臺灣博碩士論文加值系統

蛋白質雙硫異構? (protein disulfide isomerase，PDI) 家族具有氧化還原蛋白雙硫鍵的酵素，幫助新合成蛋白質的cysteine形成雙硫鍵以及幫助蛋白質正確的摺疊，也具有伴隨蛋白 (chaperone) 的功能，主要存在於內質網中，部分分布於細胞表面。
在哺乳類中，9個PDI家族成員擁有1到3個CGHC活化中心，Pdia4是唯一擁有3個CGHC 活化中心，過去文獻指出Pdia4參與內質網未摺疊蛋白質的機制；扮演鈣離子的伴隨蛋白；肝癌細胞轉移能力越好時，發現Pdia4的表現量提高；卵巢癌細胞在ionophore處理後，造成內質網壓力，Pdia4表達會增加。然而， Pdia4的生物功能卻不清楚。
遺傳基因學策略，研究基因功能被認為是最好的方式之一，但操作基因需要有適當的基因轉殖系統，本篇論文利用病毒載體，因為病毒載體可長時間表達轉殖基因。首先建立反轉錄與慢病毒載體系統，先比較兩者的感染特性，確立可生產有效價數高的病毒顆粒，再比較兩種病毒載體對不同細胞株的向性及感染率。由Jurkat T細胞有好的感染率，並表達高量Pdia4，因而利用慢病毒載體來研究Pdia4在T細胞的功能。
利用慢病毒載體帶有Pdia4干擾RNA (KD) 與空的慢病毒載體 (GK) 來感染Jurkat T細胞，流式細胞儀偵測發現感染KD與GK的細胞皆有相似的感染效率和平均螢光強度 (MFI) ；利用西方點墨法 (western blotting) ，相對於GK細胞，KD細胞約減少了80 % 的Pdia4 蛋白質表現量。這證明基因轉殖系統成功。
Pdia4的一個重要功能可能是細胞生長，進一步利用KD與GK細胞來探討Pdia4對Jurkat T 細胞生長的影響。計數細胞觀察到Pdia4減少會抑制Jurkat T 細胞的生長，但不影響死亡；AlexaFluor 647 succinimidyl ester標定細胞分裂實驗指出Pdia4減少會減緩細胞的分裂，這證據與高GFP強度的細胞減少較快一致。然而，Pdia4對TCR或Fas引發T細胞的死亡似乎參與不多。
所有初步實驗結果發現Pdia4影響T細胞生長，可能是透過調節T細胞分裂。進一步Pdia4基因的功能需被探討。

Protein disulfide isomerases (PDIs) constitute a family of enzymes which catalyze oxidation and reduction of disulfide bonds during the formation of newly synthesized proteins and help protein folding. PDIs also act as chaperone and most of the PDIs are located in the lumen of the endoplasmic reticulum (ER), but some PDIs are located on other cellular compartments.
In mammals, there are 9 PDI members with CGHC homology domains. They are involved in a broad range of cell functions. Among them, Pdia4 is the only one PDI member with 3 CGHC domains. Pdia4 was reported to be implicated in the unfolded protein response in the ER and acts as chaperone of calcium-binding properties. Upregulation of Pdia4 was found in highly metastatistic HCC cell lines. In addition, upregulation of Pdia4 also occurs when DG44 cells were treated with ionophore which causes ER stress. Little is known about the biological role of Pdia4.
In this thesis, I first established the retroviral-mediated and lentiviral-mediated systems to study gene function. We then compared the pros and cons of both systems based on our purpose. We also compared the infectivity and tropism of both viral system in different cell lines. The data showed that lentiviral-mediated system resulted in both high infection efficiency and high expression of Pdia4 in Jurkat T cells. We therefore choose the lentiviral-mediated system for Pdia4 study.
Lentiviral vector containing Pdia4 shRNA (KD) or empty sequence (GK) was used to infect the Jurkat T cells. Similar infection efficiency and mean fluorescence of intensity (MFI) were observed using FACS analysis. However, western blot data showed that level of KD cells was reduced by 80% as compared to GK cells. The data showed that the gene delivery system was successful.
Previous publications showed that PDI members are involved in T cells survival. I decided to evaluate the effect of Pdia4 on cell growth of Jurkat T cells using the RNAi knockdown approach. The results showed that knockdown of Pdia4 expression will inhibit the cell growth of Jurkat T cells but has no effect on cell death based on cell counting analysis. Besides, the data showed that Pdia4 knockdown decreased cell proliferation using AlexaFluor 647 succinimidyl ester (AFSE) analysis. This is further supported by the finding that cell subset with high GFP decreased fast. However, Pdia4 is not involved in TCR and Fas pathway.
In summary, I showed that Pdia4 affected the cell growth of T cell through modulation of T cell proliferation. We will continue to investigate the gene function of Pdia4 in cell death.

謝辭 ……………………………………………………………………i
中文摘要 ………………………………………………………………ii
英文摘要 ………………………………………………………………iv
目錄 ……………………………………………………………………vi
縮寫表 ………………………………………………………………viii

前言 ……………………………………………………………………1
一、蛋白質雙硫異構? ………………………………………………1
二、病毒基因轉殖載體 ………………………………………………4
三、T淋巴細胞 ………………………………………………………7
四、總結 ………………………………………………………………8

材料與方法 ……………………………………………………………9
一、培養基與化學藥品 ………………………………………………9
二、細胞株與質體 ……………………………………………………10
三、反轉錄病毒和慢病毒的生產、濃縮和貯存 ……………………11
四、反轉錄病毒感染 …………………………………………………12
五、慢病毒感染 ………………………………………………………13
六、細胞計數與存活測試 ……………………………………………13
七、偵測細胞分裂 (cell proliferation) ………………………………14
八、刺激細胞死亡 ……………………………………………………15
九、西方點墨轉漬法 (western blotting) ……………………………15
十、統計方法 …………………………………………………………17

結果 ……………………………………………………………………18
一、病毒基因轉殖載體的系統建立 …………………………………18
二、病毒基因轉殖載體系統和細胞平台的選擇 ……………………18
三、探討Pdia4在Jurkat T細胞的基因功能 ………………………19

討論 ……………………………………………………………………22
一、病毒基因轉殖載體和不同細胞株之間結合感染的效率 ………22
二、病毒生產、濃縮與貯存 …………………………………………23
三、Pdia4表現量對T細胞的影響 …………………………………23

總結 ……………………………………………………………………25
圖表、附錄 ……………………………………………………………26
參考文獻 ………………………………………………………………38

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