臺灣博碩士論文加值系統

介白素15 (IL-15)是一個多功能的細胞激素, 記憶CD8Ｔ細胞、自然殺手細胞、自然殺手Ｔ細胞、小腸壁細胞間隙淋巴球的活化及恆定都受其調控。我們發現老化的IL-15基因剔除鼠和IL-15受器α鍵基因剔除鼠血清中存在自體抗體 (autoantibodies) 並且胸腺細胞的陰性篩選 (negative selection)存在缺陷。胸腺表皮細胞和樹突細胞的IL-15的表達與訊號傳遞對於胸腺細胞的陰性篩選是重要的。 我的研究在於探討野生型、IL-15基因剔除鼠和IL-15受器α鍵基因剔除鼠的胸腺表皮細胞、胸腺基質細胞特性的異同, 其中包含了細胞特性、抗原呈現細胞相關的標誌和自體免疫調控因子 (Aire)。我發現全部的胸腺表皮細胞以及胸腺髓質表皮細胞在基因剔除鼠中數目下降, 但是胸腺表皮細胞的 抗原呈現細胞相關的標誌和自體免疫調控因子表現量是不變的。IL-15基因剔除鼠的 CD45- 胸腺基質細胞和胸腺皮質表皮細胞數目也有減少。為了探討自體抗原存在老化的基因剔除鼠的血清中的原因, 我們比較了老化的野生型以及基因剔除鼠的胸腺表皮細胞以及胸腺基質細胞的特性, 但是沒有發現差異。除此之外, 我們比較年輕野生型老鼠和老化野生型老鼠胸腺表皮細胞以及胸腺基質細胞的特性。在老化野生鼠的胸腺中, 胸腺基質細胞、胸腺髓質表皮細胞、 Aire+ 胸腺髓質表皮細胞數目大量的下降, 但是 胸腺皮質表皮細胞則是上升。這些老化的胸腺表皮細胞MHC-II表現量下降, 但是CD86表現量上升。這些在老化胸腺中的劇烈改變可能影響胸腺的免疫耐受性。由於調節性Ｔ細胞對於免疫耐受性的維持扮演重要的角色, 我們也分析了年輕及老化的野生型老鼠和基因剔除鼠中脾臟調節性Ｔ細胞的所佔比例、細胞數以及Foxp3表現量。在基因剔除鼠和老化的老鼠的調節性Ｔ細胞有較低的Foxp3表現量。
我的研究能幫助更進一步的了解IL-15系統如何藉由影響胸腺表皮細胞進一步調控胸腺細胞的陰性篩選。而在胸腺老化的研究可以幫助我們了解為何在老化的胸腺會出現一些功能上的缺損。

Interleukin 15 (IL-15) is a multi-function cytokine that regulates the activation and homeostasis of memory CD8 T cells, NK cells, NKT cells and intestinal intraepithelial lymphocytes (iIEL). Our lab found that aged Il15rα -/- and Il15-/- mice produce autoantibodies (ANA) and are impaired in thymocyte negative selection. The expression of IL-15 system by thymic epithelial cells (TECs) and dendritic cells (DC) are required for thymocyte negative selection. My project is to characterize TECs and stromal cells of WT, Il15rα -/- and Il15-/- mice, including cellularity, APC (antigen presenting cell)-associated marker expression, and Aire expression. I found that the number of total TECs and mTECs declines in KO thymus but not in WT thymus, while the expression of APC-associated markers and Aire were comparable between WT and KO TECs. The number of CD45- stromal cells and cTECs decreases in Il15-/- thymus. To investigate the ANA arising in aged KO mice, we compared TECs in aged WT and KO thymus, but did not find differences in the examined features. Then, we compare these properties between young and aged TECs and stromal cells. The cell number of stromal cells, mTECs and Aire+ mTECs largely decreases but cTECs increases in aged thymus than that of young thymus. The expression level of MHC-II decreases, but CD86 increases in aged TECs. The enormous changes in aged thymus may influence central tolerance formation. Besides, We also examined the frequency, cell numbers and Foxp3 expression of WT and KO splenic Tregs in young and aged mice, which are associated with the maintainence of tolerance. The expression level of Foxp3 is lower in KO and aged mice than that of WT mice.
My work will advance our understanding in how the IL-15 system regulates thymocyte negative selection via regulating properties of mTECs. The part of aging investigation provides an accurate analysis of TECs in atrophic thymus, which may explain certain dysfunction of aged thymus.

CONTENTS
Contents
Chinese Abstract………………………………………………………………………iv
English Abstract……………………………………………………………………….vi
List of Figures…………………………………………………………………………viii
Chapter 1. Introduction…………………………………………………………….…1
1.1. The development and function of the thymic microenvironment…………..1
1.1.1. The components of the hymic microenvironment…….……..…..1
1.1.2. Central Tolerance.………………………………………...……..1
1.1.3. The crucial role of Aire in the expression of peripheral tissue antigens (PTAs).…………………………………………….….2
1.1.4. Aire is a multi-functional factor………………………………...3
1.2. The IL-15 System.…………………………………………………….……3
1.2.1. IL-15 signal and IL-15 receptors …………...……………….....3
1.2.2. The defects without IL-15 expression.………………..…...……3
1.2.3. IL-15 system and thymus……………………………………….4
1.3. Aged thymus undergoes immunosenescence……………..………………..4
1.3.1. The cellularity of TECs in the aged thymus.……….…………...4
1.3.2. T cells in the aged thymus………………...…………………….5
1.4. Specific Aims……………………………………………………………....5
1.5. Significance…………………………………………………………......…6
Chapter 2. Materials and methods……………………………………………...…….7
2.1. Mice…………………………………………………………………..…….7
2.2. Preparation of thymic cells……..………………………………………….7
2.3. Bromodeoxyuridine (BrdU) treatment and staining……………………….8
2.4. Immunofluorescence and intracellular staining…..……………………….8
2.5. Statistical analysis……………………………………………………….....9
Chapter 3. Results……………………………………………………………………10
3.1. Definition of TEC populations…………….……………………………...10
3.2. The differential release order of thymus subpopulation during enzyme
digestion…………………………………...……………………….……...11
3.3. Comparison of TEC subpopulations among WT, Il15rα−/− and Il15−/− mice at
young and aged stages………………...…………………………………..11
3.4. Proliferation rates of TECs and stromal cells among WT, Il15rα−/− and Il15−/−
mice…………………………………………………………………….....12
3.5. The APC-associated markers of TEC subpopulations are comparable between
WT and KO mice at young and aged stages …………………………........12
3.6. Expression of Aire in WT and KO TECs at young and age stages……….13
3.7. Expression of IL-15 receptors in TECs and stromal cells…......………….13
3.8. The cell number of TECs and stromal cells is reduced in aged thymus…..14
3.9. Analysis of APC-associated markers and Aire expression by TECs of aged
thymus...…………………………………………………………………...14
3.10. Charaterization of Foxp3+-Tregs of WT and KO mice at young and aged
stages……………………………………………………….……………..15
Chapter 4. Discussion…………………………………………………………………16
4.1. The decline of TECs may lead to a dysfunction of Il15rα−/− and Il15−/−
thymus………………………………...……………………………………16
4.2. Both insufficient negative selection and reduced Foxp3 expression in
CD4+Foxp3+ Tregs may lead to tolerance break in KO mice……..………..17
4.3. Thymus atrophy may lead to its dysfunction..……………………………..17
4.4. Lower Foxp3 expression may lead to CD4+Foxp3+ Treg dysfunction in aged
spleen……………………………………………………………………....18
4.5. Il15rα−/− and Il15−/− mice and aged mice share certain phenotypes..…….…19
4.6. Certain surface markers were cleaved during enzymatic digestion of the
thymus………………………………………………………………………19
Chapter 5. Conclusion………….……………………………………………………...20
Chapter 6. Referances ………………………………………………………………....21


List of Figures
Figure 1 Definition of TEC populations……..…………………………………………..26
Figure 2 The differential release order of thymus subpopulation during enzymatic
digestion……………………………………...…………………………………27
Figure 3 Comparison of TEC subpopulations among WT, Il15rα−/− and Il15−/ mice at
young and aged stages…….…………………………………………………...28
Figure 4 Proliferation rates of TECs and stromal cells among WT, Il15rα−/− and Il15−/−
Mice……………………………………………………………………………30
Figure 5 The APC-associated markers of TEC subpopulations between WT and KO mice
at young and aged stages………………….…………………………………...31
Figure 6 Expression of Aire in WT and KO TECs at young and aged stages…………..32
Figure 7 Expression of IL-15 receptors in TECs and stromal cells ……………...….....34
Figure 8 The cell number of TECs and stromal cells decline in aged thymus…………..36
Figure 9 Analysis of APC-associated markers and Aire expression by TECs of aged
thymus…………………………………………………………………………37
Figure 10 Charaterization of Foxp3+-Tregs of WT and KO mice at young and aged
stages……………………………………………….…………………...…....38

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