【作者】 李薇；

【导师】 王冠军；

【作者基本信息】 吉林大学 ， 内科学， 2005， 博士

【摘要】 遗传信息不仅由DNA 序列的基因密码决定,组蛋白的共价修饰所引起的染色质重塑同样也调控基因的转录,影响基因的表达,被称为“第二遗传密码”。组蛋白的异常去乙酰化修饰与多种肿瘤的发生发展密切相关,HDACI能通过抑制HDAC、重塑染色质、阻断由于HDAC 募集功能紊乱而导致的基因表达受抑,从而治疗癌症。HDACI 治疗肿瘤的研究已经取得一定的进展,但其抗癌谱和药理机制还有待于进一步研究,尤其在抗白血病方面的研究报道较少。　本研究首次发现组蛋白去乙酰化酶抑制剂SB 上调NB4、U937、HL-60、Kasumi-1、Jurkat　5 种白血病细胞共刺激分子CD86、CD80 及胞间黏附分子CD54,并证实HDACI 通过促进组蛋白乙酰化作用促进NF-кB和CREB 的表达,后者导致白血病细胞共刺激分子和黏附分子表达增强。上调的共刺激分子能刺激淋巴细胞的增殖,本研究结果为HDACI应用于抗肿瘤治疗又添有力证据,并反证了肿瘤细胞免疫分子表达下调、免疫逃避与异常的去乙酰化有关。　本研究证实SB 能诱导NB4 细胞凋亡,进一步做了联合药物实验研究,单独应用SB 即可诱导凋亡,SB+ATRA 联合应用呈协同作用,效果最强,SB+　As₂O₃ 作用最差,呈拮抗作用,为临床合理联合用药提供了实验基础。　本文还首次使用蛋白质组学的方法,筛选出SB 诱导NB4 细胞凋亡过程中的22 种敏感蛋白,这些敏感蛋白中,有13 种已经被证实为凋亡相关蛋白,9 种蛋白还没有与凋亡相关性的报道,这些蛋白的鉴定为进一步研究HDACI诱导肿瘤细胞凋亡的机制及抗肿瘤药物靶点的筛选奠定了基础。更多还原

【Abstract】 Chromatin　remodeling　is　the　alteration　of　chromatin　localization　and　structure.　It　is　a　key　step　in　the　transcriptional　activation　of　genes,　also　a　significant　mode　of　epigenetics　modification.　Epigenetics　modification　contains　more　precise　gene　message　compared　with　genetype　.　Abnormal　chromatin　remodeling　plays　a　key　role　on　pathogenesis　and　development　of　malignant　tumor　and　leukemia.　A　couple　of　adverse　proteases——histone　acetylases(HAT)　and　histone　deacetylases(HDAC)　are　in　charge　of　th　acetylation　state　core　histone.　The　enzymes　act　when　they　associate　with　correlate　proteins　called　Coactior(CoA)　and　Corepressor(CoR)　respectively.　Abnormal　binding　of　CoR　and　DNA　may　cause　　continuing　deacetylation　of　the　gene　and　thus　depress　gene　transcription　which　may　play　key　roles　in　carcinogenesis.　Recently　many　compounds　are　found　with　the　abilities　to　inhibit　HDAC　and　a　lot　of　studies　verified　their　antitumor　abilities　such　as　inducing　tumor　cell　differentiation,　blocking　cell　cycle,　apoptosis　and　immunomodulation.　Potentializing　the　immunogenicity　of　tumor　cells　is　a　new　way　with　great　prospect.　Inducing　tumor　cells　apoptosis　refers　to　widespread　tumor　types,　and　studying　on　the　mechanism　will　provide　evidence　for　clinical　drug　development.　Here　we　will　focus　these　two　aspects　to　explore　the　anti-tumor　effects　and　mechanism　of　histone　deacetylase　inhibitor　(HDACI).　　　For　the　study　of　immunomodulation,　we　selected　five　cell　strains　as　the　study　objects　which　are　NB4,Kasumi-1,　HL-60,U937　and　Jurkat.　The　cells　were　treated　with　sodium　butyrate(SB)　at　0,　0.1,0.5,1.0,5.0mM　concentrations　and　examined　at　different　time　points　respectively.　　Cell　viability　examined　by　both　trypan　blue　dye　exclusion　and　MTT　reduction　method,　applied　dual-　or　mono-　agent　analysis　of　variance.　We　selected　concentrations　for　cell　survival　rate　>85%　and　with　obvious　growth　suppression　and　time　points　which　occur　pre-,　mid-　and　post-　growth　suppression　for　subsequent　experiments.　FACS　was　applied　to　detect　the　up-regulation　of　CD86,　CD80　and　CD54.　　Semiquantitative　RT-PCR　was　used　to　examine　the　gene　transcription　of　CD86　and　found　that　CD86　mRNA　was　obviously　enhanced　by　SB.　To　ensure　that　the　enhanced　gene　transcription　was　due　to　the　high　acetylation　state　of　core　histone,　We　applied　the　AUT　gel　electrophoresis　and　found　the　core　histone　was　highly　acetylated　by　SB,　which　was　coincided　with　the　current　reports.　The　acetylation　of　histone　results　in　a　relaxed　chromatin　structure　and　activates　the　transcription　of　CD86　by　promoting　the　access　of　transcription　factor.　To　further　explore　the　mechanism　of　augmented　CD86　transcription,　we　detected　content　changes　of　the　two　main　subunits,p65　and　p50,of　NF-κB　in　nucleic　extraction　from　pre-　and　post-　SB　treated　cells.　The　results　reveals　that　the　increases　of　p65　and　p50　are　remarkably　coherent　with　the　changes　of　CD86,which　supports　the　view　that　NF-κB　is　one　of　the　important　transcription　factors　that　take　part　in　up-regulation　of　CD86　on　tumor　cells.　However,　the　findings　that　pretreatment　with　Pyrrolidine　dithiocarbamate(PDTC),　a　inhibitor　of　NF-κB　,could　still　up-regulate　33.57%　than　control　group　indicates　that　SB　up-regulating　CD86　may　be　partially　dependent　on　the　NF-κB　signal　transduction　pathway.　We　then　detected　the　changes　of　　activated　CREB　with　a　pCREB　detection　kit　and　found　that　the　pCREB　obviously　increased　after　SB　administration.　These　results　indicate　that　CREB　may　also　be　responsible　for　the　up-regulation　of　CD86.Allogeneic　mixed　lymphocyte　reaction　found　that　NB4　cells　treated　with　SB　far　more　effectively　stimulated　proliferation　of　lymphocytes　than　untreated　cells.　This　result　supports　that　up-regulated　costimulatory　molecules　and　adhesion　molecules　are　functional.　We　also　observed　the　expression　changes　of　CD86　in　NB4　and　HL-60　treated　with　ATRA,　As2O3　and/or　SB.　Neither　ATRA　nor　As2O3　can　up-regulate　CD86　expression　along　while　SB　combined　ATRA　show　higher　CD86　expression　(P>0.05)　which　may　indicate　synergistic　effect　of　the　two　compounds.　In　SB+ATRA+As2O3　and　SB+As2O3　　groups,　positive　percentage　of　CD86　were　less　than　SB　along　group.　These　results　indicate　that　As2O3　can　partially　counteract　the　ability　of　SB　in　up-regulating　CD86　(P<0.05).　　　Our　research　verified　that　histone　deacetylase　inhibitor　SB　can　up-regulate　costimulatory　molecules　and　adhesion　molecules　of　acute　leukemia　cells　in　vitro.　And　we　primarily　investigated　the　mechanism　of　SB　up-　regulating　the　costimulatory　molecules.　One　study　showed　HDACI　can　up-regulate　MHC-Ⅰand　MHC-Ⅱmolecules　and　CD40　in　neuroblastoma　cells.　All　of　these　indicate　that　HDACI　can　reinforce　the　activation　of　the　first　and　second　signal　pathway　for　CTL　by　chromatin　remodeling　which　may　be　good　evidence　for　HDACI　being　used　for　immunotherapy　in　the　future.　On　the　other　hand,　it　also　argues　that　abnormal　deacetylation　takes　part　in　down-regulation　of　immune　molecular.　Our　research　revealed　that　NF-кB　and　CREB　are　important　transcriptional　factors　for　SB　up-regulating　CD86　molecules　on　tumor　cells,　which　will　make　for　well　understanding　of　the　complex　regulatory　network　in　cells　and　also　identifying　the　pathogenesis　of　immune　molecules　down-regulation　in　tumor　cells.　The　study　about　combination　of　SB　with　　ATRA　and　AS2O3　will　become　the　basis　for　drug　combination　in　the　future.　In　the　study　of　SB　inducing　apoptosis,　we　selected　the　NB4　cell　strain　which　contains　translocation　of　15　and　17　chromosome.　FACS　was　applied　to　detect　cell　cycle　blockage　and　apoptosis　rate.　We　compared　SB　and/or　RA　and/or　As2O3　,　and　no　significant　difference　was　observed　in　the　growth　blockage　among　these　groups.　However,　SB+RA　caused　greater　apoptosis　effect　than　SB　along　while　SB+　As2O3　had　the　opposite　results.　These　results　indicate　that　RA　has　synergistic　effect　with　SB　while　As2O3　can　counteract　with　SB　during　SB　inducing　NB4　cells　apoptosis.　　In　order　to　explore　the　overall　and　accurate　mechanism　of　SB　inducing　NB4　apoptosis　,　we　applied　2-D　gel　electrophoresis　and　21　sensitive　proteins　were　identified　with　mass-spectrum,　these　protein　include　Profilin　I 、adenylate　kinase　2　isoform　b 、annexin　I、A+U-rich　element　RNA　binding　factor、H2B　histone　family,　member　E、nuclear　chloride　ion　channel　protein、BTF3a、hsp27、transaldolase　1、TIP、FABP　and　so　on.　There　are　several　apoptosis　associated　proteins,　for　example,　transcriptional　factor　eIF5A　and　BTF3,　Thioredoxin,hsp27,　TIP　and　TCTP.　Most　of　these　proteins　were　firstly　discovered　to　be　sensitive　during　the　process　of　SB　inducing　NB4　cells　apoptosis.　These　proteins　indicate　correlated　events　take　part　in　the　apoptosis　procedure　and　establish　the　basis　for　further　analyze　accurate　mechanism　of　SB　inducing　NB4　cells　apoptosis.　cytoskeletal　protein　　profilin　1　is　sensitive　to　SB　,　indicating　that　SB　may　function　on　reversing　tumor　malignant　phenotype.　Several　sensitive　proteins(Cofilin,　FABP,Thioredoxin)　associated　with　drug　fast　show　that　SB　can　enhance　cells’　sensitivity　to　apoptosis　and　chemotherapeutics　by　regulating　correlated　proteins.　SB　also　alters　expression　of　immunomodulatory　proteins　(eg.TIP)　,　which　indicates　the　immune　escape　of　leukemia　cells　correlate　with　deacetylation　of　hisone　,　also　hints　that　SB　can　play　anti-tumor　effect　by　immunomodulation.　The　study　about　SB　combined　with　RA　and/or　As2O3　inducing　apoptosis　on　NB4　cells　and　proteomics　study　provide　a　significant　basis　for　further　study　apoptotic　mechanism　and　following　treating　APL　with　SB.更多还原