臺灣博碩士論文加值系統

前言：當個體開始老化時，免疫系統也會跟著衰弱，造成免疫衰老(immunosenescence)，也伴隨著自體免疫疾病的發生率上升。
調節性T細胞(regulatory T cell，Treg﹚對於維持體內免疫的自我耐受性（self-tolerance）以及免疫平衡（immunohomeostasis）扮演了很重要的角色。隨著年齡的上升，調節型T細胞的族群有增加的現象，而數量增加的調節型T細胞可以幫助個體對抗自體免疫疾病的發生。植物性雌激素中二苯乙烯類的白藜蘆醇已知具有多種生物活性，但目前並沒有關於其對於Treg活性影響的研究，而arachidin-1同屬富含於花生芽萃取物中的stilbenes的相關研究更少；本篇探討兩種stilbene，resveratrol、arachidin-1，對於小鼠脾臟與胸腺的Treg的活性與比例的影響。
結果：經細胞毒性試驗，決定兩種成分對於免疫細胞不具有明顯細胞毒性的試驗濃度。並且發現能夠與E2一樣有抑制ConA刺激淋巴細胞增生的現象。進一步利用流式細胞儀分析發現雖然resveratrol、arachidin-1都無法提升ConA刺激之淋巴細胞增生族群中之Treg細胞的族群比例，但卻能促進脾臟及胸腺淋巴細胞之CTLA-4的表現量及TGF-β、IL-10的分泌，利用RT-PCR偵測mRNA表現亦有相同的趨勢。使用tamoxifen作為雌激素受體拮抗劑時，發現脾臟細胞增生現象恢復，而胸腺細胞則有部分的恢復；因此瞭解resveratrol、arachidin-1能夠藉由雌激素受體路徑影響淋巴細胞。最後於飼糧中添加花生芽粉中富含兩種stilbene萃取物，長期餵食老年ICR小鼠，發現在周邊淋巴組織脾臟中Treg族群沒有顯著增加的現象，但在中樞淋巴組織胸腺中Treg族群則有些微的增加，另分析自脾臟中以磁珠分離純化濃縮的Treg細胞的Foxp3、CTLA-4與TGF-β的mRNA表達，也都有顯著提升的現象。
結論：衰老會造成個體自體免疫疾病的增加，影響壽命的重要因素，而Treg細胞所主導的免疫抑制作用可以平衡免疫系統對自我抗原的過度反應；在本篇發現花生芽粉中提取的stilbene成分包含resveratrol與arachidin-1具有與雌激素相同的作用，可以藉由雌激素受體作用路徑，增加Treg細胞的CTLA-4、TGF-β、IL-10，進而達到更理想的老化狀態，未來將有潛力作為老年保健食品的參考。

Background：Alterations in immunity that occur with aging (immunosenescence) likely contribute to the development of autoimmune diseases. CD4＋CD25＋Foxp3＋ regulatory T cells (Treg) play an immunosuppressive role in immune system. They are responsible in the maintaining of immunological self-tolerance as well as immunohomeostasis. Age-associated increases in the population of Treg protect host against autoimmune diseases. Estrogen is known to increase Treg activity in facilitating immunological tolerance to fetal antigens. Stilbenes are known possessing phytoestrogenic activities. Among stilbene family, resveratrol is the most well-known and proved to have immunosuppressive activity by inhibiting T cell proliferation. In this study, we hypothesize that resveratrol and arachidin-1 might have exhibited a phytoestrogenic property to affect Treg cells development and their activity.
Result： Immunotoxicity of both resveratrol and arachidin-1 was assessed. There was no obvious cytotoxicity of resveratrol and arachidin-1 on immune cells when concentration was under 25 M and 5 M respectively. All experiments were therefore tested below the toxic concentrations. ConA stimulated lymphoblastogenesis of either spleen or thymus was inhibited by pre-treatment with resveratrol, arachidin-1 or E2. Flow cytometric analysis revealed that the peripheral Treg population was not altered by treatments, whereas Treg cell functions including CTLA-4, TGF-, and IL-10 were all upregulated by resveratrol, arachindin-1 and E2. And similar results were also demonstrated at mRNA level. Foxp3, the specific nuclear transcription factor of Treg , was also enhanced. The phytoestrogenic activity of resveratrol and arachidin-1 in inhibition of ConA induced lymphoblastogenesis was recovered by pre-treatment of estrogen receptor blocker, tamoxifen. In general, arachidin-1 and resveratrol showed limited effect on Treg population but greatly upregulate Treg activity. The immunomodulatory effect of stilbenes was further demonstrated in aged male ICR mice by a long term dietary supplementation of peanut sprout diet enriched with stilbenes. Dietary stilbenes enhanced Treg populations as well as stimulated mRNA expression of CTLA-4, TGF- and Foxp3 in enumerated purified Treg cells.
Conclusion： Aging resulted in immunosenescence and leading to autoimmune diseases, immunosuppressive Treg dominated conditions represented a successful aging. In this study, we demonstrated that peanut sprout enriched with stilbenes including resveratrol and arachidin-1 exhibited an estrogenic activity in upregualting Treg cell functions. Peanut sprouts diet enriched with stilbenes may have beneficial effects on aging individuals as a healthy supplement.

目錄 III
圖次錄 VII
附錄目錄 IX
縮寫表 X
中文摘要 XII
英文摘要 XIV
一、 文獻探討 1
(一) 調節性T細胞 1
Regulatory T cell
(二) 雌激素 5
Estrogen
1. 雌激素在生理上的意義 5
2. 雌激素與調節性T細胞的關係 7
(三) 植物性雌激素 10
Phytoestrogen
1. 異黃酮類 10
2. 木酚素 11
3. 二苯乙烯 12
4. 植物性雌激素與調節性T細胞的關係 15
(四) Arachidin-1 16
二、文獻引用 20
三、前言 27
四、材料 32
(一) 材料來源 32
(二) 動物飼養與管理 32
(三) 流式細胞儀用表面分子抗體、ELISA套組、RT-PCR 32
(四) 藥品與耗材 33
(五) 儀器 35
五、方法 37
(一) 半體外試驗 37
1. 分離純化細胞 37
A. 脾臟單核細胞純化 37
B. 胸腺單核細胞純化 37
2. 毒性試驗 38
A. Lactate dehydrogenase release細胞毒性試驗 38
B. Alamar blue細胞毒性試驗 39
3. 增生試驗 39
4. 偵測細胞表面抗原試驗 40
5. 酵素免疫分析 41
A. TGF-β 41
B. IL-10 41
6. Reverse transcriptase-polymerase chain reaction 42
A. 純化RNA 42
B. RNA反轉錄成cDNA 42
C. PCR 43
7. 雌激素扮演的角色 46
(二) 體內試驗 47
1. 分離純化細胞 47
A. 脾臟及胸腺淋巴細胞之分離與純化 47
B. 磁硃抗體純化濃縮調節性T細胞 47
2. 偵測表面抗體 48
3. RT-PCR 48
(三) 統計分析 48
六、試劑配方 49
(一) 培養液 49
(二) 純化細胞 49
(三) 毒性試驗 49
(四) 偵測表面抗原 50
(五) 免疫酵素分析 50
七、結果 52
(一) Res、Ara細胞毒性試驗 52
(二) Res、Ara抑制ConA誘導之淋巴細胞增生 53
(三) Res、Ara對於regulatory T cell族群的影響 54
(四) Res、Ara對於regulatory T cell功能的影響 55
(五) Res、Ara提升Foxp3、CTLA-4、TGF-β mRNA的表達量 56
(六) 雌激素受體在Res以及Ara中所扮演的角色 57
(七) Stilbene對老年小鼠regulatory T cell族群的影響 58
(八) Stilbene影響regulatory T cell的mRNA的表現量 59
八、討論 60
九、參考文獻 68
十、 結果圖 73

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