基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)課件

Microarray-baseddiscoveryofhighlyexpressedolfactorymucosalgenes:potentialrolesinthevariousfunctionsoftheolfactorysystem

基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)：在嗅覺系統(tǒng)多功能作用中扮演的潛在角色基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)實(shí)驗(yàn)?zāi)康模韩@得在嗅黏膜中組織特異性基因表達(dá)的全局觀。Why？

OM呼吸道與神經(jīng)系統(tǒng)的交界處，執(zhí)行使兩個(gè)器官系統(tǒng)連為一體的功能，是脊椎動(dòng)物成體中神經(jīng)發(fā)生和神經(jīng)再生的主要位點(diǎn)。嗅覺器官的感覺神經(jīng)元負(fù)責(zé)氣味轉(zhuǎn)換，呼入的空氣中有毒媒介能夠被鼻呼吸道和嗅覺組織中的黏膜纖毛結(jié)構(gòu)和代謝酶清除。OM相對(duì)于其它神經(jīng)組織的獨(dú)特之處在于：OM有一個(gè)基底細(xì)胞群，能夠不斷地提供新的神經(jīng)元，使因受傷或毒氣損傷而丟失的神經(jīng)元得以替換?；谖㈥嚵械男狃つぶ懈弑磉_(dá)基因的發(fā)現(xiàn)

MATERIALSANDMETHODS

SECTION1基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)MATERIALSANDMETHODS：1、Animals,tissuesamples,andRNApreparation2、Fluorescentprobepreparationandmicroarrayhybridization3、Dateanalyses4、confirmatorytest

5、Identi?cationofcis-elementsincoordinatelyexpressedgenes基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)組織來(lái)源：

成鼠的嗅黏膜（OM）、嗅球（olfactorybulb）、鼻呼吸道黏膜（nasalrespiratorymucosa）組織取自6-8周齡的C57BL/6雌性小鼠。Theethmoidturbinatesandcaudalone-thirdofthenasalseptumservedasthesourceofOMRNA,andthenasoturbinates,maxillo-turbinates,andanteriortwo-thirdsofthenasalseptumwerethesourceofrespiratorymucosalRNA.Othertissuesincludingmulti-plebrainsubregions,dorsalrootganglion,heart,lung,kidney,liver,reproductiveandendocrineorgans,immuneorgans,muscle,andskinarefromageneralizedmousegeneexpressiondatabase.RNA提取：用CO2吸入法處死小鼠解剖小鼠，迅速取出組織，液氮速凍將取自八只小鼠的組織在冰上勻漿TRIReagent提取總RNA總RNA用酒精、醋酸鈉沉淀總RNA在DEPC水中溶解基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)ProbepreparationandmicroarrayhybridizationwereperformedbyIncyteGenomics(PaloAlto,CA)usingtheGEMBrightrandomprimerreversetranscriptionlabelingkit(IncyteGenomics).LabeledcDNAwaspreparedfromeachpoly(A)+RNAsampleusingnucle-otideslabeledwiththe?uorescentdyeCy5.LabeledcDNAswerethensubjectedtocompetitivehybridizationtomouseGEM1microarrays,composedofspottedcDNAscorrespondingto8,638sequence-veri?edIMAGEexpressedsequencetag(EST)clones(500–5,000nucleotidesinlength)withalowredundancyratewithrespecttotheircorrespondinggeneproducts.Forallsamplesinthedatabase,hybridizationswereperformedincompetitionwithCy3-labeledmRNAfromwholepostnatalday1(P1)mouse.基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)ForeachmRNAsample,duplicatearrayswerehybridized.Additionaldye-?ipcontrolexperimentsindicatedalowdyeeffect,particularlycomparedwithotherCy3/Cy5labelingmethods(datanotshown).Dyeeffectwas,however,subtractedfromrelativesample-speci?cgeneexpressionbasedonpergenemediannormalizationacrossalargenumberofsamples.FluorescenceintensityanalysesandbackgroundsubtractionwereperformedusinganAxonInstrumentsscannerandGenePixsoftware.基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)數(shù)據(jù)的預(yù)處理：數(shù)據(jù)分析、點(diǎn)幾何學(xué)和背景熒光用IncyteGEMTools軟件處理。1、芯片上有缺陷的點(diǎn)（不規(guī)則的幾何形狀、有刮痕、點(diǎn)的面積小于平均面積的40%）在數(shù)據(jù)集中被刪除；2、數(shù)據(jù)的對(duì)數(shù)轉(zhuǎn)化：所有數(shù)據(jù)集用Cy5測(cè)量通道中值除去Cy3測(cè)量通道中值進(jìn)行平衡系數(shù)歸一化；3、每張芯片包含192個(gè)對(duì)照基因，包括非哺乳動(dòng)物單個(gè)基因和大多數(shù)細(xì)胞類型中表達(dá)的基因構(gòu)成的復(fù)雜靶標(biāo)。4、每個(gè)實(shí)驗(yàn)中mRNA樣品用增加數(shù)量的非哺乳動(dòng)物RNA（2-fold、4-fold、16-fold，etc）進(jìn)行放大從而允許對(duì)從每張芯片中獲得的動(dòng)態(tài)范圍進(jìn)行評(píng)估?；谖㈥嚵械男狃つぶ懈弑磉_(dá)基因的發(fā)現(xiàn)第二階段的數(shù)據(jù)分析用Gene-spring軟件處理。1、基因的歸一化處理：①Cy5對(duì)數(shù)值/Cy3對(duì)數(shù)值②用LOWESS方法進(jìn)行局部加權(quán)歸一化處理③一系列探針中每個(gè)基因的歸一化每個(gè)測(cè)量值/全部測(cè)量值中值>=0.01每個(gè)基因在每個(gè)樣本中的測(cè)量值/該基因在全部樣本中的測(cè)量中值>=0.012、找出在MO中高表達(dá)的基因基因表達(dá)量與在數(shù)據(jù)庫(kù)中所有其它組織中的表達(dá)中值之比>=1.7269個(gè)基因3、用分層樹聚類方法對(duì)269個(gè)基因進(jìn)行分類基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)對(duì)芯片數(shù)據(jù)的實(shí)驗(yàn)驗(yàn)證&組織原位雜交

選取在MO中高表達(dá)的基因進(jìn)行分析定位

(Sgpl1、Cyp2f2、Dmbt1、Ltf）&免疫組織化學(xué)

抗血清、二抗的制備、組織切片&Sgpl1蛋白活性測(cè)定

材料：maleLong-Evansrats方法：用sphinganine-1-phos-phate測(cè)定活力基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)共表達(dá)基因順式作用元件的確定&geneontology&選取三個(gè)亞分類(每個(gè)亞分類選取三個(gè)基因）進(jìn)行分析&通過(guò)數(shù)據(jù)庫(kù)獲得選定的基因在人和小鼠中的全序列分析&序列比對(duì)找出保守的順式作用元件&通過(guò)TRANSFACTDatebase和DNA-bindingsitepro?les定位作用元件位點(diǎn)&找出選定基因的相同順式作用元件位點(diǎn)基因分類基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)SECTION2RESULTS基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)

RESULTS1、IdentificationofgeneshighlyexpressedinadultOM.

成熟OM組織中高表達(dá)基因的鑒定2、Sgpl1activitymeasurements.

Sgpl1基因活力的測(cè)定3、Insituhybridizationandimmunohistochemistrystudies.

原位雜交和免疫組化學(xué)分析4、Functionalclassificationofgeneshighlyexpressedintheadultmouseolfactorysystem.

成熟OM系統(tǒng)中高表達(dá)基因的功能分類5、Genediscoveryintheolfactorysystem.

嗅覺系統(tǒng)基因的發(fā)現(xiàn)6、Commoncis-elementsincoordinatelyexpressedgenes.

共表達(dá)基因共同順式作用元件的鑒出基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)GENEEXPRESSIONINOLFACTORYMUCOSAPEARSONcorrelation269/8734(1.7-foldhigher)ROW：geneCOLUMN:tissueRED:upregulatedBLUE:downregulated→temporallyandspatiallyrestrictedexpression基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)MaleLong-EvansratsNormalizedexpressionPvalue(UC-CHMCCgeneexpressiondatabase)Activity(VanVeldhovenPP.Sphingosine-1-phosphatelyase.MethodsEnzymol311:244–254,2000)→activityofSgpl1inOMwasindeedhigh基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)TheBowman’sglandstheepithelialsurfaceofthedorsalseptum&theturbinatesliningthedorsalmedialairwaysductandacinarcellsofBowman’sglandsapicalcytoplasmofsustentacularcellsantisenceCyp2f2anti-Cyp2f2antibody基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)***olfactoryepitheliallayer(olfactorysensoryneurons)subepithelialcompartmentsustentacular(supporting)cells***negativenegativeantisenseSgpl1senseSgpl1基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)perinuclearlayerofmatureolfactorysensoryneurons***olfactoryepithelialcompartmentsubepithelialstructuresanti-Sgpl1antibodyantisenseAlms1基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)lateralnasal

glandolfactoryepithelium(oftendisplayinghigh

expressionatthejunctionsofOMandrespiratory

mucosa)antisenceDmbt1antisencePlunc基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)*upstream3-kbregion*TraFaCserver基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)discussionsection3基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)特色一、充分運(yùn)用了各種數(shù)據(jù)庫(kù)資源NCBI、ENSEMBLUC-CHMCCEST→全基因序列SwissProt、Geneontology基因功能注釋功能分類BlastNTransfact序列比對(duì)結(jié)構(gòu)域確定順式作用元件確定基于微陣列的嗅黏膜中高表達(dá)基因的發(fā)現(xiàn)特色二、就OM組織解剖學(xué)與生理學(xué)對(duì)其基因表達(dá)模式進(jìn)行了細(xì)致分析1.OM中神經(jīng)細(xì)胞直接與外界接觸→高表達(dá)與解毒代謝相關(guān)的基因（與LungandLiver比較得出）2.OM有神經(jīng)細(xì)胞再生功能→高表達(dá)與神經(jīng)再生與神經(jīng)發(fā)育的基因芯片雜交結(jié)果顯示：OM、胚胎、出生一天小鼠腦都高表達(dá)一些與神經(jīng)分化、遷