臺灣博碩士論文加值系統

SARS-CoV是一種引起嚴重急性呼吸道症候群 (severe acute respiratory syndrome, SARS)的冠狀病毒。由人類檢體所分離出的SARS-CoV可能來自於果子狸或貉。比較從果子貍分離出的動物SARS-CoV-like virus和人類的SARS-CoV的基因序列，有99.8%的相似度。主要的序列差異在於人類的ORF8有29個核苷酸的減少。動物的SARS-CoV-like virus在宿主細胞內會轉譯一條連續性的ORF8蛋白質，而人類的SARS-CoV則在宿主細胞內轉譯出兩條蛋白質ORF8a及ORF8b。在本實驗室之前的研究顯示，ORF8a位於粒線體。在酵母菌雙雜合系統，發現ORF8a和細胞內的調控鈣離子濃度的蛋白質CAML有交互作用，但ORF8及ORF8b並沒有。在Hela細胞的Tet/on系統中，發現ORF8a會增強staurosporine所引起的細胞凋亡。而本論文的主要目標在於研究ORF8a及ORF8其特性及生物功能，藉此更進一步了解病毒在宿主細胞的致病機轉。
利用細胞螢光呈色及次細胞分離法，我們發現當細胞內CAML正常存在時，ORF8a主要位於粒線體，而ORF8可能位於內質網。若我們過度表現CAML在細胞內，ORF8a和ORF8會改變原來的表現位置，和CAML的分布有很明顯的共位現象。利用免疫沉澱法，證實這個現象可能是因為ORF8a或ORF8和CAML皆有交互作用。ORF8a和CAML有很強的交互作用，但ORF8和CAML則有較弱的結合能力，導致ORF8a改變原來的分布。在之前的研究發現，SARS-CoV同樣會感染肺部及腸道細胞，並可以進行複製，但肺部細胞有嚴重的細胞凋亡，腸道細胞卻沒有。我們分別利用肺部細胞株A549以及結腸細胞株Caco-2，表現ORF8a或ORF8以觀察是否會造成細胞凋亡。我們在A549表現ORF8a及ORF8，並利用Propidium iodide染色，發現有DNA斷裂的情形；利用TMRM染劑偵測粒線體膜電位變化，當A549處理Staurosporine，使細胞走向凋亡，發現ORF8a和ORF8皆會促進此凋亡的反應；只處理DMSO的控制組，ORF8a和ORF8也有較低程度的細胞凋亡，和propidium iodide染色的實驗相呼應。有趣的是，腸道細胞株Caco-2無論有無處理Staurosporine，皆不會促進細胞凋亡。
目前，我們並不清楚ORF8a或ORF8是否藉由和CAML的交互作用，而影響細胞凋亡。未來我們將建立降低CAML表現的A549及Caco-2細胞，並重複上述的實驗，以觀察ORF8a或ORF8所造成細胞凋亡，和細胞內蛋白質CAML的關連。

SARS-CoV, a coronavirus, was identified as the etiologic agent for severe acute respiratory syndrome. It is established that human SARS-CoV originated from animals, such as palm civets and raccoon dogs. SARS-CoV genome shares about 99.8% homology with its animal counterpart. The major difference of the two viruses is the 29 nucleotide deletion found in the corresponding open reading frame (ORF) 8 of human SARS-CoV, given rise to ORF8a and ORF8b. Our previous data had shown that ORF8a localizes to mitochondria. Using yeast two-hybrid screening, ORF8a, but not ORF8 and ORF8b, was shown to interact with calcium-modulating cyclophilin ligand (CAML), a protein had been known to regulate the intracellular Ca2+ concentration. Using Hela cell Tet/on system, ORF8a can promote the apoptosis initiated by staurosporine. The main goal of this dissertation is to determine the biological functions of ORF8 and ORF8a that might give better understandings to the pathogenesis of both viruses.
With immunofluorescence assay and subcellular fractionation, ORF8a and ORF8 are shown to be located in mitochondria and ER, separately, in the presence of endogenous CAML. However, when we ectopically expressed CAML, both ORF 8a and ORF8 co-localized with exogenous CAML and displayed a similar pattern of distribution, which differed from that in the presence of endogenous CAML. ORF8a was shown to have higher binding capacity for CAML than that of ORF8 in an immunoprecipitation assay, which likely contributed to the redistribution of ORF8a. In light of reports indicating that in SARS patient lung cells showed readily apoptosis than that of enterocytes, although both tissues are permissive for viral infection and growth. We proceeded to evaluate the apoptotic effect of either ORF8a or ORR8 on an alveolar epithelium cell, A549 and an enterocyte, Caco-2. A549 was showed to have DNA fragementation in the presence of ORF8a or ORF8 by propidium iodide staining, while both proteins also facilitated staurosporine-induced apoptosis using TMRM staining assay that targeting at changes in mitochondrial membrane potential. A549 was also showed low grade of apoptosis in the DMSO control group in the presence of ORF8a or ORF8, which coincided with the observed induction of DNA fragmentation by both proteins. Interestingly, Caco-2 with ectopically expressed ORF8a or ORF8 failed to exhibit apoptotic phenotype enhancement regardless of the presence or absence of staurosporine.
Currently, the contribution of interaction between CAML and ORF8a or even ORF8 in apoptosis remains unclear. In the future, we will re-examine the above stated observations in both A549 and Caco-2 cells with CAML knockdown for the evaluation of the contribution of the interactions between CAML and ORF8a or CAML and ORF8.

英文摘要........................................................................................................................4
中文摘要........................................................................................................................5
緒論...............................................................................................................................6
第一節、 前言........................................................................................................6
第二節、 SARS 疾病介紹.....................................................................................6
1. 臨床症狀........................................................................................................6
2. 傳播途徑........................................................................................................6
3. SARS 致病原.................................................................................................6
第三節、 SARS冠狀病毒(SARS-CoV)介紹.........................................................7
1. 冠狀病毒的簡介............................................................................................7
2. SARS-CoV的分類.........................................................................................7
3. 組織器官分佈................................................................................................8
4. SARS-CoV的基因體(Genome)…………………………………………….8
5. SARS-CoV的受體和進入細胞.....................................................................9
6. SARS-CoV的複製和轉錄.............................................................................9
7. 結構性蛋白質(structural proteins)………………………………………..10
8. 附屬性蛋白質(accessory proteins) ……………………………………….11
第四節、 SARS-CoV 的來源...............................................................................12
1. 跨物種傳染(Cross-species transmission) ………………………………...12
2. 儲存宿主(reservoir)……………………………………………………….12
第五節 研究動機與實驗目的..............................................................................13
1. 人類SARS-CoV 在宿主細胞所表現的ORF8a 和動物SARS-CoV-like
virus 所表現的ORF8 其特性上的差異.......................................................13
2. SARS-CoV 能感染肺部或腸道細胞，但其臨床症狀並不相同………...14
2
材料與方法..................................................................................................................16
1. 細胞培養......................................................................................................16
2. 使用之質體..................................................................................................16
3. 細胞轉染......................................................................................................17
4. 細胞螢光呈色並用共軛焦顯微鏡觀察......................................................17
5. 次細胞分離技術..........................................................................................17
6. 西方墨點分析法(western blot analysis)…………………………………..18
7. 蛋白質連結以及免疫沉澱分析..................................................................19
8. 利用Propidium iodide (PI)將DNA 染色，用FACs 觀察並分析.............20
9. 利用TMRM 偵測粒線體電位差的變化，用FACs 觀察並分析.............20
10. 萃取total RNA 及RT-PCR………………………………………………..20
11. 慢病毒(lentivirus)的製備、濃縮與純化.....................................................22
12. 統計方法(Statistical analysis)……………………………………………..23
結果..............................................................................................................................24
第一節、 SARS-CoV附屬蛋白質ORF8a 及ORF8 在細胞內分布的位置...…24
第二節、 ORF8a 或ORF8 和細胞內蛋白質的結合..........................................25
第三節、 ORF8a 或ORF8 在肺部細胞株A549 的生物功能............................26
第四節、 ORF8a 或ORF8 是否調控細胞內蛋白質CAML，使細胞走向凋亡.28
第五節、 ORF8a 和ORF8 表現在A549 及Caco-2 細胞株的生物活性差異..30
討論..............................................................................................................................31
第一節、 SARS-CoV 附屬蛋白質ORF8a 及ORF8 在細胞內分布的位置......31
第二節、 ORF8a 或ORF8 和細胞內蛋白質的結合..........................................32
第三節、 ORF8a 或ORF8 在肺部細胞株A549 的生物功能...........................33
第四節、 ORF8a 或ORF8 是否調控細胞內蛋白質CAML，使細胞走向凋亡.35
第五節、 ORF8a 和ORF8 表現在A549 及Caco-2 細胞株的生物活性差異..35
第六節、 總結與未來展望..................................................................................36
3
參考文獻......................................................................................................................37
圖表..............................................................................................................................46
Fig. 1. ORF8a and ORF8 localizes in cytoplasm………………………………...46
Fig. 2. ORF8a and ORF8 mainly localize in mitochondrial fraction…………….47
Fig. 3. ORF8a localizes to mitochondria, but ORF8 doesn’t…………………….48
Fig. 4. ORF8a interacts with CAML strongly, but ORF8 interacts with CAML
weakly……………………………………………………………………49
Fig. 5. ORF8a and ORF8 partially co-localizes to CAML………………………50
Fig. 6. ORF8a and ORF8 induce DNA fragementation in A549 cells………...…51
Fig. 7. ORF8a and ORF8 induce mitochondrial membrane potential loss in A549
……………………………………………………………………………53
Fig. 8. CAML mRNA expression in A549, VeroE6, Caco-2 and 293t…………...54
Fig. 9. Relative viral titer in A549 cells………………………………………….55
Fig. 10. ORF8a and ORF8 don’t affect mitochondrial membrane potential in
Caco-2……………………………………………………………………57
Table 1 pLKO.1-siRNA 的序列.........................................................................58

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