臺灣博碩士論文加值系統

即使在新世紀的開始，對於細菌性腦膜炎造成的死亡率與神經方面後遺症，依然沒有明顯改善。由於對細菌腦膜炎的系統性免疫機轉的變化，迄今並不完全的清楚, 因此對細菌引發腦膜炎的深入研究有其必要性與急迫性。目前關於感染性疾病免疫機制與疾病狀態探討，以及免疫細胞基因表達與生理功能相關的研究中，哺乳類動物中之小鼠動物模式，關於免疫系統調節與免疫細胞基因表達，提供了最直接且有效的實驗方式。因此為釐清當腦膜炎發生時，系統性淋巴細胞所扮演的角色及其不同時程的表現。我們分別利用購自國家動物中心BALB/c小鼠與本實驗室參與研究成功的免疫雙基因轉殖鼠(T1/T2 double transgenic mice)，探討在動物細菌性腦膜炎模式中，在不同感染時程的免疫系統變化，並以我們培育T1與T2雙基因轉殖鼠的淋巴細胞直接研究 Th1/Th2 的表現。當此鼠體內免疫細胞分泌IFN-時，於細胞表面上會表現hThy1；相反地，若免疫細胞產生IL-4時，則會在細胞表面表現出mThy1.1。為進一步探討流行性感冒嗜血桿菌，在不同來源的菌株引起腦膜炎的動態性系統免疫機制，我們分別用從兒科腦膜炎病人所分離出來的非莢模型流行性感冒嗜血桿菌(invasive nontypeable Haemophilus influenzae, INTHi)與從健康兒童鼻咽部採樣得到的非莢模型流行性感冒嗜血桿菌(colonized nontypeable Haemophilus influenzae, CNTHi)，利用經頭顱內注射法注入INTHi與CNTHi，藉以直接引發細菌性腦膜炎，並以 PBS作為對照組。此外並進一步探討由兒科腦膜炎病人所分離出來的b 型流行性感冒嗜血桿菌 (Hib)，利用相同的實驗方法直接引發 Hib細菌性腦膜炎，並與上述各組加以比較。
依此動物模式裏，綜合以上列使用的三種菌株中，所引發小鼠的細菌性腦膜炎中，分析其臨床症候包括死亡率、疾病嚴重度與腦組織腦脊膜發炎程度做比較。依時程比較，我們發現各實驗組的臨床症狀最嚴重的時間，皆發生在顱內注射細菌後第三天。尤其是在 Hib 細菌性腦膜炎的臨床症狀，比其他二組NTHi 細菌性腦膜炎更為嚴重。另外進一步分析這些老鼠在感染後其脾臟的淋巴細胞， 在不同時間點的變化包括脾臟淋巴細胞總數，以及各種主要淋巴細胞亞型的比例差異。在Hib 細菌性腦膜炎的小鼠中，分析其脾臟的總淋巴數，比其餘二實驗組來得偏低。此外由CNTHi 引發腦膜炎的小鼠分析其脾臟CD4+淋巴細胞有較高的比例表現，相對在B淋巴細胞其所佔的比例明顯降低。而在CD8+淋巴細胞方面，三組在各評估時間點，皆有偏高比例的表現趨勢。關於轉殖基因的表現方面，雖三組實驗組在感染第三日，CD4+hThy1+(Th1)淋巴細胞皆有明顯表現，但在感染後第七日在 CNTHi、INTHi與Hib，則依序漸減。雖CD4+mThy1.1+ (Th2)感染後的表現並不強，但仍有差異性。反之在感染第三日Th2 淋巴細胞以Hib這組最明顯，CNTHi這組最弱，而INTHi這組則介於二者之間。但在感染後第十四日不論Th1或Th2 淋巴細胞在CNTHi、 INTHi或Hib，皆很難偵測到了。最後我們分別萃取三種菌株的細胞膜蛋白做特別抗原淋巴細胞反應檢測，分析各實驗組的淋巴細胞經抗原刺激後的反應並收集培養之上清液，加以分析第四血球間白素(IL-4, interleukin-4)與丙型干擾素(Interferon-γ)的濃度。我們發現 Hib 有最強 的IL-4 表現，而IFN-γ則以 CNTHi表現較強。
綜合上述Hib感染的小鼠除有較嚴重的臨床症狀外，並有最明顯Th2的傾向包括在感染後第三與第七日；而NTHi，尤其是由健康兒童鼻咽腔分離出來的CNTHi 引發腦膜炎不但症狀較輕微，而且有Th1的傾向。本論文除了建立研究流行性感冒嗜血桿菌的小鼠腦膜炎實驗模式外，更進一步了解菌株本身具莢膜特性與否以及來源性質的差異，而表現出不同Th1/Th2動態性的變化。 因此藉由此研究模式，希冀對腦膜炎的免疫機制有更深入的了解，並且對未來疾病的預防與新型疫苗開發 提供有效的方針與對策。

To investigate the immunopathogenic mechanism of Haemophilus influenzae meningitis, we established an experimental meningitis model and elucidated the immune responses in T1/T2 doubly transgenic mice based on BALB/c mice background. These mice carry two transgenes that express two distinct cell-surface markers: a human Thy1 transgene (hThy1) under the control of the murine IFN-γ promoter, and a murine Thy1.1 transgene (mThy1.1) under the control of the murine IL-4 promoter, designated T1 and T2, respectively. These mice were infected with variant strains of H. influenzae including invasive and colonized nontypeable H. influenzae (NTHi). Moreover, mice inoculated with H. influenzae type b (Hib) were dissected for comparison. We disclosed mice infected with Hib, invasive NTHi (INTHi) or colonized NTHi (CNTHi) revealing most severe, moderate and less mild in disease severity in terms of clinical score, mortality rate and histopathology of brain respectively.
In addition, mice infected with Hib displayed severest symptoms and lowest total splenocyte counts on day 3 after infection. Simultaneously, we examined the significantly low percentage of CD19+ B cells, the relatively high level of CD4+ T cells and significantly high percentage of CD8+ T cells in Hib-infected mice.
We observed a rapid induction of the Th1 response, since the IFN-γ-producing CD4+ T cells significantly increased in all infected T1/T2 doubly transgenic mice. Furthermore, T1/T2 doubly transgenic mice infected with CNTHi expressed the most significant Th1 response, in terms of preferential increase of CD4+ T cells, as well as the highest percentage and longest maintenance of Th1 cells. In contrast, in transgenic mice infected with invasive Hib, the Th1/Th2 paradigm shifted to Th2 response. On day 7 after infection, the Th1 response gradually declined and the Th2 response rather sustained in Hib infected mice. This was due to a significant increase in IL-4-producing CD4+ T cells, accompanied by a rapid decline of Th1 cells. Mice infected with INTHi revealed intermediate Th1/Th2 responses between those of mice infected with CNTHi and Hib. Two weeks after infection, both Th1 and Th2 cells were barely detectable. Moreover, we demonstrated using an antigen-specific re-stimulation test to analyze the effector function of lymphocyte subsets that CD8+ T cells contributed to more predominantly production of IFN-γ than CD4+ T cells did; and CD4+ T cells partly contributed to the secretion of IL-4 from flowcytometry of intracellular cytokine staining in Hib infected mice. In conclusion, the preferential Th1/Th2 trend in H. influenzae meningitis is correlated with clinical severity as well as characteristics of the pathogens themselves.Our results support that these transgenic mice provide an available model to dissect the complex kinetic change of adaptive immunity in bacterial infectious diseases.

目錄
頁次
目錄 ……………………………………………………………………I
圖目錄 …..……………………………………………………..……VII
英文縮寫表 ……………….…………………………………………XI
中文摘要 …….…………………..……………………….…………XII
英文摘要 ……..……………………………………………………XIII

第壹章 緒論…………………….…..…………………1
一、 細菌性腦膜炎…………………….……..……….………1
二、 關於流行性感冒嗜血桿菌………………..…..…….…2
三、 細菌腦膜炎的系統性免疫機轉………….…..…….…3
四、 輔助性T淋巴細胞之功能…………..……..…………4
五、 Th1/Th2 於細菌性感染的角色…………....……...…5
六、 T1/T2雙基因轉殖鼠之運用……………..……………6
七、 實驗目標……...….….………….……..…..………………6

第貳章 探討b型流行性感冒嗜血桿菌引起腦膜炎的系統動態性調解型免疫機轉……...……….……….…8
一、 前言……………..…….………….……..…….………….…8
二、 材料與方法………..………….……...…….………….…9
1. 實驗材料與設備………………….……….…….………….…9
(1) 試劑.…..………...……..…….…..….…....………..……….…9
(2) 藥品…..………...……..…………….…...…….………….…10
(3) 培養液..………...……..……..………….……......……….…11
(4) 儀器設備……...……..………………….…….....……..……11
2. 實驗動物與T1/T2雙基因轉殖小鼠之篩選…….…..………12
3. 電泳分析……..………………….……....……….………...…13
4. 實驗菌株配置……......…….…….……....…….….……….…13
5. Hib小鼠腦膜炎實驗模式..……...……....…….…….…….…14
6. 臨床疾病嚴重度之評估……..….……....……..….……….…14
7. 腦組織切片檢查…..……..…………………….….……….…15
8. 淋巴細胞的製備…..….….………………………..……….…16
9. 以流式細胞儀分析細胞表面抗原之表現…………..…….…17
10. 酵素連結免疫吸附分析….………………….……..,…...…18
11. 專一抗原細菌外層膜蛋白製備…….………..….……….…19
12. 蛋白質濃度之測定….….…………………….…..…………19
13. SDS-PAGE電泳分析方法………………….……..….….…20
14. T1/T2雙基因轉殖小鼠後脾臟淋巴對於菌株專一抗原產生細胞增生之實驗…………………….………..………...….….…21
15. 細胞內細胞素(IL4, IFN-γ)染色……….……….….….…22
三、實驗結果……………………….………………....….….…24
1. 動物實驗…….……………….………………..........….….…24
2. 小鼠Hib腦膜炎的臨床表徵………….……….....…..…..…26
3. 小鼠Hib腦膜炎的腦組織切片病理變化…………….….…28
4. 以流式細胞儀偵測在不同時間點實驗小鼠脾臟細胞的各種淋巴細胞亞型之觀察分析…….……………….........…..…..…30
5. 以流式細胞儀偵測在不同時間點Hib感染小鼠脾臟的CD4+T淋巴細胞之觀察分析轉殖基因hThy1+/mThy1.1+(T1/T2)的表現……………………………………………………………32
6. T1/T2雙基因轉殖小鼠後脾臟淋巴對於菌株專一抗原產生細胞增生之實驗………...…………….….........………….….….…35
7. 脾臟淋巴細胞對特異抗原刺激產生Th1/Th2細胞素表現，藉酵素免疫分析法偵測……….....………....…………….…..…37
8. 以流式細胞儀偵測在經培養脾臟淋巴細胞對特異抗原刺激產生細胞內IL-4 與IFN-γ細胞素表現……………….…...…39
四、討論………………..……………….........…………….…..…41

第三章 探討非莢模型流行性感冒嗜血桿菌引起腦膜炎的系統動態性調解型免疫機轉..…………………..43
一、 前言………….……..…….………….……..……………….…43
二、 材料與方法…………………….……..…….……..…….44
1. 實驗材料與設備………..………...……….….….……..….…44
(1) 試劑.…..………...……..…….…………..…….…..……..….44
(2) 藥品…..………...……..…….…………..…………..…….…45
(3) 培養液..………...……..……..………….……......….………46
(4) 儀器設備……...……..………………….…….....…..…..…..46
2. 實驗動物與T1/T2雙基因轉殖小鼠之篩選……..…….……47
3. 電泳分析……...……..………………….……....….….…...…48
4. 實驗菌株配置……...……..……….…….…….….…….….…48
5. NTHi小鼠腦膜炎實驗模式..……….…..…….….…….….…49
6. 臨床疾病嚴重度之評估…………..…….…….….…….….…49
7. 腦組織切片檢查…..……….…….…………….……….….…50
8. 淋巴細胞的製備…..……….…….…………….……….….…51
9. 以流式細胞儀分析細胞表面抗原之表現……….…….….…52
10. 酵素連結免疫吸附分析Enzyme-linked Immunosorbent Assays (ELISAs)………………………………….….……….…53
11. 專一抗原細菌外層膜蛋白製備………………………….…54
12. 蛋白質濃度之測定….………………………………………54
13. SDS-PAGE 電泳分析方法………..…………….….….…..55
14. T1/T2雙基因轉殖小鼠後脾臟淋巴對於菌株專一抗原產生細胞增生之實驗…………………………..…………...….….…56
三、實驗結果……………………..….………………....….….…58
1. 動物實驗……….…………….………………..........….….…58
2. 不同來源NTHi的菌株引發小鼠腦膜炎的臨床表徵….….60
3. 小鼠感染NTHi (INTHi 與CNTHi)腦膜炎的腦組織切片病理變化.……………………………….…………………........…62
4. 以流式細胞儀偵測在不同時間點實驗小鼠脾臟細胞的各種淋巴細胞亞型之觀察分析…………………….….…………....64
5. 以流式細胞儀偵測在不同時間點NTHi感染小鼠脾臟的CD4+T淋巴細胞之觀察分析轉殖基因hThy1+/mThy1.1+(T1/T2)的表現………………………………………………………..…67
6. T1/T2雙基因轉殖小鼠後脾臟淋巴對於菌株專一抗原產生細胞增生之實驗…………………….………………........….….…69
7. 脾臟淋巴細胞對特異抗原刺激產生Th1/Th2細胞素表現，藉酵素免疫分析法偵測……………………………….………...72
四、討論…………………..……………….........…………….…..…74
第肆章 總結………..……….........…………….…..…74
參考文獻………………..………………...............…………….…..…87

附錄一、與本論文研究有關之個人著作及發表

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