2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659228摘要：藉由研究miRNA和mRNA之相互作用，釐清幽門螺旋桿菌相關胃瀰漫大型B細胞淋巴癌之細胞來源目的：藉由miRNA和mRNA的特性來確認胃瀰漫大型B細胞淋巴癌(DLBCL)是源自於胚中心B細胞(GCBCs)或邊緣區B細胞(MZBCs)， 並且發展尖端分子生物科技來界定miRNA和mRNA的相互作用。背景：胃粘膜相關淋巴癌（MALToma)源自於MZBCs，但和幽門螺旋桿菌相關的DLBCL其細胞來源目前尚未明瞭。我們發現GCBC所特有之轉錄因子E2A若在胃粘膜相關淋巴癌有異常表現，會導致幽門螺旋桿菌根除治療無效，同時MZBC所特有之miR-146a若在DLBCLs有過度表現，對會化學治療有較好反應。因此，將胃部的DLBCL分類成GCBC及非GCBC兩種亞型，具有臨床意義。尤其是CagA+ DLBCL的起源，和幽門螺旋桿菌根除治療可能有關，所以在臨床上也具有其重要性。假說：因為幽門螺旋桿菌會經T細胞之作用誘導出對幽門螺旋桿菌具專一性的GCBC，或不須經T細胞之作用誘導出對自體抗原具專一性的MZBC。因此與幽門螺旋桿菌有關之DLBCLs有可能源自於GCBCs或MZBCs，證實此一假說將延展幽門螺旋桿菌相關胃淋巴癌之病理及臨床範疇。方法：1. 幽門螺旋桿菌相關的DLBCLs是源自於GCBC或MZBC：我們將以mRNA和miRNA的基因體特徵來確認CagA+ DLBCLs的起源。源自扁桃腺的GCBCs 和MZBCs 將用來作為對照組。2. miRNA/mRNA的的基因體特徵於生物學上的意義：我們將會建構表現綠色螢光蛋白(EGFP)之3’端非編碼區(3’UTR)基因庫。此基因庫將會與miRNA一同轉染到細胞株中，我們將篩選出因3’端非編碼區之作用而不表現綠色螢光蛋白之細胞株，藉此篩選出miRNA於mRNA上之作用位置。後續實驗則將探討miRNA和mRNA的相互影響在生化及生物上的意義。3. GCBCs或MZBCs癌化形成DLBCLs：將小鼠長期感染Helicobacters，使GCBCs或MZBCs癌化形成DLBCLs。再利用雷射捕捉顯微切割（LCM）分離GCBCs或MZBCs，比對其和DLBCLs具有相同的免疫球蛋白基因序列，藉此證實早期病變中之GCBCs或MZBCs癌化轉型成晚期病變之DLBCL。4. 經由單鏈抗體之建構，證實B細胞抗原受體對幽門螺旋桿菌或自體抗原之專一性：跟據CagA+ DLBCLs的免疫球蛋白基因序列，建構出單鏈抗體，並篩出其抗原專一性。源自GCBC 或 MZBC的CagA+ DLBCLs應具有分別對抗幽門螺旋桿菌相關抗原及T細胞自體抗原之抗原受體。主要預期成果：(1)結合細胞起源、免疫上的特性及臨床表現來區分DLBCLs亞型。(2) 並且發展尖端分子生物科技來界定miRNA和mRNA的相互作用。與主計畫關聯性： 提供一個病理上的及分子上的基準，來擴展幽門螺旋桿菌相關之淋巴癌的概念。相對的，從其他子計畫得到的臨床上及免疫上的數據可以更進一步驗證分子病理的研究理念。<br> Abstract: Cellular origin of Helicobacter pylori-related gastric diffuse large B-celllymphoma: Identifications of miRNA/mRNA interactions through a novelapproachObjective: To identify a diffuse large B-cell lymphoma (DLBCL) derived fromgerminal center B cells (GCBCs) or marginal zone B cells (MZBCs) throughmiRNA/mRNA signatures, and characterization of miRNA/mRNA interactions througha novel approach.Background: MALT lymphoma is derived from MZBCs, but the origin of Helicobacterpylori (HP)-related DLBCL is unclear. We found that aberrant E2A expression, a GCBCmarker, in MALT lymphomas implied a worse response to HP eradication therapy(HPET), and over-expression of miR-146a, a MZBC marker, in DLBCLs implied abetter response to chemotherapy. Gastric DLBCLs are thus heterogeneous, andclassifying them into GCBC and non-GCBC subtypes might be significant. In particular,the origin of CagA+ DLBCL, a new subtype of gastric DLBCL potentially curable withHPET, might be clinically important.Hypothesis: Because HP might induce a T-cell-dependent GCBC pathway involvingHP-specific antigens or a T-cell-independent MZBC pathway involving autoantigens,HP-related DLBCLs might be derived from GCBCs or MZBCs, thus broadening thecurrent consensus that identifies only HP-related MALToma of MZBCs.Methods:1. GCBC vs MZBC origin through genome-wide characterization of HP-relatedDLBCLs: A combined mRNA/miRNA signature will be used to identify CagA+DLBCLs of GCBC or MZBC origin. CagA− DLBCLs, normal GCBCs and MZBCsfrom the tonsils, and CagA+ or CagA− DLBCL cell lines will be used as controls.2. Biological significance of miRNA/mRNA signatures: We will construct a 3’UTRlibrary into the 3’ end of EFGP codong region. The EGFP library will becotransfected with the miRNA of interest into a cell line, and screened forextinguishing of EGFP expression. The 3’UTR clone whose EGFP expression isinhibited will be the target of miRNA. Further experiments will be done tocharacterize biochemical and biological significance of the miRNA/mRNAinteractions.3. Transformation of GCBCs or MZBCs into DLBCLs with GCBC or MZBCphenotype: BALB/c mice chronically infected with Helicobacters will be used todemonstrate the development of DLBCLs. GCBCs or MZBCs isolated by lasercapture microdissection from early lesions shall have identical immunoglobulin genesequences to those of DLBCLs of GCBC or MZBC origins from late lesions.4. HP-specific vs autoantigen-specific B-cell receptors through single-chainantibody production: Single-chain antibodies will be constructed from sequences ofimmunoglobulin genes of CagA+ DLBCLs, and screened for antigen specificity.CagA+ DLBCLs of GCBC or MZBC origin shall have antigen receptor againstHP-related antigen and T-cell autoantigen, respectively.Major anticipated achievements: (1) Identifications of subtypes of DLBCLs withdistinct HP-status, cellular origins, immunological features, and clinical courses. (2) Anovel method for characterizing miRNA/mRNA interactions.Relevance to the main project: This subproject provides a pathological and molecularbasis to extend the concept of HP-related lymphoma. In turn, clinical and immunologicaldata from the other subprojects would consolidate the molecular pathological approach.幽門螺旋桿菌胃瀰漫大型B細胞淋巴癌治療策略diffuse large B-cell lymphomagastricantibiotic treatmentHelicobacter