臺灣博碩士論文加值系統

感染寄生蟲時嗜伊紅性白血球(eosinophils)為具有殺死寄生蟲幼蟲能力的作用細胞(effector cells)之一。目前由於免疫反應在體外的研究，嗜伊紅性白血球的來源，大多以寄生蟲感染實驗動物誘發產生嗜伊紅性白血球的模式來取得所須的嗜伊紅性白血球，但寄生蟲的生活史在實驗室維持不易，而使得利用非寄生蟲外來藥物或試劑，誘發宿主產生嗜伊紅性白血球的發展值得探討。因此在本研究中，我們試圖在C57BL/6(B6)鼠注射環磷醯胺(cyclophosphamide)的模式下合併注射卵蛋白素(ovalbumin)及副腎皮質素(adrenal cortex hormones)後，觀察對環磷醯胺的加成或抑制作用。
環磷醯胺在調節免疫反應方面，主要是扮演著免疫抑制的角色；它作用於抑制性T淋巴細胞(T-suppressor cells)族群的分化及降低B淋巴細胞活化等等。另一方面，它則是在臨床醫學上常適用於治療癌症、腫瘤的藥物。
環磷醯胺經腹腔(intraperitoneal)注射鼷鼠後，會引發實驗動物造成血液中嗜伊紅性白血球增加(eosinophilia)，所以在我們實驗中發現，B6鼠經注射環磷醯胺後的十到十四天，血液中與骨髓內的嗜伊紅性白血球數量比未注射的B6鼠明顯有增加，但是白血球之總量卻是稍微的明顯減少。
另外，我們利用酵素連結免疫吸附法(ELISA)去偵測促嗜伊紅性白血球生成激素(eosinophilogenic cytokines)；白血球間質-3 (IL-3)、白血球間質-5 (IL-5)及顆粒性-巨嗜性族群刺激因子(GM-CSF)的濃度變化情形，由結果發現，在注射環磷醯胺後的B6鼠，其脾臟細胞培養液經培養後，在第十天開始促嗜伊紅性白血球生成激素的濃度已有明顯上升，持續到十八天後才下降，而頸部淋巴結細胞培養液中則有白血球間質-3 (IL-3)上升。另外，合併卵蛋白素經皮下(subcutaneously)注射後，發現在血液中及骨髓內的嗜伊紅性白血球數量則更為促進性增加，相對其促嗜伊紅性白血球生成激素的濃度，也是比對照組明顯的上升，而且白血球的數量也稍微增多，但發現在注射副腎皮質素後，血液中及骨髓內的嗜伊紅性白血球數量卻是比注射卵蛋白素的B6鼠明顯減少，相對其促嗜伊紅性白血球生成激素的濃度也降低，並且與對照組沒有明顯差異性。
本研究結果顯示：在實驗B6鼠經注射環磷醯胺藥物後，其體內免疫反應造成白血球數量稍微減少，但是促嗜伊紅性白血球細胞激素的濃度卻有明顯上升，而在合併注射卵蛋白素的B6鼠，其促嗜伊紅性白血球細胞激素更有加成效應性的反應上升，卻發現在注射副腎皮質素的B6鼠，因拮抗而降低此免疫反應作用。總結以上之結果，B6鼠經合併注射環磷醯胺及卵蛋白素可以刺激促嗜伊紅性白血球生成激素的分泌並促進嗜伊紅性白血球的分化及成熟。

Eosinophil is one of important effector cells in the immune responses to the infection of parasites. Eosinophils were usually obtained from experimental animals by inoculation of parasites for an in vitro study on this field. However, it is still unconvinient to maintain the life cycle of parasites for the inoculation purpose. Thus, it is worth to develop a non-parasitic infection model to evoke the production of eosinophils in experimental animals. It is known that the injection of cyclophosphamide (CY) in experimental animals can elevate the concentration of eosinophils in peripheral blood. In this study, eosinophilogenic cytokines will be assayed in C57BL/6 (B6) mice after injection with CY to understand the mechanism of production of eosinophils by this drug. In addition, the effect of CY on the production of eosinophils promoted by ovalbumin (OVA) and suppressed by adrenal cortex hormones (ACH) will be probed.
CY is a cytotoxic drug that effect the differentiation of T-suppressor cells and inhibit the activation of B lymphocytes profoundly. Besides, it is also used to treat many inflammatory and malignant diseases. CY is known to produce a marked eosinophilia in mice after intraperitoneal challenge. Indeed, a marked eosinophilia was observed in blood and bone marrow of B6 mice during 10 to 14 days after injection with CY in this study, even if the total count of w.b.c. significant decreased than that of untreated control mice.
On the other hand, eosinophilogenic cytokines including IL-3, IL-5 and GM-CSF in culture medium of spleen cells and cervical lymphoid cells of B6 mice were assayed for dynamic changes by ELISA technique with commercial kits in this study. Those three eosinophilogenic cytokines in the culture medium of spleens from B6 mice injected with CY showed significant increase after injection and then gradually returned to normal levels. However, significant increase was only observed on IL-3 in the culture medium of cervical lymphoid cells.
In B6 mice subcutaneously injected with CY and OVA at the same time, higher concentration of eosinophils in blood and bone marrow were observed than those of mice singly injected with CY. All eosinophilogenic cytokines in spleen cells and cervical lymphoid cells from B6 mice of this group showed synchronously increase as that of eosinophils in blood and bone marrow. On the contrary, lower number of eosinophils in blood and bone marrow as well as eosinophilogenic cytokines in culture medium of spleen cells and cervical lymphoid cells were obtained from B6 mice simultaneously injected with CY and ACH. Thus, to induce eosinophilogenic cytokines in B6 mice and then to promote the proliferation of eosinophil colony in bone marrow and maturation in peripheral blood can be achieved by injection with CY especially combined with OVA and this model can recommended to replace a traditional parasite infection model.

目 錄
中文摘要………………1
英文摘要.………………4
緒 論…...………………6
材料與方法………….12
Ⅰ. 實驗動物…………..13
Ⅱ. 藥物的製備……………..…13
(1) 環磷醯胺……………….…….………..……………13
(2) 卵蛋白素………………..…..………………………13
(3) 副腎皮質素………………...…………………….…14
Ⅲ. 實驗動物體內試驗……………………………………14
A. 藥物的注射…..………...……………………………14
B. 血清的收集.……………...….…….………………...15
C. 血液中白血球的計算與分類………....…………....16
D. 骨髓內白血球的計算與分類…..…..….……….…..16
E. 脾臟與頸部淋巴結細胞的收集…..………..……....17
F. 脾臟與頸部淋巴結細胞培養液的製備…..……..…18
G. 細胞激素的測定………..…………………..……....19
(1) 白血球間質-5……………..…………………........19
(2) 白血球間質-3………………..………………..…..20
(3) 顆粒細胞-巨嗜細胞族群刺激因子…...………….21
H. 統計與分析….………………….………….………..21
結果………………………………...………….…….…….…22
一. 週邊血液中之白血球總量……………...……………23
二. 週邊血液中之嗜伊紅性白血球總量….………..……24
三. 週邊血液中之嗜中性白血球總量….……..…………24
四. 週邊血液中之淋巴球總量….………………..………25
五. 骨髓內之白血球總量…….…………….…...…….….26
六. 骨髓內之嗜伊紅性白血球總量……….…. …………26
七. 骨髓內之嗜中性白血球總量….…………………..…27
八. 骨髓內之淋巴球總量………….…………….…….…28
九. 實驗B6鼠脾臟細胞的細胞激素之測定….……..….28
(1) 白血球間質-5………….………..………….…..…..28
(2) 白血球間質-3……………………………...….……29
(3) 顆粒細胞-巨嗜細胞族群刺激因子..………...…....30
十. 實驗B6鼠頸部淋巴結細胞的細胞激素之測定……31
(1) 白血球間質-5……………………….….……..…....31
(2) 白血球間質-3………………………………..……..31
(3) 顆粒細胞-巨嗜細胞族群刺激因子…..…………...32
十一.實驗B6鼠注射不同藥劑後其血液中之嗜伊紅性白血球總量與脾臟細胞之細胞激素的關係…………….33
十二.實驗B6鼠注射不同藥劑後其血液中之嗜伊紅性白 血球總量與頸部淋巴結細胞之細胞激素的關係….33
討論………………….……………………...……………......35
附圖……………………...…….…….……………………….42
環磷醯胺之化學結構式…………………………….…..….43
參考文獻………………..……………………………..….…66

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