【作者】 彭丽秀；

【导师】 周昌菊；

【作者基本信息】 中南大学 ， 妇产科学， 2009， 博士

【摘要】 目的:检测雌、孕激素受体不同表达状态子宫内膜样腺癌的基因表达谱特征及雌、孕激素受体作用的相关基因改变,探讨雌、孕激素受体在子宫内膜样腺癌发病中的作用并为特异性的靶向治疗提供理论基础。方法:(1)采用人17kcDNA表达谱芯片,抽取ER(+)PR(+);ER(-)PR(-);ER(+)PR(-)子宫内膜样腺癌组织及正常子宫内膜组织RNA,经逆转录成cDNA分别进行芯片杂交实验,筛选比值大于、等于2或等于、小于.2的差异表达基因并进行差异基因功能分类;(2)通过生物信息学分析、筛选不同雌、孕激素受体子宫内膜样腺癌差异基因表达谱、凋亡信号通路及雌、孕激素受体调节相关基因;(3)RT-PCR、免疫组化验证部分基因。结果:(1)ER(+)PR(+);ER(-)PR(-);ER(+)PR(-)子宫内膜样腺癌分别与正常子宫内膜比较,差异表达基因各有1653个、1756个、2215个;其中上、下调基因分别有902个、751个;907个、849个;1029个、1186个;基因功能分类主要有:细胞结合、凋亡、信号转导、细胞周期、转录子激活等16类。(2)ER(+)PR(+);ER(-)PR(-);ER(+)PR(-)子宫内膜样腺癌特异性差异表达基因各有571个、645个、1125个;上、下调基因分别有399个、172个;443个、202个;608个、517个。(3)雌激素受体相关差异表达基因1177个,其中上调493个,下调684个;上调的基因功能分类主要为细胞有丝分裂、胚胎发育及形态、免疫反应调节、糖代谢、DNA复制调节等31类;下调的基因类别为:T细胞增殖、血管形成、细胞迁移、细胞粘附、创伤反应、信号转导等共27类。孕激素受体相关的差异表达基因1413个,其中上调814个,下调549个;上调基因功能类别主要有巨噬细胞激活、病毒防御反应、肽及酪氨酸磷酸化正调节、氨基酸代谢调节、抗原递呈与处理等73类;下调基因类别主要有翻译、生物合成、大分子代谢3类。雌、孕激素受体相关的差异表达基因838个,其中上调466个,下调372个;雌、孕激素受体相关的上调基因类别主要有:淋巴细胞负调节、B细胞增殖、多肽抗原递呈与处理、补体经典途径、体液免疫反应、抗原递呈等65类;下调的基因类别有:气体运输、分泌、消化等4类。(4)ER(+)PR(+);ER(-)PR(-);ER(+)PR(-)子宫内膜样腺癌在经典凋亡信号通道中差异表达基因分别是:BIRC3、TNFSF10、NFκBIA;PDCD8、BIRC3、PRKACB、NFκBIA、PIK3R1、PIK3R3;ER(+)PR(-)TP53、PDCD8、BIRC3、CASP7、NFκBIA、CHUK、PIK3CD、PIK3R1、PIK3R3、IL3RA、ILlA。(5)RT-PCR实验:INHBA在正常内膜组为26.37±15.35;在ER(+)PR(+)、ER(-)PR(-)、ER(+)PR(-)内膜癌组分别为117.68±44.15,45.96±29.02,37.14±31.03,分别与正常组比较,结果除与ER(+)PR(+)内膜癌组有显著差异外(P＜0.05),余均无显著差异(P＞0.05)。FYN在正常内膜组为83.24±32.30;在ER(+)PR(+)、ER(-)PR(-)、ER(+)PR(-)内膜癌分别为48.01±38.29,18.01±19.67,20.46±16.51与正常内膜组比较,结果除与ER(+)PR(+)内膜癌组无明显差异(P＞0.05):外,余两组差异显著(P＜0.05)。Cyr61在正常内膜组织为102.63±34.83,在内膜癌ER(+)PR(+)、ER(-)PR(-)、ER(+)PR(-)组分别为25.28±28.99、55.35±44.82,10.23±11.06与正常内膜组比较,结果除与ER(-)PR(-)内膜癌组无明显差异(P＞0.05)外,余两组差异显著(P＜0.05)。(6)免疫组化:Cyr61蛋白定位于细胞膜、细胞浆,阳性表达为淡黄到棕褐色不等;Cyr61在正常组织的表达为5.10±2.64,在内膜癌手术-病理分期Ⅰ、Ⅱ、Ⅲ期分别为3.38±1.69、3.25±1.96、1.60±1.35。组间两两比较除内膜癌Ⅰ期与Ⅱ期间无显著差异外(P＞0.05),余组间均差异明显(P＜0.05);将Cyr61在不同组织学分级比较,G1为3.55±1.53,G2为3.30±1.71均高于G3的1.82±1.17(P＜0.05),但内膜癌G1与G2级间无明显差异(P＞0.05)。结论:(1)多基因表达的变化参与了子宫内膜样腺癌的发病;(2)不同类型激素受体内膜癌不同的差异基因表达谱及凋亡信号通路是其预后差别的分子基础;各型受体特异的差异表达基因可能成为基因靶向治疗的有效靶点;(3)雌、孕激素受体能调节多种基因表达,单一雌激素受体主要通过促进有丝分裂,下调T细胞增殖分化反应;单一孕激素受体主要经刺激巨噬细胞激活,下调核糖体蛋白的转录;而雌、孕激素受体交互作用主要上调淋巴细胞负调节,下调气体运输等参与内膜癌的发病;(4)Cyr61可能成为指导临床治疗的分子指标。更多还原

【Abstract】 Objective:Study on the profile of gene expression association with the distinct estrogen and progestogen receptor in endometrioid adenocarcinoma.so as to reveal the role of the estrogen and progestogen receptor in the pathogenesis of endometrioid adenocarcinoma and provid a special targeted therapy in rationale.Methods:(1)By drawing the RNA from the endometrioid adenocarcinoma with estrogen receptor and progestogen receptor all positive(ER+)(PR+),estrogen receptor and progestogen receptor all negetive(ER-)(PR-),estrogen receptor positive and progestogen receptor negetive(ER+)(PR-),and normal endometrium,respectively, reversetranscription RNA to cDNA and hybridization with 17kcDNA human gene chip,screen the gene ratio equal 2 or higher than 2 and ratio eqeal -2 or lower than -2 as the difference expression genes.(2) Analysis the genetic expression and the apoptosis signal pathway and the related gene in the distincted expression of ER and PR in endometrioid adenocarcinoma by bioinformatics.(3) Verification the genes by reverse transcriptase PCR and immunohisochemistry.Results:(1) There differential expression genes were 1653,1756, 2215 strips when the(ER+)(PR+),(ER-)(PR-),(ER+)(PR-) endometrioid adenocarcinoma comparedwith the nomor endometrium,respectively,in each group the up-regulated genes and down-regulated gens were 902、751,907、849,1029and 1186.According to the Gene Ontology(Go) classified the gene into 16 kinds:binding,apoptosis,metabolism,cell cycle,signal transduction,transcription activation,etc.(2) There were specially differential expression genes 471 in the(ER+)(PR+) endometrioid adenocarcinoma,which up-regulated genes 399 and down-regulated genes 172.The special differential expression genes 645 in(ER-)(PR-) endometrioid adenocarcinoma,the up-regulated genes were 443,the down-regulated were 202.The special differential expression genes 1125 in(ER+)(PR-) endometrioid adenocarcinoma,the up-regulated genes were 608,the down-regulated were 567.(3)The difference expression genes 1177 were relatived with the estrogen receptor in which up-regulated genes 493 and down-regulated genes 684.According to the Gene Ontology(Go) classified the up-regulated genes into 31 kinds:cell mitosis,embrynomic,cranial skeleton morphogensis,immune response regulation,glycometabolism,DNA duplicate,and so on.The down-regulation genes kinds 27:T cell proliferation,angiopioesis,cell migration,cell adhesion,wound responds,signal transduction,etc.The difference expression genes 1413 were relatived with the progestrogen receptor in which up-regulated genes 814 and down-regulated genes 549. The up-regulated genes classified into 73 kinds:macrophage activied, defence virus,positive regulated the peptide,tyrosine phosporylation, amino acid metabolism,antigen presentation,etc.The down-regulated genes classified into 3 kinds:translation,biosynthesis,macromolecule biosynthetic prosess.There were difference expression genes 838 relatived to the expression of the ER and PR,in which up-regulated genes were 466 and down-regulated genes 372.The up-regulated genes classified into 65 kinds:negative regulation of lymphocyte activation,B cell proliferation,antigen processing and presentation of peptide antigen via MHC calss I,antigen processing and presentation of peptide antigen, etc.The down-regulated genes were classified kinds 4:gas transport, excretion,digestion,etc.(4) The differencial expression genes in the apoptosis pathway in endometrioid adenocarcinoma with(ER+)(PR+) were BIRC3,TNFSF10,NFκBIA,and in endomtrioid adenocarcinoma with(ER-)(PR-) were PDCD8,BIRC3,PRKACB,NFκBIA,PIK3R1, PIK3R3.but in endometrioid adenocarcinoma with(ER+)(PR-) were TP53,PDCD8,BIRC3,CASP7,NFκBIA,CHUK,PIK3CD,PIK3R1, PIK3R3,IL3RA,IL1A.(5) Vertifercail the genes by RT-PCR.The expression of INHBA were 26.37±15.35 in the normal endometrium, and were 117.68±44.15,45.96±29.02,37.14±31.03 in the (ER+)(PR+),(ER-)(PR-),(ER+)(PR-) endometrioid adenocarcinoma, respectively,When compared the endometrioid adenocarcinoma with the normal endometrium,there were significant defference between the endometrioid adenocarcinoma with(ER+)(PR+) and the normal(p＜0.05). There were no difference in the others(p＞0.05).The expression of FYN in normal were 83.24±32.30,but in endometrioid adenocarcinoma with (ER+)(PR+);(ER-)(PR-),(ER+)(PR-) were 48.01±38.29,18.01±19.67, 20.46±16.51,respectively.Compared each other,exceptes had no difference between(ER+)(PR+) endometrioid adenocarcinoma with the normal(p＞0.05),There were significant differernce in the others (p＜0.05).The expression of Cyr61 were 102.63±34.83 in normal endometrium and in the endometrioid adenocarcinoma with (ER+)(PR+),(ER-)(PR-),(ER+)(PR-) were 25.28±28.99,55.35±44.82, 10.23±11.06,respectively.Compared with each others,excepted no significant difference between the(ER-)(PR-) endometrioid adenocarcinoma with the normal endometrium(p＞0.05),there were significant difference in the others(p＜0.05).(6) The expression of Cyr61 were located in cell membrane and cytoplasm by immunohistochemistry. The positive appearance were color changed frome yellow to brown.The expression of Cyr61 in normal endometrium were 5.10±2.64 and in endometrioid adenocarcinoma with FIGO stageⅠ,stageⅡ,stageⅢwere 3.38±1.69,3.25±1.96,1.60±1.35,respectively.There were no difference when between the stageⅠand stageⅡ(p＞0.05),but there were significants difference not only in the stageⅠwith normal but also in the stageⅡwith normal endometrium(p＜0.05).The expression of Cyr61 in the endometioid adenocarcinoma with grade 1,grade 2 and grade 3 were 3.55±1.53,3.30±1.71,1.82±1.17.There no significant difference between the G1 and G2(p＞0.05),but there were significant defference not only between G1 and G3 but also between G2 and G3(p＜0.05).Conclusions:(1)There are many genes participated the etiopathogenisis of endometrioid adenocarcinoma(2) The various genes and the distinct apoptosis pathway along with the dictinct expression of the ER and PR played a crital roles in the prognosis of the endometrioid adenocarcinoma.(3) The ER or PR could regulate the variants gene expression.The ER up-regulated mitosis and negatived the T cell proliferation at the same time in favour the genesis of the endometrioid adenocarcinoma,the PR by up-regulated actived macrophage and down-regulated the transcription of ribosomal protein and the up-regulated negative regulate lympholyte and down-regulate the gas transport by ER and PR all participate the pathogenesis of the endometrioid adenocarcinoma.(4) The Cyr61 maybe regard as a target of the therapy in endometrioid adenocarcinoma.更多还原