多药耐药基因Mdr1 RNA探针的构建及初步临床应用

检测Mdrl基因表达水平可预测白血病化疗效果,用原位杂交的方法可检测Mdrl在单个细胞的表达水平。本文用Rt-PCR的方法获得了一段特异的cDNA片段,将其克隆到PGEM4Z载体中,经DNA序列分析证明与文献报道一致,采用地高辛素（DIG）RNA标记试剂盒制备反义RNA探针,已初步用于临床骨髓涂片标本的原位杂交检测。     

血管内皮细胞MICA基因mRNA和蛋白质表达

MICA系MHC-I类相关多态性基因A,其表达细胞膜蛋白质分子与器官移植免疫排斥有关.新生儿脐带静脉血管内皮细胞(HUVEC)被分离和培养至4～6代.11例新分离培养的HUVEC和7种人细胞株的MICAmRNA转录水平用RT-PCR检测,应用细胞流式检测细胞膜表面的MICA分子的表达情况,同时应用免疫印迹的方法对整个细胞MICA蛋白质的表达水平进行检测和分析.结果显示MICAmRNA在所有检测细胞中有表达,HUVEC细胞膜或胞浆中MICA蛋白质表达量很高,而在人体淋巴细胞膜表面和胞内未检出MICA分子的表达.提示HUVEC和淋巴细胞在MICA表达的差异性可能与不同细胞的内部调节机制有关.而供体器官血管内皮细胞表达多态性MICA分子有可能成为移植抗原发生免疫排斥反应.     

Kin17表达促进乳腺上皮细胞MCF-10A增殖

Kin17是一个与DNA复制、DNA修复有关的蛋白质,在人类的各种组织中表达均很低.乳腺上皮细胞生长增殖的分子机制尚未阐明.为了探讨Kin17与乳腺上皮细胞增殖的关系,检测了Kin17在不同增殖状况下的MCF-10A细胞中的表达情况,并把KIN17基因插入真核表达载体pCDNA3.1-(+)中,构建重组质粒pCDNA3.1-Kin17,通过转染MCF-10A细胞,检测Kin17的表达对MCF-10A细胞的增殖、DNA复制活性及信号分子表达的影响;同时在转染Kin17特异性小干扰RNA（siRNA_Kin17）后,分析MCF-10A细胞的Kin17表达及细胞生长状况.实验结果显示,经高浓度血清刺激后,细胞中Kin17表达升高,而且生长越快的细胞,Kin17表达越强;转染重组质粒pCDNA3.1-Kin17明显提高了MCF-10A细胞中Kin17的表达,同时Kin17的上调表达促进了细胞的增殖速度与DNA复制活性,增强了cyclin D1的表达水平.当转染siRNA_Kin17时使Kin17含量下调,MCF-10A细胞生长速度的抑制不显著.实验结果表明,Kin17与乳腺上皮细胞的DNA复制及生长增殖密切相关.对Kin17在乳腺上皮细胞增殖中的作用及分子调控机制的深入探讨,将有助于揭示乳腺癌细胞快速增殖的潜在机制.     

在体分离小鼠小肠隐窝、绒毛上皮细胞的生化完整性研究

目的:检测低温条件下用螯合剂沉淀法分离的小鼠小肠上皮隐窝和绒毛细胞是否具有生化完整性.方法:使用螯合剂在低温(冰浴)条件下分离和富集小肠上皮绒毛和隐窝细胞;抽提DNA、RNA和总蛋白,用电泳的方法检测完整性;用Real-time PCR检测溶菌酶Lysozyme的表达以判断隐窝、绒毛细胞富集程度.结果:低温条件下分离的肠上皮隐窝、绒毛细胞形态完整;基因组DNA完整,未出现明显的DNA ladder现象;富集细胞的RNA完整;富集隐窝、绒毛细胞的蛋白未降解,两组总蛋白具有表达谱差异性;隐窝细胞富集物溶菌酶mRNA表达水平较绒毛细胞富集物高30倍以上.结论:小肠隐窝绒毛的生物学性状可在低温螯合剂沉底法分离过程中得到保存,提示此方法可以用来分析生理和创伤痛理条件下小肠上皮基因和蛋白表达改变.     

化学发光法检测体外HBV复制

目的:建立化学发光法检测体外HBV复制水平的方法,研究其灵敏度和稳定性.方法:用HBV DNA重组质粒pCH9转染到人肝癌细胞株HepG2和Huh7中,5d后收集细胞并抽提其HBV复制中问体DNA,转印后以地高辛标记HBV DNA为探针进行杂交,用化学发光法检测杂交结果,同时进行探针灵敏度的检测.结果:转染后HepG2和Huh7细胞提取HBV DNA中检测出较强的复制中间体的信号,分别为松散环状DNA(rcDNA),双链线性DNA(dslDNA),单链DNA(ssDNA),探针检测的灵敏度可达到lpg,接近同位素法检测的灵敏度.整个实验重复3次获得同样结果.结论:成功建立了稳定的化学发光法检测体外HBV复制水平的方法.     

CKS1 表达与食管癌细胞辐射敏感性的关系研究

目的:探讨CKS1表达对食管癌细胞辐射敏感性的影响,初步研究其分子机理.方法:用Western-blotting方法筛选CKS1低表达和高表达的食管癌细胞系;构建CKS1正义表达载体p-pcDNA 3.1/myc-His A-CKS1和RNA干扰载体CKS1 siRNA,分别转染CKS1低表达细胞和高表达细胞,用不同剂量γ-射线照射各组细胞,克隆形成实验检测细胞辐射敏感性的差异.结果:CKS1在四种食管癌细胞中的表达水平依次为EC9706＞ KYSE510＞KYSE450＞ KYSE150.用p-pcDNA 3.1/myc--His A-CKS1表达载体转染KYSE150细胞后CKS12表达升高,不同剂量γ-射线照射后细胞的克隆形成能力显著高于母系对照组(P＜0.01).RNA干扰载体转染KYSE510细胞后CKS1表达水平降低,不同剂量γ-射线照射后细胞的克隆形成能力显著低于母系对照组(P＜0.01).敲降CKS1表达后DNA损伤修复相关蛋白RAD51表达下降,KU70表达没有变化.CKS1过表达后RAD51表达升高,KU70表达没有变化.结论:CKS1表达与食管癌细胞的辐射敏感性密切相关,可能通过影响DNA损伤修复发挥作用.     

miR-21与DNA甲基化在不同乳腺癌细胞中的相互作用

目的:探究不同乳腺癌细胞(MCF-7、MDA-MB-231)中miR-21与DNA甲基化相互调节作用。方法:将荧光标记的miR-21抑制剂及阴性对照瞬时转入MCF-7、MDA-MB-231细胞中,用荧光显微镜观察其转染效率,以Real-time PCR检测miR-21的敲低水平,并以bisulfite-q MSP法检测基因组DNA甲基化水平。同时,以2.5μmol/L DNA甲基化酶抑制剂5-AZA处理细胞72 h,以单纯二甲基亚枫(DMSO)处理做为阴性对照,观察DNA甲基化改变对miR-21表达水平的影响,接着以Western blot检测miR-21下游基因人第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)、蛋白激酶B(AKT)蛋白的表达水平。结果:miR-21抑制剂可敲低MCF-7细胞中miR-21的表达水平(P0.01),并引起基因组DNA甲基化水平的显著升高以及DNA甲基化转移酶Dnmt1、Dnmt3a以及Dnmt3b的普遍升高(P0.05,P0.01)。而在MDA-MB-231细胞中瞬转miR-21抑制剂则引起miR-21表达水平的小幅度升高(P0.01)以及整体DNA甲基化水平的降低(P0.05),并伴随有Dnmt3a的升高及Dnmt3b的降低。使用5-AZA处理后发现,其可显著上调MCF-7以及MDA-MB-231细胞中miR-21的表达(P0.01),并引起其下游基因PTEN在MCF-7细胞内的表达升高,进而下调AKT的蛋白水平。结论:瞬转miR-21抑制剂对MCF-7与MDA-MB-231细胞DNA甲基化水平的调节截然相反,而DNA甲基化的降低则可使miR-21的表达一致上调。本研究可为以后不同类型乳腺癌的临床治疗提供一定的实验依据。     

胎盘型谷胱甘肽S－转移酶基因在胃癌中的表达

用Dig-GST-πcDNA探针分子杂交方法,检测了正常胃组织、胃癌及相应癌旁正常组织中GST-πDNA和GST-πRNA水平.发现GST-πDNA水平没有明显变化,而CST-πRNA在8例胃癌组织中有6例高于正常胃组织,在12例低分比腺癌中有7例癌旁正常组织高于相应癌组织.表明GST-π基因表达增加与胃癌有关,而且早于细胞形态的变化.     

PTEN通过抑制PI3K-AKT信号通路上调Rad51基因的表达

前期研究发现pten缺陷细胞的自发DNA双链断裂损伤水平显著增加.本研究探讨了抑癌基因pten对参与DNA同源重组修复的rad51基因表达的影响和机制．用实时定量PCR技术检测了PTEN野生型和缺陷型细胞rad51的表达水平.结果发现,PTEN缺失会导致rad51的表达降低．PI3K激酶为PTEN的下游负调节靶分子,使用PI3K激酶抑制剂LY294002处理缺陷型细胞后,其rad51表达升高．在PTEN野生型细胞中分别转染Flag-Akt WT（野生型）和Flag-Akt AC（组成型激活）,或在PTEN缺陷型细胞中分别转染野生型PTEN和Akt-DN（失去激酶活性的Akt）. 利用RT-PCR技术检测上述细胞rad51的表达水平,同时利用Western印迹检测上述细胞RAD51蛋白的表达水平.结果发现,转染Flag-Akt WT和Flag-Akt AC后,均能促使PTEN野生型细胞中rad51在mRNA和蛋白水平降低;在PTEN缺陷型细胞中转染野生型PTEN或Akt-DN后,rad51在mRNA和蛋白水平均升高．在PTEN缺陷型细胞中使用siRNA沉默akt后,同样导致RAD51表达升高．结果提示,PTEN可以正向调节RAD51基因表达,PI3K/Akt是其信号通路机制之一．     

LRP16通过调控E-钙粘合素的表达促进MCF-7细胞的侵袭生长

LRP16在原代乳腺癌组织中表达水平与雌激素受体α（ERα）表达状态以及腋窝淋巴结侵袭数目密切相关.为研究LRP16基因对乳腺癌MCF-7细胞侵袭生长的影响,并探讨涉及的分子机制,采用基质胶黏附实验与Transwell方法,检测内源性LRP16表达抑制MCF-7细胞的体外黏附、侵袭生长与迁移特征.结果表明,抑制LRP16在MCF-7细胞中的表达,降低了细胞的体外黏附、侵袭与迁移能力;采用FVB小鼠进行的实验性转移试验结果显示,抑制LRP16显著降低了MCF-7细胞的肺转移结节数目;为探索可能的分子机制,采用Western印迹方法,检测了LRP16对乳腺癌转移相关分子MMP-2, MMP-9, CD44和E-钙黏着蛋白表达的影响,结果在LRP16抑制的MCF-7细胞中只有E-钙黏着蛋白蛋白表达上调.进一步的Northern印迹与免疫组化实验结果表明,抑制LRP16可上调MCF-7细胞中E 钙黏着蛋白基因的mRNA与蛋白表达水平;共转染与双荧光素酶方法检测LRP16对E-钙黏着蛋白基因启动子的表达调控效应,结果显示,LRP16抑制E 钙黏着蛋白基因基因5′-近端启动子的转录激活,该抑制效应选择性存在于内源性ERα阳性细胞,并且依赖于雌激素的存在;染色质免疫共沉淀方法（ChIP）检测ERα与E-钙黏着蛋白基因启动子的相互作用,结果显示,在LRP16基因表达缺陷的MCF-7细胞中,ERα抗体沉淀到E-钙黏着蛋白基因启动子的DNA序列;上述研究结果表明,抑制LRP16基因表达,削弱了激素依赖型乳腺癌细胞的侵袭生长能力,其分子机制涉及了LRP16通过ERα介导对E-钙黏着蛋白基因基因转录激活的调控.     

Enhanced expression of human cDNA by phosphoglycerate kinase promoter-puromycin cassette in the mouse transthyretin locus

To produce a humanized mouse, it is critical to obtain a correct expression of a human gene/cDNA after insertion into a mouse locus. We previously generated a targeted allele in which the PGK-neo cassette, flanked by lox71 and loxP, was inserted into the first exon of the mouse endogenous transthyretin (Ttr) gene in ES cells. Using these ES cells, we showed that a human transthyretin (TTR) cDNA with the PGK-puro cassette can be efficiently inserted into this locus by Cre-mediated recombination, and that the human TTR cDNA was expressed in a tissue-specific manner under the control of the mouse endogenous Ttr promoter. To examine whether the PGK-puro cassette or IRES could affect the expression of human TTR cDNA, we generated four mouse lines using Cre and Flp-mediated recombination. The mouse line containing the PGK-puro cassette, but not IRES, exhibited quantitatively and temporally similar expression of human TTR cDNA. Removal of the PGK-puro cassette significantly downregulated the expression of the cDNA. The insertion of IRES sequence upstream of the human TTR cDNA resulted in decreased expression, even in the presence of the PGK-puro cassette. The mouse line containing IRES, but not PGK-puro, showed the lowest level of expression. These results suggest that the PGK-puro cassette is necessary to obtain the enhanced expression of a co-existing human cDNA in the mouse Ttr locus, even though the expression of co-existing cDNA was under the control of the mouse endogenous promoter.     

Molecular cloning, identification and characteristics of a novel isoform of carbamyl phosphate synthetase I in human testis

A gene coding a novel isoform of carbamyl phosphate synthetase I (CPS1) was cloned from a human testicular library. As shown by cDNA microarray hybridization, this gene was expressed at a higher level in human adult testes than in fetal testes. The full length of its cDNA was 3831 bp, with a 3149 bp open reading frame, encoding a 1050-amino-acid protein. The cDNA sequence was deposited in the GenBank (AY317138). Sequence analysis showed that it was homologous to the human CPS1 gene. The putative protein contained functional domains composing the intact large subunit of carbamoyl phosphate synthetase, thus indicated it has the capability of arginine biosynthesis. A multiple tissue expression profile showed high expression of this gene in human testis, suggesting the novel alternative splicing form of CPS1 may be correlated with human spermatogenesis.     

Evaluation of experimental designs for two-color cDNA microarrays.

The major goal of two-color cDNA microarray experiments is to measure the relative gene expression level (i.e., relative amount of mRNA) of each gene between samples in studies of gene expression. More specifically, given an N-sample experiment, we need all N(N - 1)/2 relative expression levels of all sample pairs of each gene for identification of the differentially expressed genes and for clustering of gene expression patterns. However, the intensities observed from two-color cDNA microarray experiments do not simply represent the relative gene expression level. They are composed of signal (gene expression level), noise, and other factors. In discussions on the experimental design of two-color cDNA microarray experiments, little attention has been given to the fact that different combinations of test and control samples will produce microarray intensities data with varying intrinsic composition of factors. As a consequence, not all experimental designs for two-color cDNA microarray experiments are able to provide all possible relative gene expression levels. This phenomenon has never been addressed. To obtain all possible relative gene expression levels, a novel method for two-color cDNA microarray experimental design evaluation is necessary that will allow the making of an accurate choice. In this study, we propose a model-based approach to illustrate how the factor composition of microarray intensities changed with different experimental designs in two-color cDNA microarray experiments. By analyzing 12 experimental designs (including 5 general forms), we demonstrate that not all experimental designs are able to provide all possible relative gene expression levels due to the differences in factor composition. Our results indicate that whether an experimental design can provide all possible relative expression levels of all sample pairs for each gene should be the first criterion to be considered in an evaluation of experimental designs for two-color cDNA microarray experiments.     

人Six1基因真核表达载体的构建及其生物学功能研究简

目的：构建带myc标签的Sixl基因的真核表达载体,获得myc-Sixl融合蛋白,并对其生物学功能进行初步检测。方法：以本实验室保存的乳腺文库为模板,采用PCR技术扩增Sixl编码序列,将其插入pXJ-40-myc载体,West-ern印迹检测其在人293T细胞中的表达;将重组质粒-9空载体分别转染乳腺癌细胞ZR75-1,通过qRT-PCR检测细胞中VEGF-C在mRNA水平的变化。结果：双酶切和测序结果表明myc-Six1真核表达质粒构建成功;Western印迹证明转染293T细胞后成功表达;qRT—PCR结果表明myc-Sixl可升高乳腺癌细胞ZR75-1的VEGF-C转录水平。结论：构建了带myc标签的Six1表达载体,为进一步研究Six1在乳腺癌转移中的作用奠定了基础。     

Identification and characterization of a novel cancer/testis antigen gene CAGE

We applied serological analysis of cDNA expression library technique to identify cancer-associated genes. We screened cDNA expression libraries of human testis and gastric cancer cell lines with sera of patients with gastric cancers. We identified a gene whose expression is testis-specific among normal tissues. We cloned and characterized this novel gene. It contains D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. It showed wide expression in various cancer tissues and cancer cell lines. The corresponding gene was named cancer-associated gene (CAGE). PCR of human x hamster Radiation Hybrids showed localization of CAGE on the human chromosome Xp22. Transient transfection of CAGE showed predominantly nuclear localization. Both Western blot and plaque assay indicated seroreactivity of CAGE protein. We found that demethylation played a role in the activation of CAGE in some cancer cell lines that do not express it. Cell synchronization experiments showed that the expression of CAGE was related with cell cycle. This suggests that CAGE might play a role in cellular proliferation. Because CAGE is expressed in a variety of cancers but not in normal tissues except testis, this gene can be a target of antitumor immunotherapy.     

TPO内含子Ⅴ可显著增强人血小板生成素基因在乳腺细胞中的表达

人血小板生成素（thrombopoietin,TPO）基因组包括6个外显子和5个内含子,内含子在其表达过程中可能扮演着重要作用.为了研究人TPO基因组中不同内含子对TPO表达的影响,构建可在转基因动物乳腺中高水平表达人TPO的乳腺特异性表达质粒.本研究以65 kb的山羊β-casein启动子为调控元件,构建了包括人TPO cDNA(pTPOA)、TPO intronⅠ-TPO cDNA (pTPOB)、ΔTPO intronⅠ-TPO cDNA (pTPOC)、TPO intronⅤ-TPO cDNA (pTPOD)和TPO gDNA (pTPOE)等5种TPO乳腺特异性表达质粒,并在人乳腺细胞HC-11细胞上进行了瞬间表达研究.在转染48 h后,通过双抗体夹心的ELISA方法定量分析上述质粒在HC-11细胞上的表达水平.结果表明,含有内含子Ⅴ的 pTPOD的表达量最高,而含有整个基因组TPO的pTPOE表达水平最低.为了进一步证实pTPOD可在乳腺细胞中高水平表达,将pTPOD经脂质体包埋后注射到泌乳期山羊的乳腺中.结果显示,在山羊乳汁中可连续14 d检测到人TPO的表达.上述实验证实,人TPO基因组中的内含子V可显著提高TPO在HC-11细胞内的表达水平,并提示内含子Ⅴ中可能含有特殊的调控序列.