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Author: 陳桂芳
Thesis Title: 第十介白質在去氫表雄固酮調節免疫球蛋白分泌時的角色
Role of interleukin-10 in dehydroepiandrosterone-mediated immunoglobulin production
Advisor: 曾哲明
Tseng, Jer-Ming
Degree: 碩士
Master
Department: 生命科學系
Department of Life Science
Academic Year: 87
Language: 中文
Number of pages: 148
Keywords (in Chinese): 去氫表雄固酮BALB/c mice老鼠脾臟淋巴球細胞免疫球蛋白第十介白質抗老鼠單株抗體
Keywords (in English): DHEA, BALB/c mice, mouse spleen lymphocytes, immunoglobulin, interleukin 10, anti-mouse interleukin-10 monoclonal antibody
Thesis Type: Academic thesis/ dissertation
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  • 中文摘要
    DHEA為腎上腺皮質網狀層所分泌的類固醇荷爾蒙,它是製造性荷爾蒙的中間產物,參與哺乳類體內多種生理活動。在文獻報導中,DHEA及DHEAS被證實能多方面調節血清性及細胞性免疫反應。由前人歷年對DHEA及DHEAS所做的研究結果,顯示在離體的狀況下,DHEA及DHEAS可促進老鼠脾臟B淋巴球細胞、人類周邊血液單核球細胞(PMNC)及B淋巴球細胞株(Dakiki及SKW6.4)分泌免疫球蛋白。本實驗進一步在活體的狀況下進行實驗,發現將BALB/c老鼠依每公克體重為單位,皮下注射不同劑量的DHEA或DHEAS溶液後,脾臟淋巴球細胞的生長受到抑制,存活率降低,但顯著地促進免疫球蛋白(IgG、IgA、IgM)的分泌。DHEA及DHEAS的最佳作用劑量為250mg/gw,在老鼠體內作用48小時後作用最為顯著。相關的文獻報導中指出,細胞激素包括 IL-10對免疫球蛋白的製造有顯著地調節作用。本實驗隨後同樣以DHEA或DHEAS施打老鼠,探討其對IL-10的製造是否有調節作用,結果顯示DHEA及DHEAS可以顯著地促進老鼠IL-10的分泌,進一步的研究結果也發現DHEA及DHEAS對IL-10的調控作用,乃是經由對IL-10基因轉錄的調節。為了證實DHEA對免疫球蛋白的促進作用,是經由對IL-10的調節,在施打DHEA的同時,腹腔注射抗老鼠IL-10單株抗體(anti-mouse IL-10 mAb),即以anti-mouse IL-10 mAb中和老鼠體內由DHEA所誘發增加的IL-10及老鼠內生性的IL-10,結果顯示anti-mouse IL-10 mAb的中和作用造成IL-10的分泌量降低,也抑制了DHEA對免疫球蛋白分泌的促進作用,由此結果可推論DHEA可能經由活化Th2途徑增加IL-10的分泌,進而促進免疫球蛋白的製造。

    英文摘要
    DHEA is a predominant androgen secreted by the adrenal cortex zona reticularis. DHEA appears as an intermediate of the androgen biosynthesis pathway and play a multifunctional role in regulating physiological system in mammals. The accumulated documents have demonstrated that DHEA and DHEAS are the potential immunomodulator that regulate both humoral and cellular immune response. Previous reports showed that DHEA and DHEAS were able to enhance the immunoglobulin production by murine spleen B lymphocytes、human peripheral blood mononuclear cells (PMNC) and B-cell lymphoid cell lines (Dakiki and SKW6.4) under in vitro condition. The present study extended the previous finding to in vivo system. In this study, the male Balb/c mice were injected subcutaneously with various doses of DHEA or DHEAS based on the body weight. The mice were then sacrified at different time point after the drug treatment and both spleen lymphocytes and serum were collected. Results indicated that both the growth rate and viability of the spleen lymphocytes were reduced after 6 days of in vitro culture. However, the amounts of immunoglobulin (IgG、IgA and IgM) secreted by the cells and the serum level of immunoglobulin were increased. The optimal dose for enhancing immuno-globulin secretion was 250 mg per gram body weight and the period for the maximal effect was 48 hrs after the treatment. The previous reports suggested that cytokine including interleukin-10 (IL-10) were able to modulate the immuno-globulin production. In this study, the role of IL-10 in DHEA/DHEAS-mediated increase in immunoglobilin production was investigated. Results indicated that both DHEA and DHEAS were able to enhance the production of IL-10 by the conA-stimulated murine spleen cells. This regulatory activity of DHEA and DHEAS appeared on the messenger RNA level. To further confirm the role of IL-10, the mice were injected with both DHEA and an anti-mouse IL-10 mAb simultaneously. Result showed that the amount of IL-10 produced by the spleen cells was reduced and the serum level of IL-10 was decreased after the antibody treatment. In consistent with the IL-10 level, the amount of immunoglobulin secreted by spleen cells and the serum level of the immunoglobulin were significantly decreased. In conclusion, DHEA might enhance the immunoglobulin production by activing Th2 pathway and increasing IL-10 production.

    目 錄 中文摘要…………………………………………………………….I 英文摘要………………………………………………..………….III 圖次………………………………………………………………….V 壹、前言………………………………………………………….….1 I、文獻探討…………………………………………………...……..1 1.去氫表雄固酮(Dehydroepiandrosterone)……………….…….......1 2.免疫球蛋白(Immunoglobulin)………………………………......8 3.第十介白質(Interleukin 10)………………………………....….12 II、待答問題………………………………………………….….…15 貳、實驗材料與方法…………………………………………..…..17 一、試劑、抗體及藥品…………………………………….….…..17 1.老鼠處理試劑…………………….……………………….............17 2.分離及培養老鼠脾臟淋巴球細胞所需試劑……..…..........….….18 3.酵素免疫分析法所需抗體與溶液………………..… …......…….18 4.抽取cDNA所需試劑………………………..………………...........20 5.反轉錄反應所需試劑………………………..…………....... …….20 6.聚合酵素鏈反應所需試劑…………………..………….......… ….21 二、研究方法………………………………………….…….…..….22 1.細胞培養液的配置………………………………………….....….22 2.實驗動物……………………………………………………...…...22 3.分離老鼠脾臟淋巴球細胞…………………………………...…...23 4.取得老鼠血液之血清…………………………………………......23 5.細胞之計數……………………………………………………......24 6.動物處理及細胞處理……………………………………….....….24 (A) 動物處理………………………………………………........ 24 (B) 細胞處理………………………………………………......... 25 7.酵素免疫分析法……………………………………………...…...27 (1) 測IgG………………………………………………….............27 (2) 測IgA………………………………………………...........…..28 (3) 測IgM………………………...…………………….…........... .29 (4) 測IL-10…………………………………………...…..........…..30 8.脾臟細胞中IL-10 mRNA的製造量 ………………………............. 31 (1) 萃取脾臟淋巴球細胞中mRNA ………………… ...........…. 31 (2) 合成cDNA……………………………………………….........33 (3) 聚合酵素鏈反應…………………….……….…………........ 33 9.統計分析方法………………………………………….…...….......35 參、實驗結果……………………………………….………….…...36 一、DHEA及DHEAS對老鼠脾臟淋巴球細胞生長的影響...............36 1.DHEA抑制老鼠脾臟淋巴球細胞之生長及存活率…………........36 2.DHEAS抑制老鼠脾臟淋巴球細胞之生長及存活率………..........37 二、DHEA及DHEAS對免疫功能的影響………..…………............38 A、DHEA………………………………………………….…….......38 1.DHEA促進老鼠脾臟淋巴球細胞分泌免疫球蛋白(IgG、IgA及IgM)…...............................................................38 2.DHEA提昇老鼠血清中免疫球蛋白(IgG、IgA及IgM)的濃度…......…..................................................................40 3. DHEA在不同的體內作用時間下,對老鼠脾臟淋巴球細胞分泌免疫球蛋白(IgG、IgA及IgM)的影響 … 41 4. DHEA在不同的體內作用時間下,對老鼠血清中免疫球蛋白(IgG、IgA及IgM)濃度的影響 …………… 43 B、DHEAS ………………………………………...................………………..................................................................44 1. DHEAS促進老鼠脾臟淋巴球細胞分泌免疫球蛋白IgG、IgA及IgM ………............................................................44 2. DHEAS提昇老鼠血清中免疫球蛋白(IgG、IgA及IgM)的濃度……......................................................................46 3. DHEAS在不同的體內作用時間下,對老鼠脾臟淋巴球細胞分泌免疫球蛋白(IgG、IgA及IgM)的影響..... 47 4. DHEAS在不同的體內作用時間下,對老鼠血清中免疫球蛋白(IgG、IgA及IgM)濃度的影響..................…. 49 三、DHEA及DHEAS對IL-10的製造及基因表現程度的影響……………………….....................................................51 A、DHEA…………………………………………………………......................................................................................51 1. DHEA促進老鼠脾臟淋巴球細胞分泌IL-10………… ….................................................................................................51 2. DHEA提昇老鼠血清中IL-10的濃度 …………………….................................................................................................51 3. DHEA在不同的體內作用時間下,對老鼠脾臟淋巴球細胞分泌IL-10的影響……………………........................52 4. DHEA在不同的體內作用時間下,對老鼠血清中IL-10濃度的影響………………………..…….... ......................53 5. DHEA促進老鼠脾臟淋巴球細胞IL-10 mRNA的表現量..................................................................................................54 6..DHEA在不同的體內作用時間下,對老鼠脾臟淋巴球細胞IL-10 mRNA表現量的影響…………… ….............. 54 B、DHEAS…………………………………………………… …........................................................................................56 1. DHEAS促進老鼠脾臟淋巴球細胞分泌IL-10………………...........................................................................................56 2. DHEAS提昇老鼠血清中IL-10的濃度……………………...............................................................................................57 3. DHEAS在不同的體內作用時間下,對老鼠脾臟淋巴球細胞分泌IL-10的影響………………………….............57 4. DHEAS在不同的體內作用時間下,對老鼠血清中IL-10濃度的影響…………………………………..................58 5. DHEAS促進老鼠脾臟淋巴球細胞IL-10 mRNA的表現量................................................................................................59 6. DHEAS在不同的體內作用時間下,對老鼠脾臟淋巴球細胞IL-10 mRNA表現量的影響……… ……..….......... 60 四、Anti-mouse IL-10 mAb對DHEA免疫能力的影響……...................................................................................................61 1.Anti-mouse IL-10 mAb抑制DHEA對老鼠脾臟淋巴球細胞分泌IL-10的促進作用……….………………................. 61 2.Anti-mouse IL-10 mAb抑制DHEA對老鼠血清中IL-10濃度的提昇作用………………………………....................... 62 3.Anti-mouse IL-10 mAb抑制DHEA對老鼠脾臟淋巴球細胞分泌免疫球蛋白(IgG、IgA及IgM)的促進作用…... 62 4.Anti-mouse IL-10 mAb抑制DHEA對老鼠血清中免疫球蛋白(IgG、IgA及IgM)濃度的提昇作用……................ 65 肆、討論………………………………………………………………................................................................................68 一、 DHEA及DHEAS對老鼠脾臟淋巴球細胞生長的影響.................................................................................................68 二、 DHEA及DHEAS對免疫功能的影響……………….…................................................................................................ 70 三、 DHEA及DHEAS對分泌第十介白質的影響………….. ...............................................................................................74 四、 Anti-mouse IL-10 mAb的給予對老鼠免疫球蛋白製造上的影響……………………………………………….. 76 五、 總結……………………………………………….……. ...............................................................................................78 六、 結語………..…………….…………………….……… ................................................................................................. 83 伍、參考資料………………………………………………………......................................................................................85 附錄一、圖片說明…………………………………………………....................................................................................100

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