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7250
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狀態 NC095FJU00105020
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輔仁大學
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生命科學系
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學號 493546168
研究
生 孫子麒
(中)
研究
生 Sun Tzu Chi
(英)
論文
名稱 免疫抑制型細胞激素培養之樹突細胞對胎鼠骨髓移植之效用評估
(中)
論文
The potential application of immune suppressive cytokine conditioned-dendritic
名稱
cells on fetal mice bone marrow transplantation
(英)
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指導
教授 周秀慧
(中)
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教授 Chou Shiu Huey
(英)
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學位
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學年 95
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字 子宮內骨髓移植 樹突細胞
(中)
關鍵
字 in utero transplantation Dendriric cell TGF-beta1 IL-10
(英)
子宮內骨髓移植是指在懷孕中期，將健康之骨髓細胞植入胎兒受體的技
摘要 術，用於治療先前骨髓移植可以治療的疾病。但現階段所遇到的問題為
植入率過低(?0.1%)，植入的細胞不足以治療疾病及無法引發免疫耐受
(中)
力。樹突細胞是一種抗原呈現細胞，具有活化 T 細胞及引發 T 細胞對外
來細胞產生耐受性的雙重特性。排斥反應是由宿主 TH1 所主導的，TGF-
?1 與 IL-10 為免疫抑制型的細胞激素，可以抑制 T 細胞的增生，對於樹
突細胞具有抑制其輔助刺激因子的表現使樹突細胞成為一種不成熟的狀
態，而不成熟的樹突細胞具有引發免疫耐受力的功用。本實驗利用
MHC-mismatched 小鼠模式，將 C57BL/6 小鼠樹突細胞或經過 TGF-?1 或
TGF-?1+IL-10 培養後的樹突細胞與捐贈者(C57BL/6 GFP+ H-2b)之造血幹
細胞共同植入 13~14 天 BALB/c 胎鼠中，以評估對於胎鼠骨髓移植的效
用。首先評估 13~15 天的胎鼠在 TGF-?與 IL-10 受器的表現與 TGF-?1 分
泌的能力，結果顯示 13 天的胎鼠即具有 40% TGF-? RII 與 3.2% IL-10 R?
的表現，該時期免疫細胞具有被活化的能力，而免疫細胞也具產生及釋
放 TGF-?1 的能力。接著經由 C57BL/6 小鼠骨髓在 GM-CSF 培養下分化出
一群 CD40low、CD80 low、CD86 low、B7-H1 hi 與 B7-DC med 的樹突細
胞，該細胞呈現微弱的吞噬能力並具有被外來抗原活化的能力，將該細
胞經過 TGF-?1 培養後發現其細胞表面輔助刺激因子 CD40、CD80、
CD86、B7-DC 及 MHC II 的表現顯著的下降，而經過 TGF-?1+IL-10 培養
後也會降低 CD40、CD86 及 B7-DC 的表現，進一步將此兩組處理的樹突
細胞與異體 T 細胞做單向淋巴球混合實驗，顯示具有抑制異體 T 細胞增
生的能力，進一步分析發現經過與 TGF-?1 或 TGF-?1+IL-10 培養後的樹突
細胞作用後的 T 細胞表面上 CD28 分子會下降而 PD-1 分子的表現會上
昇，同時也使 CD4+CD25+調節性 T 細胞族群有少量的增加。分析其上清
液中細胞激素的表現，發現 TGF-?1 與 TGF-?1+IL-10 培養的樹突細胞均會
抑制 T 細胞 IL-2、IL-4、IL-10、IFN-?的分泌，但前者卻會提昇 TNF-?的
分泌；而後者卻是提昇 TGF-?1 的分泌。在活體胎鼠骨髓移植發現共同注
射處理與未處理之樹突細胞與造血幹細胞均具有提昇胎鼠骨髓移植的植
入率，但經抑制型細胞激素處理的樹突細胞並未提昇植入細胞的數目。
綜合以上的結果，體外實驗顯示經過 TGF-?1 或 TGF-?1+IL-10 培養可降低
樹突細胞表面 CD40、CD80 與 CD86 輔助刺激因子的表現，進而降低與
之接觸後異體 T 細胞 CD28 的表現與提昇 PD-1 與 B7-H1 的作用，因而引
發抑制異體 T 細胞的增生反應。該負向調節反應雖可增加活體胎鼠移植
的植入率但卻無法顯著的提昇植入細胞的數目。
In utero hematopoietic stem cell transplantation (IUHSCT) was used as strategy to
replace bone marrow transplantation without the need of toxic conditioning
therapy. The major limitation of IUT to date is limited chimerism and lack
tolerance to alloantigen. Dendritic cells (DCs) are considered very potent antigenpresenting cells for T cell activation, but strongly tolerogenic as precursor DC. The
摘要 major problem on failure of transplantation is that implant is rejected by recipient.
Rejection of implant is mediated by type I T helper cells of host. TGF-β1 and IL10 are kind of suppression cytokines. Related researches have reported that both
(英)
cytokines inhibited the Th1 cell activation and DC maturation. Herein, we firstly
investigated the expression level of TGF-β1 and IL-10 in 13-14-day fetus. Then,
we measured the effects of TGF-?1 or TGF-?1+IL-10 cultured donor derived DCs
on allogenic T cell responses by in vitro mix lymphocyte reaction assay (MLR).
Furthermore, by using a MHC-mismatched mouse model, we examine the effects
of TGF-?1 and TGF-?1+IL-10 DCs on in utero mice bone marrow transplantation.
Results indicated that 13~15 days of pregnancy fetus posses the normal expression
of TGF-?? receptor (40%) and IL-10 receptor (3.2%) on the surface of immune
cells and production of TGF-?1 cytokine (120pg/ml). Furthermore, 13-15 days of
fetus exhibited the proliferate capability in response to mitogen stimulation. In this
study, GM-CSF-derived bone marrow CD11c positive cells with expression of
CD40 low, CD80 low, CD86 low, B7-H1 hi, and B7-DC med were acquired and
used as a source of dendritic cells . Those cells had shown a weak phagocytic
activity and can be activated by LPS stimulation. After culturing with immune
suppressive cytokine, TGF-?1 cultured DCs showed less expression on costimulatory molecules CD40, CD80, CD86, B7-DC and MHC class II, while TGF?1+IL-10 cultured DC showed less CD40, CD86 and B7-DC expression. By using
mix lymphocyte assay, TGF-?1 and TGF-?1+IL-10 treated DCs inhibited the
allogenic T cell proliferation through decreased CD28 expression and increased
PD-1 on T cells. In addition, small numbers of CD4+CD25+ Foxp3 T cells were
induced in co-cultured TGF-?1 and TGF-?1+IL-10 treated DCs with allogenic T
cells. Further analysis of T cell cytokine production in cultures of MLR had
showed that both TGF-??1 and TGF-?1+IL-10 treated DCs decreased the
production of IL-2, IL-4, IL-10, and IFN-???however, the production of TNF-?
was increased in former and production of TGF-??1 was increased in later,
respectively. In animal experiment, co-injected immune suppressive cytokine
treated-DCs with donor bone marrow cells had increased engraftment of donor
cells, but did not increase the number of donor cells in fetus recipients. In
conclusion, TGF-??1 or TGF-?1+IL-10 conditioned DCs have induced the
negative regulation in response to allogenic T cells. Although this negative
regulation increased the engraftment of donor cells in fetal recipients, but the
percentage of donor cells in recipients was not improved in this application.
目錄----------------------------------------------------------------------------------------------------I 表目錄-----------------------------------------------------------------------------------------------VI 圖目錄----------------------------------------------------------------------------------------------VII 中文摘要---------------------------------------------------------------------------------------------1 英文摘要---------------------------------------------------------------------------------------------3 第一章 研究背景 第一節 骨髓移植與子
宮內骨髓移植 1.1.1 骨髓移植的定義、應用與重要性----------------------------論文 -------------------5 1.1.2 骨髓移植的瓶頸與解決策略-----------------------------------------------------5 1.1.3 子宮內骨髓移植的定義、應用與重要性--------------------------------------7 1.1.4 子宮內隨移植的瓶頸與動物研究模式-----------------目次
------------------------8 第二節 免疫排斥、免疫耐受性與移植 1.2.1 移植的排
斥反應---------------------------------------------------------------------9 1.2.2 免疫排斥
反應的理論基礎-------------------------------------------------------10 1.2.3 免疫耐受
性-------------------------------------------------------------------------11 1.2.4 免疫耐受
性與骨髓移植----------------------------------------------------------12 第三節 樹突
細胞 1.3.1 樹突細胞的特性與生物活性---------------------------------------------------13 1.3.2 樹突細胞在器官以及骨髓移植上的應用------------------------------------15 1.3.3 樹突細胞與子宮內骨髓移植的應用------------------------------------------16 第四節 轉型生長因子-? (Transforming Growth Factor-beta) 1.4.1 TGF-
?的生化特性-----------------------------------------------------------------18 1.4.2 TGF?受器與訊息傳導-----------------------------------------------------------19 1.4.3 TGF?的免疫活性-----------------------------------------------------------------20 1.4.4 TGF?在移植上的應用-----------------------------------------------------------22 第五節 合
併免疫抑制型細胞激素介白質-10 (Interleukin-10) 與轉型生長因子-β1
(Transforming Growth Factor-beta；TGF-β1) 1.5.1 IL-10 簡介---------------------------------------------------------------------------22 1.5.2 IL-10 的免疫活性-----------------------------------------------------------------23 1.5.3 IL-10 與 TGF-β1 的合併
在移植上的應用-------------------------------------24 第二章 研究目的與重要性-----------------------------------------------------------------26 第三章 實驗材料及方
法 第一節 實驗設計 part A：胎鼠體內環境的確認 (TGF-?1 的分泌與 TGF?1 receptor 的表現) ------27 part B： IL-10 與 TGF-?1 共同培養樹突細胞的體
內與體外實驗-------------------28 第二節 實驗材料與方法 一、實驗小鼠--------------------------------------------------------------------------------29 二、實驗藥
品、培養基與各類試劑-------------------------------------------------------29 三、儀
器設倍----------------------------------------------------------------------------------37 第
四節實驗方法 (I) 胎鼠、新生鼠和成鼠體內 TGF-?1 的分泌與 TGF-?1
receptor 的表現 1、胎鼠、新生鼠和成鼠免疫器官初代細胞之製備--------------------------39 2、胎鼠、新生鼠與成鼠免疫細胞對抗原增值能力評估--------------------39 3、胎鼠與成鼠免疫細胞細胞激素分析-----------------------------------------40 4、胎鼠與成鼠免疫細胞表面 TGF-β 與 IL-10 receptor 蛋白表
現的偵 測------------------------------------------------------------------------------------40
5、胎鼠與成鼠免疫細胞 TGF-β 與 IL-10 receptor mRNA 的偵測----------41
(II) TGF-?1 培養骨髓樹突細胞定性及免疫活性分析 1、 骨髓樹突細胞的
置備-----------------------------------------------------------42 2、 骨髓樹突細胞
TGF-β1 與 IL-10/TGF-β1 最佳培養濃度的備置--------42 3、 骨髓樹突細
胞免疫活性分析 3-1 骨髓樹突細胞表面抗原分析----------------------------------------------43 3-2 骨髓樹突細胞釋放一氧化氮能力評估----------------------------------43 3-3 骨髓樹突細胞細胞激素釋放之測定-------------------------------------44 3-4 單向異體淋巴球混合實驗 (One-Way MLR Assay)--------------------44
3-5 樹突細胞與 T 細胞表面分子的改變--------------------------------------45 3-6
異體 T 細胞經由骨髓樹突細胞刺激後所分泌細胞激素的改變-----46 3-7 異
體 T 細胞經由骨髓樹突細胞刺激後 Foxp3 的改變-----------------46 (III) TGF?1 培養後骨髓樹突細胞對於胎鼠骨髓移植植入效用的評估 1、骨髓 lin-造
血幹細胞純化------------------------------------------------------46 2、子宮內骨髓移
植手術----------------------------------------------------------47 (IV) 骨髓移植植入率
的評估 1、接受者體內捐贈細胞之追蹤 1-1 血液檢體之取得------------------------------------------------------------48 1-2 細胞表面抗原（H-2Kb）之染色----------------------------------------48 1-3 檢體 DNA 萃取與聚合酵素連鎖反應----------------------------------49 (V) 統計分析-------------------------------------------------------------------------------49 第四章 結果 第一節 小鼠不同發生時期免疫細胞
活性與 TGF-β1 及 IL-10 受器表現的評估 1. 胎鼠、新生鼠與成鼠免疫細
胞活化增殖之活性評估---------------------------50 2. 胎鼠免疫組織中 TGF-β
receptor 以及 IL-10 receptor 表現的評估------------51 3. 胎鼠及成鼠免疫器官
釋放 TGF-β1 細胞激素能力之評估----------------------51 第二節 骨髓樹突狀
細胞的定性及其活性測定 1. 骨髓樹突細胞表面抗原之表現------------------------------------------------------52 2. 樹突細胞的活性分析 骨髓樹突細胞釋放
NO 以及細胞吞噬能力評估-----------------------------52 骨髓樹突細胞釋放細
胞激素之能力--------------------------------------------53 第三節 免疫抑制型細胞
激素 (TGF-β1 與 IL-10) 培養後之骨髓樹突細胞免疫功能的分析 1. 免疫抑
制型細胞激素培養後對骨髓樹突細胞表面抗原表現影響之分析---54 2. 免
疫抑制型細胞激素培養之骨髓樹突細胞釋放 NO 能力的評估-------------54
第四節 免疫抑制型細胞激素 TGF-β1 或/和 TGF-β1+IL-10 培養後之骨髓
樹突細胞對異體 T 細胞的影響評估 1. 免疫抑制型細胞激素培養前後之骨
髓細胞對於異體骨髓接受者之抗原性-----------------------------------------------------------------------------------------------54 2. 免疫抑制型細胞激素培養後之樹
突細胞對於異體接受者 T 細胞表面分子表驗影響的評估--------------------------------------------------------------------------55 3. 免疫抑制型細胞激素培養後之
樹突細胞誘導異體 T 細胞產生細胞激素的估-----------------------------------------------------------------------------------------56 第五節 TGF-β1 與 TGF-β1+IL-10
培養後的樹突細胞對胎兒和新生鼠骨髓造血幹細胞移植的評估 1. 子宮內
骨髓移植------------------------------------------------------------------------56 第五章
討論-----------------------------------------------------------------------------------------58
參考文獻--------------------------------------------------------------------------------------------64
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