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輔仁大學
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生命科學系
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研究
姜文婷
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論文
名稱 第十介白質提升羊水幹細胞
(中)
論文
Evaluation of amniotic fluid stem cells improving GvHD prevention mediated by
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interleukin-10
(英)
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指導
教授 周秀慧
(中)
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全文 2020.9.7
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封面 目錄 摘要 第一章 第二章 第三章 第四章 第五章 參考文獻 附錄
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介白質 10 羊水幹細胞 移植物抗宿主病
字(中)
關鍵
IL-10 amniotic fluid stem cells GvHD
字(英)
宿主排斥(rejection)或移植物對抗宿主疾病(graft-versus-host disease；GvHD)
是臨床進行異體造血幹細胞移植過程中主要常見的問題。近來研究結果
顯示，進行造血幹細胞移植時，合併注入成體幹細胞具有減輕 GvHD 反
摘要 應以及提高造血幹細胞植入率的效用。實驗室先前的動物實驗證明來自
羊水的(AFSC)與胎盤的(PDSC)成體幹細胞均可提升捐贈造血幹細胞植入
(中)
的效率，其中 PDSC 也具有減緩急性 GvHD 的效果，然而 AFSC 細胞並
未能如預期在活體內有減緩急性 GvHD 的排斥現象。另外，現今有關成
體幹細胞為何可提升骨髓移植植入率的機制並不清楚。IL-10 是一種免疫
抑制細胞激素，可抑制第一型輔助 T 細胞(Th1)表現的活性，因此，本實
驗室利用慢病毒作載體將 IL-10 的基因轉染至羊水幹細胞(AF)中，期望經
轉染 IL-10 後的 AFSC 細胞能提升其免疫抑制的活性並於活體上達到減緩
GvHD 排斥反應的療效，針對以上的論點，本研究的目的有二：[1]探討
羊水及胎盤幹細胞增加骨髓移植效率的相關分子機制；[2]評估轉染 IL-10
基因的羊水幹細胞對 GvHD 的抑制效用。 在評估羊水或胎盤幹細胞調控
造血幹細胞分子機制的實驗結果顯示，(1)以 Flow cytometry 分析其幹細
胞表面分子的表現，發現 AFSC 和 PDSC 均能表現不等程度的黏附型表
面分子 CD29、CD44、CD106，但均不會表現 CD49a 分子；(2)以 colonyforming unit(CFU)方法發現羊水及胎盤幹細胞可以產生可溶性因子促進造
血幹細胞增生或分化，收集 CFU 培養中的 colony 細胞，以 RT-PCR 方法
評估發現，AFSC 和 PDSC 均可促使造血幹細胞中造血過程所需之轉錄因
子 Cdx4 基因的表現；(3)以 RT-PCR 方法檢測發現，AFSC 細胞會表現:
SDF-1、IL-6、IL-11、OSM、G-CSF、M-CSF、GM-CSF 等 mRNA，PDSC
細胞會表現：SDF-1、IL-11、M-CSF、GM-CSF 等造血相關調節因子的
mRNA，但兩者均不會表現 TPO mRNA。 在評估轉染 IL-10 的
AFSC(AF/IL-10)是否能夠減緩 GvHD 反應的實驗結果顯示，(1)以 RT-PCR
與 ELISA 方法檢測 IL-10 基因轉染後的羊水幹細胞顯示 AF/IL-10 細胞可
表現 IL-10 基因並且可分泌 IL-10 細胞激素；(2)利用 mixed lymphocyte
reaction(MLR)的方式確認經轉染 IL-10 後的 AFSC 細胞在高密度作用下，
具有提升抑制異體 T 細胞增生的活性；(3)轉染 IL-10 後並不影響 AFSC
細胞表面上 CD29、CD49a、CD44、CD106、MHC I 和 MHC II 等分子的
表現量，並且分化證明 AF/IL-10 細胞仍具有分化成脂肪細胞的能力，但
分化能力會降低；(4)將 AF/IL-10 細胞和 C57BL/6J 小鼠的造血幹細胞一
起植入 BABL/c 的 GvHD 小鼠體內進行抗 GvHD 的驗証，依據評估 GvHD
score、骨髓接受者存活率、捐贈細胞植入狀況與血液細胞族群恢復程度
等實驗結果發現，AF 轉染 IL-10 後可提升其在活體中減緩 GvHD 排斥反
應的療效。
Rejection or graft-versus-host disease (GvHD) is major problems of allogenic
hematopoietic stem cell transplantation (HSCT). Human pre-clinical studies have
showed that co-transplantation of hematopoietic stem cells with mesenchymal
stem cells enhanced donor cell engraftment and reduced the GvHD occurrence.
Our lab has established the adult stem cells from amniotic fluid (AFSC) and
placenta (PDSC). The in vivo results of allogenic bone marrow transplantation
摘要 have confirmed that infusing AFSCs or PDSCs can enhance the engraftment of
hematopotic stem cells. Furthermore, the PDSC can reduce the severity of GvHD
after allogenic BMTx, but AFSC cells did not. However, the mechanism of AFSC
(英)
and PDSC contribute on supporting the hematopoietic stem cell engraftment and
differentiation is remained unclear. IL-10 is a suppressive cytokine that expresses
the inhibition on the Th1-mediated immune responses. We previously have
delivery the mIL-10 gene into AFSCs (AF/IL-10) by using lentiviral transduction
system, expecting the AF/IL-10 cells can enhance the immunosuppressive activity
and reduce the GvHD responses. According to the contention, the aims of this
study have two goals: First, to rule out the possible molecular mechanism(s) on
hematopoietic supporting activities of AF and PDSC. Second, to examine whether
the use of IL-10-transduced AFSCs (AFSCs/IL10) could reduce the severity of
acute GvHD after HSCT. Results of mechanism(s) on hematopoietic supporting
activities of AF and PDSC have showed that (1) AFSC and PDSC express CD44
and CD106, an adhesion molecule, and CD29, a cellular junction matrix, but both
cells did not express CD49a molecule on cell surface. (2) the AFSCs and PDSCs
can secrete the several growth factors to enhance the proliferation and
differentiation of hematopoietic stem cells (HSCs) in CFU assay. Hematopoietic
colony cells express Cdx 4 and GATA-2 genes after cultured in AFSC or PDSC
derived conditional medium in CFU assay. (3) by RT-PCR assay, AFSCs possess
capable of hematopoietic growth factors included IL-6, IL-11, OSM, G-CSF, MCSF, and GM-CSF, as well as, PDSC produces IL-11, M-CSF, and GM-CSF.
Both cells do not express mRNA of TPO. Results of evaluate the therapeutic
efficacy of IL-10-transduced AFSCs (AFSCs/IL10) on attenuation the severity of
acute GvHD have showed that (1) AF/IL-10 cells capable expressed the IL-10
gene and IL-10 proteins. (2) AF/IL10 cells exhibit strong allogenic T cell
immunosuppression in high cell number density after MLR culture. (3) The
expression level of CD29, Cd49a, CD44, CD106, MHC I, and MHC II on AFSC
did not change after IL-10 transduction. However, the ability of adipogenesis on
AF/IL-10 cells was attenuated after IL-10 transduction. (4) A mice acute GvHD
model by infusing allogeneic hematopoietic stem cells with donor-derived T cells
in transplantation, and co-transfusion with AF/IL-10 was tested for it potential
effects to reduce GvHD while comparing with the control group. We found that
AF/IL10 could inhibit lethal GvHD after allo-HSCT based on the GvHD score,
recipient survival rate, donor cells engraftment and blood cells reconstitution.
目錄 目錄 I 附表目錄 V 附圖目錄 VI 英文縮寫表 IX 中文摘要 1 英文摘要
3 第一章 研究背景 第一節 異體骨髓移植 1.1.1 異體骨髓移植的定義、應
用和重要性 5 1.1.2 異體骨髓移植的瓶頸與解決策略 6 1.1.3 移植物抗宿主
疾病 7 第二節 間葉系幹細胞的特性與應用 1.2.1 間葉系幹細胞的特性 8
1.2.2 間葉系幹細胞在醫學上的研究和應用 9 1.2.3 間葉系幹細胞的免疫抑
制性 11 第三節 羊水幹細胞的特性及其應用 1.3.1 羊水幹細胞 12 1.3.2 羊
水幹細胞在臨床上的應用 13 第四節 胎盤幹細胞的特性及其應用 1.4.1 胎
論文 盤幹細胞 14 1.4.2 胎盤幹細胞在臨床上的應用 14 第五節 間葉系幹細胞與
造血幹細胞的調控機制 1.5.1 間葉系幹細胞與造血幹細胞的應用 16 1.5.2
間葉系幹細胞具有支持造血幹細胞相關因子的表現 17 第六節 免疫抑制
目次 型細胞激素介白質-10(Interleukin-10, IL-10) 1.6.1 IL-10 簡介 18 1.6.2 IL-10
在移植上之應用 19 1.6.3 IL-10 基因轉染在幹細胞的應用 20 第二章 實驗
目的和重要性 21 第三章 實驗材料及方法 第一節 實驗設計 23 第二節 實
驗材料 3.2.1 實驗動物 26 3.2.2 胎盤和羊水細胞株 26 3.2.3 pTY-EF-DsRedLv 與 pTY-EF-IL-10-Lv 慢病毒系統感染羊水細胞的製備與培養 27 3.2.4 實
驗藥品、培養基與各類試劑 27 第三節 儀器設備 36 第四節 實驗方法
3.4.1 羊水和胎盤幹細胞之繼代培養 38 3.4.2 群落形成單位實驗(colony
forming unit assay) 38 3.4.3 造血幹細胞表面上吸附分子的鑑定 39 3.4.4 以
反轉錄聚合酶連鎖反應(RT-PCR)技術鑑定 CFU 群落中造血前驅細胞的表
現以及羊水或胎盤幹細胞和轉染 DsRed 或 IL-10 之羊水幹細胞中造血相
關生長因子的表現 39 3.4.5 酵素連結免疫反應 (Enzyme link immunosorbent
assay, ELISA)評估細胞培養 中或動物血清中細胞激素的含量 41 3.4.6 經轉
染 IL-10 之羊水幹細胞分化成脂肪細胞的分化能力評估 41 3.4.7 異體淋巴
球混合實驗 (One-Way allogenic mixed lymphocyte reaction, one-way MLR) 42
3.4.8 小鼠急性 GvHD 活體動物模式的建立 43 3.4.9 移植後異體造血幹細
胞造血重建的評估 44 3.4.10 統計分析 45 第四章 實驗結果 第一節 體外評
估羊水及胎盤幹細胞促進骨髓移植植入效率之相關分子機制 4.1.1 群落形
成單位實驗 (Colony forming unit assay, CFU) 46 4.1.2 評估羊水及胎盤幹細
胞表面吸附分子的表現 48 4.1.3 評估羊水或胎盤幹細胞中造血活性相關
生長因子的表現 48 第二節 經帶有 IL-10 基因慢病毒感染後之羊水幹細胞
之特性分析 4.2.1 評估經慢病毒感染後，羊水幹細胞在表現 IL-10 基因的
成效 49 4.2.2 IL-10 慢病毒感染後羊水幹細胞中 IL-10 基因與蛋白質表現
的檢測 49 4.2.3 利用異體 T 淋巴細胞混合實驗評估轉染 IL-10 基因後之羊
水幹細胞的免疫抑制活性 50 4.2.4 IL-10 慢病毒感染後羊水幹細胞在異體
T 淋巴細胞混合實驗中 Th1 細胞激素 IFN-γ 的檢測 51 4.2.5 IL-10 基因感
染後羊水幹細胞中 GFP 螢光表現的檢測 51 4.2.6 評估 IL-10 基因轉染後羊
水幹細胞表面抗原之分布 52 4.2.7 AF/IL-10 細胞脂肪分化能力評估 53 第
三節 活體評估轉染 IL-10 基因之羊水幹細胞對抑制急性 GvHD 反應的治
療成效 4.3.1 IL-10 基因轉殖羊水幹細胞後再 GvHD 的治療效果 53 4.3.2
IL-10 基因轉殖的羊水幹細胞減緩 GvHD 的作用機制 54 第四節 體外和活
體評估轉染 IL-10 之羊水幹細胞對異體骨髓移植與造血活性的影響 4.4.1
IL-10 基因轉殖後對接受者免疫細胞活性的影響評估 55 4.4.2 評估 IL-10
轉染後羊水與胎盤幹細胞的表面貼附分子的表現 55 4.4.3 AF/IL-10 群落形
成單位實驗(Colony forming unit assay, CFU) 56 4.4.4 IL-10 基因轉染後對羊
水幹細胞調控造血幹細胞分化活性的檢測 56 4.4.5 評估 IL-10 基因轉染後
羊水幹細胞中產生與造血相關生長因子的檢測 57 第五章 討論 58 參考文
獻 68
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