臺灣博碩士論文加值系統

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE) 是最近發現的新分子，屬於TNF的成員之一，主要是表現在活化的T細胞上。RANK (receptor activator of NF-kB)，為TRANCE的受體，主要是表現在棘細胞(dendritic cell)上。TRANCE被認為是棘細胞的生長因子，並且對於噬骨細胞形成及噬骨母細胞的分化有關。而棘細胞與T細胞之間，TRANCE-RANK的相互作用，可以增進T細胞的活化及生存。而本篇研究中，使用由桿狀病毒（Baculovirus）所表現純化的可溶性人類RANK-Fc融合蛋白，來阻斷TRANCE-RANK的作用，並評估其在小鼠異體胰島移植排斥反應的影響。結果中顯示，所純化的hRANK-Fc可結合至老鼠活化的T細胞表面上。在混合淋巴球試驗中，使用可溶性人類RANK-Fc並沒有明顯抑制T細胞的增生反應，但對於細胞激素的產生上有所影響。另外在測試hRANK-Fc在抑制異體胰島排斥反應上，我們使用streptozotocin（STZ）引起Balb/C小鼠的糖尿病後，植入C57/BL6小鼠的胰島細胞於腎臟膜下。並在移植後的第0，1，3，5天以靜脈注射的方式給予200ug 的hRANK-Fc或是同時在移植後的第0，2，4，6天給予200ug 的hCTLA-4 Ig。我們發現單獨使用hRANK-Fc 能夠讓小鼠的血糖在正常範圍維持約5天，而同時使用hCTLA-4 Ig及hRANK-Fc，則能夠讓血糖在正常範圍維持約6至9天。另外，使用hRANK-Fc及沒有處理的老鼠，在組織學上的分析並沒有太大的差異。雖然單獨使用hRANK-Fc不能有效的抑制排斥反應的發生，但是阻斷TRANCE-RANK的作用，對於異體排斥的免疫反應，尤其是IFN-γ的分泌仍有影響。顯示除了hRANK-Fc的使用之外，合併其他途徑的阻斷作用，尤其是anti-CD40L及hCTLA-4 Ig對於異體胰島移植，將可提供更有效的治療效果。

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a novel member of the TNF family is recently identified in activated T cells. RANK (receptor activator of NF-kB), a receptor of TRANCE, is expressed on dendritic cell. TRANCE is known as a dendritic cell survival factor and essential for osteoclastogenesis and osteoclast activation. Interaction between dendritic cells and T cells, TRANCE-RANK enhances T cell activation and survival. In this study, we purified soluble human RANK-Fc (hRANK-Fc) from Baculovirus expression system. We tested the effect of hRANK-Fc on the rejection of allogenic islet transplant. Purified hRANK-Fc can be detected in Western blot and binds to murine activated T cells on flow cytometry. In mixed lymphocyte reaction, hRANK-Fc is not able to inhibit the T cell proliferation significantly; however, it can affect the T cell cytokines secretion pattern after allostimulation in vitro. We tested the ability of hRANK-Fc in suppression of rejection in islet transplantation. We used streptozotocin-induced diabetes in Balb/C mice as recipients. These diabetic mice received allogenic islet cells form C57/BL6 mice under the subrenal capsules. Purified hRANK-Fc were administrated on day 0, 1, 3, 5 after transplantation (200ug, i.v.) or combined with hCTLA-4 Ig on day 0, 2, 4, 6 after transplantation (200ug, i.v.). The results indicated that using hRANK-Fc alone could prolong the islet graft survival and keep normal blood glucose for 5 days. When we combined hRANK-Fc and hCTLA-4 Ig recombinant proteins, it prolonged islet graft survival and keep the blood glucose normal for 6 to 9 days. hRANK-Fc administration didn’t affect the histology of graft tissues during rejection. Although using hRANK-Fc alone is not sufficient to protect graft form rejection, blockage of TRANCE/RANK does affect the allogenic immune response, in particular on the IFN-γsecretion by T cells. Our results suggest that hRANK-Fc administration in combination with other costimulation pathway blockage, for example, anto-CD40L and CTLA-4 Ig, may be more efficient in suppression allogenic immune response in islet transplantation.

目 錄
中文摘要………………………………………………………………….. 1
英文摘要………………………………………………………………….. 3
第一章 序論
前言……………………………………………………………… 4
第一節 棘細胞與器官移植…………………………………….. 7
第二節 TRANCE與其受體……………………………….…… 8
TRANCE…………………………………….……. 8
RANK……………………………………………... 9
OPG……………………………………………….. 10
第三節 TRANCE與RANK的功能……………………………. 10
TRANCE與RANK對棘細胞的作用……………. 10
TRANCE對T細胞的作用………………………. 13
TRANCE缺失老鼠………………………………. 14
第四節 研究目的及動機……………………………………….. 16
第二章 實驗材料與方法
第一節 實驗材料……………………………………………….. 18
1. 實驗小鼠………………………………………….. 18
2. 細胞珠﹑抗體及酵素…………………………….. 18
3. 試劑與藥品配方………………………………….. 19
4. 儀器與耗材……………………………………… 25
第二節 實驗方法
1. 次選殖人類CTLA-4 Ig基因至Baculovirus表現載體………………………………………………... 28
2. 利用Baculovirus系統表現重組蛋白…………… 28
3. 西方點墨法蛋白質偵測………………………….. 32
4. 分離老鼠脾臟細胞……………………………….. 34
5. 以Nylon Wool Column分離T細胞……………. 34
6. 使用hRANK-Fc測定活化T細胞表面TRANCE 36
7. 混合淋巴球試驗………………………………….. 37
8. 建立糖尿病小鼠模式…………………………….. 38
9. 小鼠糖尿病的測試……………………………….. 38
10.胰島細胞之分離………………………………….. 39
11.胰島細胞移植手術……………………………….. 40
12.組織病理切片分析……………………………….. 41
13.利用胞內染色分析IFN-γ的產生………………. 42
第三章 實驗結果
第一節 產生人類可溶性RANK-Fc融合蛋白 ……………………………………………………… 44
第二節 產生人類可溶性CTLA-4 Ig融合性蛋白 ……………………………………………………… 45
第三節 人類可溶性RANK-Fc融合蛋白與老鼠活化T細胞表面TRANCE的結合………………………………. 46
第四節 人類可溶性CTLA-4 Ig融合蛋白與Raji細胞上B7的結合………………………………………………… 47
第五節 人類可溶性RANK-Fc融合蛋白對於混合淋巴球試驗的影響……………………………………………… 48
第六節 人類可溶性RANK-Fc融合蛋白對於小鼠異體胰島移植的影像…………………………………………… 50
第七節 共同使用人類可溶性CTLA-4 Ig及RANK-Fc融合蛋白對於小鼠異體胰島移植的影響………………… 52
第八節 人類可溶性RANK-Fc融合蛋白對於混合淋巴球試驗中細胞激素的影響………………………………… 53
第四章 討論……………………………………………………………… 55
第五章 參考文獻………………………………………………………… 69
第六章 圖表及說明……………………………………………………… 76

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