臺灣博碩士論文加值系統

雙極性情感性躁鬱症是一個非常普遍的精神疾病，在美國大約有1.3-1.5％的人口罹患。它的症狀包含憂鬱和躁症兩個時期。鋰鹽和valproic acid為治療雙極性情感性躁鬱症的首選藥物，但他們的作用機轉並不清楚。在神經系統上，鋰鹽可以保護神經系統及防止凋亡；在免疫系統上，雙極性情感性躁鬱症服用情緒穩定藥的病人，被檢測出較容易罹患自體免疫病人，而服用鋰鹽治療的病患罹患的機率比較小。因此，我們有興趣研究鋰鹽在免疫系統之鑰—樹突狀細胞分子及功能上所扮演的角色。自人類周邊血液分離出單核球，將之培養七天成未成熟樹突狀細胞，並在培養第一天便給予不同濃度的鋰鹽。實驗發現給予鋰鹽2.5-10 mM可有意義的增加樹突狀細胞CD86和CD83的表現約2-3倍。但對其他樹突狀細胞表面記號如HLA-DR, CD80, CD40和CD14並無影響。鋰鹽刺激的未成熟樹突狀細胞與控制組比起來有意義的增加IL-6, IL-8, IL1-β和TNF-α的分泌約6-30倍。接下來我們研究經由鋰鹽刺激過的樹突狀細胞它刺激異體淋巴球的影響，發現鋰鹽對於異體淋巴球的增生並沒有影響。在T細胞的分化階段，我們測量細胞激素發現鋰鹽對於T細胞的分化與控制組沒有太大的不同。更進一步地，我們研究了它的訊號傳遞機制，給予Glycogen Synthase Kinase -3β (GSK-3β)抑制劑 (SB415286, SB216763, GSK-3β inhibitor I 和GSK-3β inhibitor VII)及mitogen- activated protein kinase kinase (MEK)抑制劑(PD98059)後發現樹突狀細胞表面記號並沒有顯著差異。反之，在給予phosphatidylinositol-3-kinase (PI3K)抑制劑LY294002後發現樹突狀細胞表面記號CD83和CD86有意義顯著降低。這些實驗數據顯示鋰鹽在調控樹突狀細胞的功能和免疫調節中可能扮演重要的角色。

Bipolar disorder is a common psychiatric disease in the world, its symptoms include alternating depressive and manic episodes. It affects 1.3-1.5％ of population in the U.S. lithium (Li) and valproic acid have been chosen as the first line medicine for bipolar disorder. However, their mechanisms remain unclear. Lithium could protect and prevent apoptosis of neuron cells. Patients with bipolar disorder have higher prevalence of autoimmune disease, but patients treated with LiCl have lower incidence. Therefore, we are interested in the effect of lithium on the functions of dendritic cells (DC), which is a key regulator of immune responses. Peripheral blood mononuclear cell (PBMC) derived DCs were generated and treated with lithium for examination of change of surface molecules, mixed lymphocyte reaction and cytokine production. We found that lithium could significantly increase the expression of CD86 and CD83 expression on DCs to 2-3 folds as compared to control. Furthermore, IL-8, IL-6, IL-1β and TNF-α secretion were increased by lithium approximately 6-30 folds. We found that lithium had no effect on allogeneic T lymphocytes proliferation in a mixed lymphocyte reaction. To assay of culture supernatant for T cell polarization, we found that no apparent secretion of Th1 or Th2 cytokines was observed. DC Treated with GSK-3β inhibitors (SB415286, SB216763, GSK-3β inhibitor I and GSK-3β inhibitor VII) and mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059) did not alter the profiling of surface markers expression in DCs. In contrast, phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 significantly decreased the expression of CD86 and CD83 in DCs. These data suggest that lithium could modulate DC functions, and might have potential role in immunomodulation.

中文摘要.............................................................................................................. I
英文摘要..................................................................................................... II
圖表目次..................................................................................................... i
附圖目錄......................................................................................................
附錄目錄..……………………………………………………………….……… ii
iii
縮寫表..........................................................................................................
前 言...................................................................................................... a
1
實驗目的..............................................................................................................
實驗材料與方法..................................................................................................
1. 常用溶液..............................................................................................
2. 樹突狀細胞培養..................................................................................
3. 藥物處理..............................................................................................
4. 流式細胞儀分析樹突狀細胞表面分子..............................................
5. 異體混合淋巴球反應試驗..................................................................
6. 酵素連結免疫吸附分析試驗..............................................................
7. 細胞激素磁珠分析...............................................................................
8. 統計方法.............................................................................................. 6
7
7
8
9
9
10
11
12
13
實驗結果.............................................................................................................. 14
1. 鋰鹽可增加單核球分化成未成熟樹突狀細胞聚集現象...................
2. 鋰鹽可以增加單核球分化成未樹突狀細胞CD86之表現................
3. VPA增加單核球分化成未樹突細胞聚集現象...................................
4. VPA無法增加單核球分化成未樹突狀細胞表面分子.......................
5. 鋰鹽對於樹突狀細胞的影響在於未成熟樹突狀細胞分化階段.......
6. 鋰鹽可以增加單核球分化成未樹突狀細胞IL-6, IL-8, TNF-, IL-1β之表現.........................................................................................
7. VPA可增加IL-6之表現，但抑制IL-8、TNF- 之表現...............
8. 鋰鹽刺激過的未成熟樹突狀細胞並不影響異體T淋巴球增生.......
9. 鋰鹽刺激T細胞分化趨向於Th0........................................................
10. PI3K在鋰鹽增加樹突狀細胞表面分子中所扮演的角色..................
11. MEK在鋰鹽增加樹突狀細胞表面分子中所扮演的角色.................
12. GSK-3在鋰鹽增加樹突狀細胞表面分子中所扮演的角色............. 14
14
15
16
16

17
18
19
19
19
21
21
討 論............................................................................................................... 23
附 圖............................................................................................................... 27
附 錄.............................................................................................................. 44
參考文獻............................................................................................................... 49


附圖目錄
Fgure 1. Morphology of iDC with different concentration of LiCl........... 27
Figure 2. Effects of LiCl on the generation of monocyt -derived iDC....... 28
Figure 3. Morphology of iDC with different concentration of VPA.......... 30
Figure 4. Effects of VPA on the generation of monocyt-derived iDC........ 32
Figure 5. Effects of LiCl on the maturation of iDC.................................... 33
Figure 6. LiCl enhanced cytokine production on iDCs.............................. 34
Figure 7. Effects of VPA on IL-6, IL-8 and TNF- production................. 35
Figure 8. Effects of LiCl on allogeneic T cell proliferation by MLR......... 36
Figure 9. Effects of LiCl on the capacity of iDC to promote Th1-Th2 differentiation………………………………………………......
37
Figure 10. Roles of PI3K pathway in PBMC-derived DC differentiation by LiCl treatment........................................................................
38
Figure 11.

Figure 12. Roles of MEK pathway in PBMC-derived DC differentiation by LiCl treatment........................................................................
Roles of PI3K pathway in PBMC-derived DC differentiation by LiCl treatment........................................................................
40

42





附錄目錄
Appendix 1. The process of dendritic cell maturation.................................. 44
Appendix 2. Features that changes during DC maturation........................... 45
Appendix 3. Characterization of the phenotype of the monocyte-deriverd DC via CD marker expression.................................................
46
Appendix 4. Difference of inhibitor and effect target sites........................... 47
Appendix 5 A proposed scheme for neuroprotective effects of lithium...... 48

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