维甲酸对培养的猪支气管上皮细胞β-catenin2f某cf信号途径的影响

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1、华中科技大学同济医学院硕士毕业论文Objectives：ToinvestigatethechangesofretinoicacidonB—cateninmRNAandproteinlevel，andtheeffectsofretinoicacidon13-catenin／TCFpathwayinculturedporcinlracheobronchialepithelialcelI(TBEC)．Methods：1．IsolationandcultureofporcinetracheobronehialepithelialcellsTracheaandbronchu

2、swereobtainedfromfreshlyslaughteredpig．AmodifiedmethodofWuetalforisolatingcellshasbeendescribedpreviously．About105eells／cm2w眦platedincultureflaskscoatedwithrattallcollagen．TheculturemediumwasDMEM／F·12supplementedwith51xg／mlinsulin,10)xg／mltransferrin，O．5l_tg／mlhydrocortisol，lOn#rntepi

3、dermalgrowthfactor(EGF)，5％fetalbovineserunl，andantibioticsf100U／mlpenicillin，10099／mlstreptomycin)．Cellsareculturedat37。Cwith5％C02．Microscopically,cellsconfluentandconnectedtightly，showingconspicuouslytiltappearance．Anti-cytokeratinproteinwaspositiveandanti-vimentinproteinwasnegeafive

4、．Weconcludedthat99percentculturecellsweretracheobronchialepithelialcells．Afterretinoicacidtreatment，cellularvacuolesincreased，cellularconnectionWasstilltight．Thefirstpassagesofcellwereused．Experimentswererepeatedindifferentisolationsofcells．2．TreatmentofexperimentgroupThisexperimentdi

5、videdintothreegroups-Firstgroup：controlgroup；Secondgroup：addedretinoidacid(1gg／91)intoculturemedium；Thirdgroup：addedretinoidacid(299／l_d)intoculturemedium．AfterRAtreatmentfor12hoursand24hours，weisolatedcells．7坐主堕查兰旦堕堕堂堕堡主皇些兰3．ImmunocytoehemistryAfterexposureoftheculturedtracheobmnehia

6、lepithelialcellsfortwelvehoursandtwenty-fourhours011coverslipstoretinoicacid．Thecellswerefixedinfixedsolution(coldacetone：ethanol=1：1)，slideswereincubatingwitllamonocolonalanti-p—catenin(1：500)overnightat4℃．followedbydetectingusingstriptavidin／peroxidase(SP)immunoeytochemicalkit．Theho

7、rseradishperoxidasesubstrate，namely3,3．diaminobenzidineWaSused．Heamtoxylinwasusedasacouterstain．4RT—PCRArialysisAfterexposureoftheculturedtracheobronehialepithelialcellsfortwelvehoursandtwenty-fourhoursoncoverslipstoretinoicacid．TotalRNAsureoftheculturewasisolatedusingTRIZOLreagent．Re

8、verse