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《曲古抑菌素對小細(xì)胞肺癌細(xì)胞系nci-h446的誘導(dǎo)分化及凋亡作用研究》由會員上傳分享,免費(fèi)在線閱讀,更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。
1、江蘇大學(xué)碩士學(xué)位論文曲古抑菌素對小細(xì)胞肺癌細(xì)胞系NCI--H446的誘導(dǎo)分化及凋亡作用研究姓名:劉侃申請學(xué)位級別:碩士專業(yè):內(nèi)科學(xué)指導(dǎo)教師:崔國興20120608江蘇大學(xué)碩士學(xué)位論文摘要研究目的:觀察人小細(xì)胞肺癌(SCLC)NCI-H446細(xì)胞的干細(xì)胞特性(Stemness),探討組蛋白去乙?;敢种苿┣乓志?TSA)對NCI—H446細(xì)胞的誘導(dǎo)分化及凋亡的作用機(jī)制。為進(jìn)一步應(yīng)用組蛋白去乙?;敢种苿?HDACI)或其它細(xì)胞誘導(dǎo)分化劑治療小細(xì)胞肺癌提供了理論和實(shí)驗(yàn)基礎(chǔ)。研究方法:(1)NCI.H446細(xì)胞經(jīng)RPMI1640完全培養(yǎng)基培養(yǎng)傳代后,用干細(xì)胞標(biāo)志蛋白巢蛋白(nestin),
2、CDl33,波形蛋白(vimentin)和CD44的抗體對進(jìn)行免疫熒光染色,鑒定該細(xì)胞的干細(xì)胞特性。(2)NCI.H446細(xì)胞經(jīng)TSA誘導(dǎo)培養(yǎng)后,用神經(jīng)標(biāo)志蛋白神經(jīng)絲蛋白200(NF.200),pIII.Tubulin,微管相關(guān)蛋白2(MAP2)和BM88及細(xì)胞增殖核抗原ki67的抗體進(jìn)行免疫熒光染色和免疫印跡測定(WesternBlotting法),觀察NCI.H446細(xì)胞向神經(jīng)細(xì)胞分化的成熟程度和細(xì)胞增殖指數(shù)的變化。(3)用TUNEL染色法計算細(xì)胞的凋亡指數(shù)并用免疫印跡法測定細(xì)胞的凋亡相關(guān)蛋白Caspase3,Bax,Bcl.2表達(dá)水平的變化,觀察TSA對NCI.H446細(xì)胞的誘導(dǎo)凋
3、亡作用。研究結(jié)果:(1)在小細(xì)胞肺癌NCI.H446細(xì)胞株中,nestin,CDl33,vimentin和CD44高表達(dá)。曲古抑菌素對小細(xì)胞肺癌細(xì)胞系NCI.H446的誘導(dǎo)分化及凋亡作用研究(2)經(jīng)TSA誘導(dǎo)后的NCI.H446細(xì)胞株,NF一200,[3111.Tubulin,MAP2和BM88高表達(dá)。(3)經(jīng)TSA誘導(dǎo)后的NCI.H446細(xì)胞株,Caspase3明顯激活,Bax表達(dá)水平增高,Bcl.2表達(dá)水平降低,細(xì)胞的增殖指數(shù)明顯降低,凋亡指數(shù)明顯增高。結(jié)論:小細(xì)胞肺癌NCI.H446細(xì)胞株具有干細(xì)胞特性,TSA可誘導(dǎo)小細(xì)胞肺癌NCI—H446細(xì)胞向神經(jīng)細(xì)胞分化成熟,并激活Caspa
4、se3導(dǎo)致細(xì)胞凋亡。關(guān)鍵詞:人小細(xì)胞肺癌;曲古抑菌素;誘導(dǎo)分化;凋亡II江蘇大學(xué)碩士學(xué)位論文Objective:ABSTRACTToexplorethesternnessofsmallcelllungcancerNCI—H446celllineandtheeffectsofhistonedeacetylaseinhibitor(HDACi),trichostaninA(TSA),onthedifferentiationandapoptosisofthecancercells.Forfurtherapplicationofhistonedeacetylaseinhibitors(HDACI
5、)orothercelldifferentiationinducertreatmentofsmallcelllungcancerprovidesatheoreticalandexperimentalbasis.Methods:(1)NCI—H446cellswereculturedandproliferatedinRPMI1640mediumcontaining10%FBS,thenidentifiedtheexpressionsofstemcellmarkersonthemincludingnestin,CD133,CD44andvimentinwithimmunofluorescen
6、ce.(2)TheNCI.H446cellsweretreatedwithTSA,thenexpressionsofneuronmarkers,NF一200,[3III—Tubulin,MAP2andBM88,inthecellsweredetectedbyimmunofluorescenceandWesternblotting.TheproliferationcellnuclearantigenKi67immunofluorescence。stainingindex(proliferationindex)wasusedtoeveluatetheeffectofTSAonprolifer
7、ationofNCI-H446cells.(3)TheTUNEL(terminaldeoxynucleotidyltransferasedUTPnickendlabeling)methodwasappliedtocaculeteapoptoticindexoftheTSA.treatedcellsandexpressionsofapoptosisasociatedproteinsIII曲古抑菌素對小細(xì)胞肺癌細(xì)胞系NCI.H446的誘