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研究生:沈凱倫
研究生(外文):Shen Kai-Lun
論文名稱:建立IL-10或TGF-β1慢病毒系統並感染小鼠樹突狀細胞與骨髓基質細胞
論文名稱(外文):Established the lentiviral IL-10 or TGF-β1 vectors for murine dendritic cells and mesenchymal stroma cells transduction
指導教授:周秀慧周秀慧引用關係
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:120
中文關鍵詞:慢病毒載體樹突狀細胞骨髓基質細胞
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骨髓移植為治療血液或代謝性遺傳疾病的主要治療方式,造成移植失敗的主因為MHC分子的不相容所引起的異體T淋巴球的免疫排斥反應和植入細胞數目過低而無法引發免疫耐受力。排斥反應是由Th1淋巴細胞所主導的免疫反應所造成,而Th1細胞的活性會受到抗原呈現細胞的調控。另外,植入細胞的數目過低是常受到骨髓環境中基質細胞所提供的微環境影響。相關的研究顯示,抑制型細胞激素interleukin-10(IL-10)和transforming growth factor -β1(TGF-β1)均具有降低 Th1反應和影響造血過程等活性,可運用於抑制免疫排斥反應的發生。研究室先前發現以體外培養方式將樹突狀細胞先以IL-10或TGF-β1前處裡後具有顯著抑制異體T細胞的活性。但將該IL-10或TGF-β1處理的樹突狀細胞應用於胎鼠骨髓移植模式上時,卻無法提升造血幹細胞的植入率,初步推論是經IL-10或TGF-β1處理後的樹突狀細胞所具有的免疫抑制活性不夠持久與穩定。因此本研究的目的是藉由慢病毒載體感染的方式使樹突狀細胞或骨髓基質細胞能長期穩定的表現IL-10或TGF-β1細胞激素,以利未來運用於骨髓移植上。實驗中首先建立慢病毒的生產平台,這包括構築慢病毒系統中的轉導植體pTY-EF-IL-10與pTY-EF-TGF-β1,外膜質體 pVSVG 與 轉導質體 pTY-EF 的生產,與pTY-EF-IL-10-Lv以及pTY-EF-TGF-β1-Lv慢病毒的生產。接著以純化後lin-造血幹細胞作為宿主細胞進行感染,發現感染效率並不好,而在感染IL-4與GM-CSF共同誘導分化出的樹突狀細胞也觀察到感染效率不高,但卻可以偵測到IL-10以及TGF-β1的釋放,但是在異體淋巴球混合實驗中卻無法有效的提昇抑制異體T淋巴球增生的活性。進一步將骨髓基質細胞進行感染,發現具有20%的感染率,並且可在感染的骨髓基質的細胞培養上清液中偵測到IL-10或TGF-1,分析感染後的骨髓基質細胞的免疫抑制活性顯示,經過IL-10-Lv或TGF-β1-Lv 感染後的骨髓基質細胞可以顯著降低異體T細胞被活化的增生能力。綜合以上結果,本研究已建立 IL-10-Lv或TGF-β1-Lv慢病毒的生產平台,已建立eGFP、IL-10或TGF-β1感染之三株骨髓基質細胞,這些感染的細胞可以被偵測到IL-10或TGF-β1細胞激素的釋放,而且受到感染的細胞也具有促進抑制異體T細胞的活性。未來可將該類細胞運用於免疫抗排斥反應的調控研究上。
Bone marrow transplantation (BMT) was used as therapeutic way to correct a wide variety of hematological and metabolic genetic disorders. The major problem on failure of transplantation is caused by MHC-mismatch induced GvHD, limited donor cell chimerism, and lack tolerance to alloantigen. Rejection of implant is mediated by type I T helper cells. However, T cell activation is also mediated by antigen-presenting cells, such as dendritic cells (DCs). In addition, the limited donor cell implantation may be induced by improper BM microenvironment. TGF-β1 and IL-10 are kinds of suppression cytokines. Related researches have reported that both cytokines inhibited the Th1 cell activation and affects the hematopoietic process. Our previously results indicated that IL-10 or TGF-β1 conditioned dendritic cells significantly enhanced the suppression of allogenic T lymphocyte proliferation in vitro, but there did not increase the donor stem cell engraftment in fetal mice recipients in vivo. The possible explanation is that the suppressive effects of IL-10 or TGF-β1 conditioned dendritic cells were not long lasting and unstable. Therefore, the purpose of this study is to establish IL-10 or TGF-β1 transgenic dendritic cells and bone marrow stroma cells by using lentiviral transduction system. Herein, we firstly developed the IL-10 or TGF-β1 lentiviral transduction platform which included the pTY-EF-IL-10 and pTY-EF-TGF-β1 plasmids construction, pVSVG and pHP plasmids generation, and IL-10 or TGF-β1 lentivirus preparation. Then, we transduced the IL-10-Lv or TGF-β1-Lv into lin-hematopoietic stem cell-derived dendritic cells, bone marrow-derived dendritic cells, and bone marrow stroma cells. The transduced efficiency, gene expression and protein production were estimated by flow cytometer, immunofluorscence staining, ELISA, and mixed lymphocyte reaction (MLR) assays. Results indicated that the lentiviral transduced rate for lin-hematopoietic stem cell-derived dendritic cells and bone marrow-derived dendritic cells are poor. However, the expression and production of IL-10 and TGF-β1 were detectable in supernatants of lentiviral transduced dendritic cell culture, but the results of MLR indicated that IL-10 and TGF-β1 lentiviral transduced dendritic cells did not increase the suppressive effect of DCs on allogenic T cells. Results of IL-10 and TGF-β1 lentiviral transduced stroma cells indicated that the lentiviral transduced rate was nearly 20%. The expression and production of IL-10 and TGF-β1 were detectable in supernatants of lentiviral transduced bone marrow stroma cells culture. Importantly, the result of MLR indicated that IL-10 and TGF-β1 lentiviral transduced bone stroma cells did increase the suppressive effect of bone marrow stroma cells on allogenic T cells. In summary, the IL-10 or TGF-β1 lentiviral transduction platforms were well established. Three cell lines of eGFP, IL-10 or TGF-β1 transgenic bone marrow stroma cells were developed, respectively. Both IL-10 and TGF-β1 transgenic bone marrow stroma cells possess more IL-10 or TGF-β1 production and higher suppressive activities than mock-transduced bone marrow cells. These cells can be used for GvHD prevention in future application.
目錄

目錄----------------------------------------------------------------I
圖目錄 -------------------------------------------------------------IV
中文摘要------------------------------------------------------------1
英文摘要------------------------------------------------------------3

第一章 研究背景
第一節 骨髓移植的定義、應用與重要性
1.1.1 骨髓移植的定義、應用與重要性 ---------------------------------5
1.1.2 骨髓移植的瓶頸與解決策略--------------------------------------6
第二節 移植後的免疫排斥及免疫耐受性
1.2.1 移植的排斥反應-----------------------------------------------7
1.2.2 免疫排斥反應的理論基礎----------------------------------------8
1.2.3 免疫耐受性與骨髓移植------------------------------------------9
1.2.4 免疫耐受性引發的相關研究--------------------------------------10
第三節 免疫抑制型細胞激素介白質-10 (Interleukin-10, IL-10) 與轉型生長因子-β1 (Transforming Growth Factor-beta, TGF-β1)
1.3.1 IL-10簡介---------------------------------------------------11
1.3.2 IL-10 在移植上之應用-----------------------------------------13
1.3.3 TGF-β的簡介-------------------------------------------------14
1.3.4 TGF-β在移植上的應用------------------------------------------17
第四節 樹突細胞
1.4.1 樹突狀細胞的簡介---------------------------------------------18
1.4.2 樹突狀細胞在器官以及骨髓移植上的應用---------------------------20

第五節 骨髓基質細胞
1.5.1 基質細胞----------------------------------------------------23
1.5.2 基質細胞在成體骨髓移植的研究----------------------------------23
第六節 基因傳遞系統(Gene delivery system)
1.6.1 基因傳遞系統-------------------------------------------------24
1.6.2 人類免疫缺陷病毒---------------------------------------------25
1.6.3 慢病毒載體的演進---------------------------------------------27
1.6.4 慢病毒載體的應用---------------------------------------------30

第二章 研究目的與重要性----------------------------------------------31

第三章 實驗材料及方法
第一節 實驗設計-----------------------------------------------------32
第二節 實驗材料與方法
3.2.1 實驗小鼠----------------------------------------------------33
3.2.2 實驗藥品、培養基與各類試劑------------------------------------33
第三節 儀器設備------------------------------------------------------46
第四節 實驗方法
3.4.1 慢病毒之備製與純化 ------------------------------------------48
3.4.2 IL-10 與 TGF-β1 感染之骨髓樹突狀細胞的培養與製備---------------53
3.4.3 骨髓基質細胞的培養與感染--------------------------------------56
3.4.4 細胞表面分子的測定-------------------------------------------57
3.4.5 細胞激素的測定-----------------------------------------------57
3.4.6 MTT 測量----------------------------------------------------58
3.4.7 單向異體淋巴球混合實驗(One-Way Mix Lymphocyte Reaction Assay:MLR) --------------------------------------------------------------58
3.4.8 統計分析----------------------------------------------------60

第四章 結果
第一節 建構 IL-10 以及 TGF-β1 的慢病毒感染系統
4.1.1 pTY-EF-IL-10 與 pTY-EF-TGF-β1 質體構築與評估基因的表現--------61
4.1.2 pTY-EF-IL-10-Lv 與 pTY-EF-TGF-β1-Lv 慢病毒備製、感染與轉質基因表現的評估------------------------------------------------------------63
第二節 以慢病毒感染樹突狀細胞
4.2.1 感染骨髓 lin- 造血幹細胞-------------------------------------64
4.2.2 感染骨髓樹突狀細胞-------------------------------------------65
4.2.3 感染 pTY-EF-IL-10-Lv 或 pTY-EF-TGF-β1-Lv 骨髓樹突狀細胞對於異體T淋巴球之抗原性-------------------------------------------------------66
第三節 以慢病毒感染骨髓基質細胞
4.3.1 以 慢病毒感染骨髓基質細胞-------------------------------------67
4.3.2 骨髓基質細胞感染 pTY-EF-IL-10-Lv 後表面抗原表現影響之分析以及測量細胞激素的釋放-------------------------------------------------------67
4.3.3 骨髓基質細胞感染 pTY-EF-TGF-β1-Lv 後表面抗原表現影響之分析以及測量細胞激素的釋放-------------------------------------------------------68
4.3.4 感染 pTY-EF-IL-10-Lv 或 pTY-EF-TGF-β1-Lv 骨髓基質細胞對於異體T淋巴球之抗原性---------------------------------------------------------68

第五章 討論---------------------------------------------------------70

參考文獻------------------------------------------------------------76
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