velikost textu

Vlastnosti myších buněk, které byly modifikovány geny pro imunomodulační faktory a endostatin

Upozornění: Informace získané z popisných dat či souborů uložených v Repozitáři závěrečných prací nemohou být použity k výdělečným účelům nebo vydávány za studijní, vědeckou nebo jinou tvůrčí činnost jiné osoby než autora.
Název:
Vlastnosti myších buněk, které byly modifikovány geny pro imunomodulační faktory a endostatin
Název v angličtině:
Characteristics of HPV16 transformed mice cells, transduced with genes for immunomodulating factors and endostatin
Typ:
Disertační práce
Autor:
Mgr. Monika Lakatošová, Ph.D.
Školitel:
prof. MUDr. Vladimír Vonka, DrSc.
Oponenti:
RNDr. Milan Reiniš, CSc.
doc. RNDr. Jitka Forstová, CSc.
Id práce:
108441
Fakulta:
Přírodovědecká fakulta (PřF)
Pracoviště:
Katedra genetiky a mikrobiologie (31-140)
Program studia:
Molekulární a buněčná biologie, genetika a virologie (P1519)
Obor studia:
-
Přidělovaný titul:
Ph.D.
Datum obhajoby:
16. 6. 2011
Výsledek obhajoby:
Prospěl/a
Jazyk práce:
Čeština
Abstrakt:
Souhrn Vt6toprAcijsemzkoumalavlastnostimy3lchbun6ktransformovanfchviremHPV16'kfer6 pro cytokiny' chemokin a jeden angiostaticki gen' Byly byly geneticky modifikovfny vnesenim gent testovAnyhlavnlcharakteristikytransdukovanfchbun€kvporovnAnisrodidovkjminddorovfmi buikami in vitro a in viYo VprvnldristijsempouZilabunddnouliniidefrcientnlnabunddnoutymidinkin6zu'ozratenou jakol23IA,kter6bylaodvozenazmy5ich(c57BLl6)ledvinnjchbun€ktransformovarrjchonkogeny E6lETHPV16aaktivovanfmonkogenemH.rasomaEenlchjakoMK16.Builkyl23IAjsem geny pro herpetickou tymidin kin6zu (HSv-Tn, transfekovala bicistronickfmi plazmidy nesoucimi - protein 1 chemoatrahujici monocyty (MCP-L)' kostimulaini molekulu B7-,1(cD 80) nebo chemokin jen gen pro I/SY-IK' Do testfijsem zahrnula tak€ ho kontrolni idely jsem pouZila plazmid obsahujicl geny pro faktor stimulujicl tvorbu kolonii diive izolovan6 bufiky Bg, kterd byly transdukov6ny granulocytiamonocytft(GM-cSnaHSV-TK'VSechnyizolovan6liniebylycitliv6kegancikloviru' po inokulaci syngennim mystm, byly bunky exprimujici GM- prokazujice tak piitomnost HSV-TK. produkujicichMCP-I vfvofily t6m6l v5echnymy3i CSFa B7-l neonkogenni. odkov6nibunEk Po GM-CSF a B7-l byla chr6ndna pled n6slednou fl(dory. Zvl?rltaimtmizovani bunkami produkujici delenZibunkamiMKl6.Pokudjsemalepiicelen}ipouZilajinouonkogennlbundlnouliniiodvozenou liSi viadd zmyli C57BLi6 a transformovanou HPV16, zvanou TC-l' kter6 se od bunEk MKl6 mnohem menii' parametru,pak byl protektivni ridinek transdukovanfch bunEk Vdruh6d6stim6precejsemtransfekova|abuikyMKl6aTC-lgenemproangiostatickfmysi produkujici endostatin' Oznadila jsem je proteir endostdtin. Zobou linil jsem izolovala dva klony ME3aMEg(odvozendodbunEkMKl6);aTE2aTE5(odvozen6odbundkTC-l).PoodkovAnldo myxibylybuiikyME3neonkogennl.T6m6lv5echnymy3iodkovarr6bunkamiMEgvyvinulynridory' alebylaunichsiln€omezenatvorbametastazvporovn6nlsrodidovskgmibunkamiMKl6.TE2a TE5lykazovalyt6m6tstejnionkogennipotenci6tjakorodicovsk6bufikyTC-l.Zvilataimtlnilzovanf buikamiME3bylachr6n6nip|edEe|enLirodidovskjmibufikamiMKl6.Testovalajsembun6dn6 |yz6tyzevSechSestisledovanj,chtinii,abychzjistilaplitomnost25.faktorftovlivilujicichangiogenezi. BunkyMKl6seliSilyodbun6kTc-latak6odv5echsubliniiprodukujlcichendostatinzejm6na prok6zaly'tn snLeni produkceIL-lo u zvf3enouprodukclinterleukinu-lc (IL-14)' DalSiexperimenty mechanizmem' bundk MK16 vlivem endostatinubyla zptsobena autokrinnim
Abstract v angličtině:
Summary Genesfor two cytokines, one chemokine and genecoding for one angiostatic factor were used in the present work for tansfection ofmouse IIPV-16- transformed tumor cells. Main characteristics oftransduced cells werc t€stedrn raTo and, vivo and,cnrnpared in with the parentaltumor cells. In the first part I used a thymidine-kinase deficient (cTK) cell line designated 123IA, which had been derived from IIPVI6 transformedmouse (C57BL/6) cells MK16. To obain genetically modified cells, 1231A cells were transfected with bicistronic plasmids carrying the herpes simplex type 1 thymidine kinase (HSV-TK) gen,e either the gene for the mouseB7-l (CD80) co-stimulatory and molecule or the gene for the monocyte-chemoattractant protsin 1 (MCP-|). For control putposes, a plasmid vector carrying only the HSV-TK gene was used. For comparative purposesI also used 89 cells, previously isolated in our laboratory, which expressthe mouse granulocyte-macrophage colony stimulation factor (GM-CSF) and HSV-TK gene. All the cell lines testedwere found to be sensitive to minute amounts of ganciclovir, revealing the production of functional HSV-TK. When inoculated into syngeneic mice, cells expressing either GM-CSF or B7-1 were non-oncogenic. Nearly all mice inoculatedwith MCP-l-producing cells developedtumors.Animals injectedwith GM-CSF or B7-l- producing cells were protected against challenge with the parental MK16 cells. When another mouse (C57BU6) HPVl6transformed oncogeniccell line, TC-l, which differs from the MK16 cells in a number ofproperties, was used for the challenge,the protective effect was much less pronounced. Lr the secondpart, MK16 cells and TC-l cells, which had been also derived from HPVI6 transformed mouse (C57BL/6) cells, were transducedwith the gene for angiostatic mouse endostatin. Two clones constitutively expressingendostatinwere isolated from each cell line. They were denoted ME3 and ME9 (derived from MKl6 cells), and TE2 and TE5 (derived form TC-1 cells), respectively. When inoculated into mice, ME3 cells were non-oncogenic.Nearly all mice inoculated with ME9 cells developedhrmors, but metastasisformation was strongly reducedin theseanimals. TE2 and TE5 cells displayed oncogenicpotential similar to that of the parental TC-l cells. Animals immunized with ME3 cells were protected against challenge with the parental MKl6 cells. Cell lysates of all six lines studied were tested for the content of 25 factors *nown to be involved in angiogenesis.The MK16 cells differed from the TC-l cells and also from all endostatin producing sublines by a markedly higher productionofinterleukin-lc (IL-la) Additional experiments indicatedthat the suppression of the production of IL-la by the parental MK16 caused by endostatin was due to an autocrine mechanism.
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