNA表达谱芯片进行杂交。结果:小鼠缺血后有33条基因表达下调,70条基因表达上调;服用四逆汤后,相对单纯缺血组而言,有23条基因表达下调,52条基因表达上调。结论:运用基因芯片技术能快速地检测出缺血心肌及四逆汤治疗后心肌基因表达谱的改变,对差异表达基因的研究将有助于进一步了解心肌缺血及四逆汤治疗的分子机制。     

In Vivo Effects of Ovarian Steroid Hormones on the Expressions of Estrogen Receptors and the Composition of Extracellular Matrix in the Anterior Cruciate Ligament in Rats

Female athletes have a significantly higher rate of anterior cruciate ligament (ACL) injury than their male counterparts. Sex steroid hormones are considered to have an influence as risk factors for female ACL injuries. We hypothesized that estrogen and progesterone have specific and synergistic influences on the composition of extracellular matrix in ACL. By ovariectomy (OVX) followed by subcutaneous estradiol (E2) and/or progesterone (P4) replacement, 40 female rats were divided into 5 groups: E2, P4, combined E2 and P4 (EP), OVX control, and sham group. After 30 days, using undecalcified sections of knee joints in conjunction with immunofluorescence staining of estrogen receptor α and β (ERα and ERβ), collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP), the immunoreactivities of these proteins in two distinct parts of ACL, proximal and middle portions, were compared semiquantitatively among experimental groups. By E2 replacement, the expressions of ERα in ACL fibroblasts were elevated compared to the OVX group. At the proximal portion, the immunoreactivities of type 1 collagen by E2 replacement, type 3 collagen by P4 replacement, and COMP by E2 or P4 replacement were significantly reduced. At the middle portion, the immunoreactivity of type 3 collagen was significantly elevated by E2 replacement. However, no differences were observed between the sham and OVX groups. These findings suggest that ACL is ER-dependent and that ovarian hormones alter ligament tissue composition, especially at the proximal portion. Female hormonal influences are partly involved in the biological properties of ACL.     

非诺贝特对高甘油三酯血症并骨质疏松大鼠骨代谢的影响

目的探讨非诺贝特对去卵巢骨质疏松并高甘油三酯（TG）血症大鼠股骨中细胞核因子κB受体活化因子配体/护骨素（RANKL/OPG）mRNA表达的影响。方法将40只3月龄雌性SD大鼠,用果糖饲养复制高TG模型,大鼠共分为去卵巢＋果糖组、去卵巢＋果糖＋非诺贝特（FF）组、去卵巢＋普食组、假手术＋果糖组。12周后取股骨检测细胞核因子κB受体活化因子配体（RANKL）mRNA、护骨素（OPG）mRNA表达水平。结果去卵巢＋果糖组的TG水平高于去卵巢＋普食组（P〈0.01）,也高于去卵巢＋果糖＋FF组（P﹤0.01）;去卵巢＋果糖组RANKL mRNA/OPG mRNA水平,均高于去卵巢＋果糖＋FF组、去卵巢＋普食组和假手术＋果糖组（P〈0.01或〈0.05）。结论非诺贝特可通过降低TG而保持RANKLmRNA/OPGmRNA的平衡。     

Microarray analysis using amplified mRNA from laser capture microdissection of microscopic hepatocellular precancerous lesions and frozen hepatocellular carcinomas reveals unique and consistent gene expression profiles

The indirect labeling cDNA microarray technique was used to evaluate gene expression profiles of pure cell populations from frozen sections of carcinomas and adenomas harvested from precancerous hepatocellular lesions by using laser capture microdissection (LCM). The levels of differentially expressed genes were investigated using a cDNA microarray with 9,984 features with only 2 ug of two-round amplified aRNA, equivalent to 35 cells from LCM-adenomas and frozen samples of carcinomas from simian virus 40 (SV40) large T antigen transgenic rats. A total of 855 genes were identified as being 3-fold or more differentially expressed in carcinomas or adenomas as compared to normal tissue controls. Among these 855 genes, 71 genes were differentially expressed in both carcinomas and adenomas. Commonly up-regulated genes in both carcinoma and adenomas were 28 while 41 of the 71 genes were commonly down-regulated. Two genes, Igh1 (immunoglobulin heavy chain 1(Serum IgG2a), Image clone ID: 875880) and EST clone (AI893585, Image clone ID: 596604) were more than 7-fold up-regulated in carcinomas and 6-fold down-regulated in adenomas. In Cy5 and Cy3 reciprocal experiments for screening out false positive signals, the amplified carcinomas showed higher Pearson Correlation Coefficient values (-0.94 and -0.92) than the LCM-amplified adenoma samples (-0.79 and -0.84). LCM-amplified samples provided higher signal intensities over backgrounds and a greater average of Cy5:Cy3 ratios. Expression levels of mRNAs from selected genes, determined by using traditional dot blot analysis, revealed that 36 of 40 tested expression profiles were consistent with the microarray data. Thus, amplified aRNA harvested from homogeneous cell types using LCM can be applied to study gene expression profiles by use of microarray analysis.     

cDNA microarray-based analysis of differentially expressed genes in transgenic brains expressing NSE-controlled APPsw

cDNA microarray technique has been widely used for the detection and elucidation of differentially expressed genes on a large scale and at a speed never before possible. The aim of this study was to gain insight into the potentially overexpressed effects of APPsw on the modulation of genes for Alzheimer's disease (AD), which is central to understanding the complexity of AD. APPsw transgenic mice, which we previously produced, provide an important resource for identifying differentially expressed genes since this transgenic line was shown to have cognitive deficits along with Abeta-42 deposits at 12 months of age. To identify differentially expressed genes, cDNA microarray technique was conducted to get a large-scale screening of brain mRNA from 18 month-old NSE/APPsw transgenic and non-transgenic mice. A total of 52 differentially expressed genes, 10 up-regulated and 42 down-regulated, were found in the brains of moderately transgenic mice compared to non-transgenic littermates. Thus, the results suggest the need for future studies on gene functions, pathology, toxicogenomics, and pharmacogenomics.     

大鼠ARDS基因表达谱中信号传导基因的表达

 目的 对大鼠急性呼吸窘迫综合征(ARDS)基因表达谱中的信号传导基因进行研究与分析。方法 从正常和ARDS大鼠肺组织中提取总RNA,分离纯化mRNA,经反转录合成掺入生物素标记的cDNA探针,然后与基因芯片杂交,扫描芯片荧光信号图像,用Sam3.0进行统计处理,用博奥生物分子功能注释系统V4.0进行功能分析。随机选择3个差异表达基因 ,用荧光定量RT- PCR验证。结果 在ARDS大鼠肺组织中,信号传导相关基因上调的有2个,下调的有9个,涉及到的相关信号通路11个。结论 大鼠ARDS基因表达谱中涉及到许多信号传导基因和通路的差异表达。     

应用基因芯片技术筛查人子宫内膜容受性相关基因

目的探讨人子宫内膜容受性相关基因的差异表达。方法应用含14000条基因的cDNA表达谱基因芯片分析分泌早期与分泌中期子宫内膜基因的差异表达。结果所检测的14000个基因中,分泌早期子宫内膜与分泌中期子宫内膜之间存在显著差异表达基因313个。其中,分泌中期子宫内膜表达上调基因数为175个,下调基因数为138个。结论子宫内膜容受性的建立受许多因素的调控,应用基因芯片技术可以快速、高通量的筛查出相关基因,从而有可能找到合适的分子标志物作为子宫内膜容受性的临床诊断指标。