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研究生:郭懿瑩
研究生(外文):Yih-Ying
論文名稱:第二型轉麩胺酶在吞噬過程中所扮演的角色之研究
論文名稱(外文):The role of transglutaminase 2 in phagocytosis
指導教授:蔡嘉哲蔡嘉哲引用關係
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:免疫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:48
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自體免疫疾病的致病機轉到目前為止仍然不是很清楚,而凋亡小體無法被正確的辨識及清除一直被認為是造成自體免疫疾病的主要原因。因此,研究自體抗原的產生以及分析影響吞噬作用及清除凋亡細胞的因子,是本實驗主要的研究動機。Transglutaminase 2 (TG2)是一種多功能酵素,能連結催化仰賴鈣離子的反應而造成蛋白質轉譯後修飾,最近的研究發現,TG2是參與細胞凋亡的一個重要成員,甚至影響巨噬細胞辨識及清除凋亡小體。
實驗中利用UVB 1650 J/m2照射人類角質細胞株 (HaCaT) 製備凋亡小體,觀察細胞形態,發現細胞質皺縮,細胞表面出現小泡 (blebs) ,隨時間點增加細胞呈現懸浮態,由DNA ladder的實驗發現UVB 1650 J/m2作用12小時後,染色質出現DNA片段化現象,以流氏細胞儀分析UVB作用後24小時發現有56.2 % 細胞進行細胞凋亡。TG2的mRNA則在UVB 1650J/m2處理後1小時減少,之後隨時間增加,在12小時達到最高點,於24小時後再度減少。進一步分析吞噬過程中巨噬細胞吞噬能力和TG2之表現,結果發現,吞噬螢光乳膠微珠 (latex-beads) 時吞噬指數 (phagocytosis index ; PI) 和吞噬指數 (phagocytosis ratio ; PR) 在各時間點皆小於5 %,且TG2 mRNA無顯著差異;吞噬apoptotic cells時,phagocytosis index於4小時達到最高(PI=43.5%),phagocytosis ratio則隨著時間增加直到6小時(PR=43.5%),且TG2 mRNA於4小時達到最高點,顯示凋亡小體會刺激巨噬細胞中TG2的表現。Recombinant TGF-β (rTGF-β1) 與吞噬作用關係,研究結果發現 (1) rTGF-β1於0~20ng/ml劑量下不會造成細胞凋亡,(2) rTGF-β1會增加巨噬細胞中TG2 mRNA表現,其表現並與rTGF-β1劑量成正比 (p<0.05), (3) rTGF-β1的劑量會增加phagocytosis ratio (p<0.05),但rTGF-β1對於吞噬作用時TG2 mRNA的表現無顯著差異。我們的結論認為凋亡小體會刺激巨噬細胞中TG2的表現。另外rTGF-b1可增加TG2的表現且增加phagocytosis ratio,並可能抑制凋亡小體誘發TG2產生的生物功能,全身性紅斑性狼瘡的致病機轉可能是其巨噬細胞對apoptotic cells無反應,使得TG2沒有增加而吞噬能力下降,導致清除凋亡細胞的能力下降。

The mechanism of autoimmune disease remains unclear. Moreover, the deficiency of phagocytic cells to recognize and clear apoptotic body is a main factor of the induction of autoimmune responses. Therefore, it becomes important to study the mechanism involved in the production, recognition and clearance of apoptotic cells. Transglutaminase 2 (TG2) is a multifunctional enzyme with the function of cross-linking Ca2+-dependent reactions which result in post-translational modification of substrate proteins. Results from several independent laboratories indicated that TG2 plays a potential role in cell apoptosis and also in the clearance of apoptotic body. In this study, we investigated the role of TG2 in the clearance of apoptotic cells in RAW264.7. After keratinocytes were irradiated with UVB 1650 J/m2, cell apoptosis was analyzed by light microscopy, DNA fragmentation and flow cytometry. We observed the structure of small surface blebs in keratinocytes, and condensation of nuclear chromatin. DNA fragmentation was observed at 12h and 24h after keratinocytes irradiation. The cells at sub-G1 phase (56.2%) were detected at 24h by FACS. After irradiation, TG2 mRNA level was declined at 1hr, and increased at later time points (6-12h), but was repressed after 12 h. In the experiment of phagocytosis, it was found that RAW264.7 had higher ability to ingest apoptotic cells than latex-beads. TG2 mRNA level was also higher in phagocytosis with apoptotic cells than with latex-beads. The roles between TGF-β1 and TG2 expression in phagocytosis were also further studies. Our results showed that rTGF-β1 did not induce apoptosis of RAW264.7 at concentration of 0-20 ng/ml,and rTGF-β1 was able to increase the level of TG2 expression in a dose -dependent way in RAW264.7 cells. Overall, our results demonstrated that apoptotic cells and rTGF-β1 could induce TG2 expression and increase phagocytosis with apoptotic cells in RAW264.7 cells. The defect response of apoptotic cells to phagocytic cells following impairment of phagocytosis and clearance of apoptotic cells may play a role in the pathogenesis of systemic lupus erythematosus.

壹、中文摘要............................................................................................1
貳、英文摘要···························································································3
參、前言···································································································5
一、細胞凋亡···················································································6
二、細胞凋亡與吞噬作用、抗原清除在自體免疫疾病中所扮
演之角色···················································································6
三、組織型轉麩胺脢tissue Transglutaminase (tTG)······················9
肆、研究動機·························································································11
伍、實驗材料及方法
一、材料
1. 材料及藥品··································································12
2. 細胞株來源··································································12
二、方法
1. 細胞培養······································································13
2. UVB誘發細胞凋亡·····················································14
3. DAPI 螢光染色···························································15
4. 細胞凋亡DNA片段化實驗········································15
5. 流式細胞儀分析··························································16
6. RNA製備·····································································16
7. 反轉錄聚合酶反應(Reverse transcription ···············17
Polymerase chain reaction;RT-PCR)
8. 凋亡細胞染色及螢光顯微鏡觀察······························18
9. 吞噬實驗······································································19
10. 吞噬指數(phagocytosis index)與吞噬比例
(phagocytosis ratio)················································19
11. Recombinant Human TGF-β1的製備························20
陸、實驗結果
一、UVB能量1650 J/m2照射誘發細胞凋亡································21
二、分析人類角質細胞株經UVB照射引起細胞凋亡,其
細胞之TG2 mRNA的表現···················································24
三、觀察經UVB 1650J/m2 照射人類角質細胞後之上清液對
巨噬細胞型態的影響及巨噬細胞中TG2 mRNA的表現····25
四、利用螢光顯微鏡觀察老鼠巨噬細胞株(RAW264.7) 吞
噬Latex-Beads及凋亡細胞的情形及吞噬能力···················26
五、以RT-PCR分析老鼠巨噬細胞株(RAW264.7)吞噬
latex-beads及凋亡小體時,巨噬細胞之TG2 mRNA的
表現·························································································29
六、分析老鼠巨噬細胞株在經由不同劑量的重組型TGF-β1
處理下對細胞的影響·····························································31
柒、討論·································································································33
捌、圖表
圖一、UVB能量1650 J/m2照射誘發細胞凋亡····································39
圖二、RT-PCR分析人類角質細胞株經UVB照射引起細胞凋亡,
其細胞之TG2 mRNA的表現···················································40
圖三、觀察經UVB 1650J/m2 照射人類角質細胞後之上清液對
巨噬細胞型態的影響及巨噬細胞中TG2 mRNA的表現········41
圖四、利用螢光顯微鏡觀察老鼠巨噬細胞株(RAW264.7) 吞噬
Latex-beads及凋亡細胞的情形及吞噬能力·····························42
圖五、以RT-PCR分析老鼠巨噬細胞株(RAW264.7)吞噬latex-
beads及凋亡小體時,巨噬細胞之TG2 mRNA的表現··········43
圖六、分析老鼠巨噬細胞株經不同劑量的重組型TGF-β1處理下
對細胞的影響·············································································44
玖、參考文獻·························································································45

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