臺灣博碩士論文加值系統

Noxa基因所表現的蛋白質只含一個BH3 domain且歸類於Bcl-2家族。不同的Noxa蛋白表現量已被證實在神經或骨髓癌細胞中會影響細胞死亡的情況，但在胃腺癌細胞還未被證實有這種影響。另外，Noxa蛋白在不同的細胞中，所啟動促進細胞凋亡的分子機制（無論與p53蛋白相關與否），也由於承受不同的壓力而有所差別。因此，我建立了含有不同Noxa表現量的胃腺癌細胞，來分析Noxa的分子機制。
五個針對不同片段的Noxa傳信核醣核酸 (21個核�˙�)，所建立的干擾性核醣核酸載體(編號一~五)，以及一個對照組載體(未含Noxa干擾型核醣核酸)，購自中央研究院國家型干擾性核醣核酸設施。此病毒載體同時具有抵抗嘌呤霉素以及安比西林兩個標的基因以供篩選用。除了編號一號的干擾性核醣核酸載體外，其他皆經由序列比對確定無誤，挑選其中編號三與四的干擾性核醣核酸，以及對照組載體，成功放入胃腺癌細胞。其中一個針對Noxa中的MTD domain片段所建立編號四的干擾性核醣核酸載體，放入胃腺癌細胞後，經實驗顯示幾乎沒有Noxa蛋白表現量。相反地，含有對照組載體的胃腺癌細胞，經實驗顯示不會改變Noxa蛋白表現量。更進一步，我建立了含有缺乏P53蛋白表現量的胃腺癌細胞，並經實驗證實比原始胃腺癌細胞的P53蛋白表現量降低了70 %。因此，在缺少P53蛋白表現的胃腺癌細胞中，藉著觀察不同壓力處理，對Noxa蛋白表現量的影響，期望能進一步了解P53與Noxa蛋白之間的關連性。
在這篇論文中，我們檢查了不同Noxa表現量的胃腺癌細胞在亞砷酸鈉、X射線以及順雙氨雙氯鉑處理是否產生不同的細胞毒性反應。暴露於X射線的胃腺癌細胞，不同的Noxa表現量在細胞存活率並沒有統計上的差別性，但缺少Noxa表現的細胞X射線下顯示出比對照組較少的細胞凋亡率，並且只有在對照組的細胞中明顯有多核的族群產生。因此推論，降低Noxa表現量，的確能降低細胞凋亡率。經過亞砷酸鈉處理24小時後，缺乏Noxa表現的胃腺癌細胞，存活率較對照組增加了10 %，細胞凋亡情況也比對照組減少了10 %。Noxa的確主宰了亞砷酸鈉處理所造成的胃腺癌細胞凋亡現象。另一方面，我們在順雙氨雙氯順雙氨雙氯鉑的處理實驗中，發現與亞砷酸鈉處理相反的細胞毒性反應。缺乏Noxa表現的胃腺癌細胞，在順雙氨雙氯鉑處理後，反而含有比對照組低10 %的存活率，以及多10 %的細胞凋亡率。造成這種反效果，可能是因為降低Noxa蛋白表現量，受到順雙氨雙氯鉑的影響，導致Bcl-2家族的蛋白間聯繫失衡，反而加速細胞凋亡。
在這篇論文中，我建立了含有缺乏P53蛋白表現量的胃腺癌細胞，這個細胞可以幫助未來觀察P53與Noxa蛋白的關連性對細胞存亡的影響。至今仍未有文獻顯示Noxa在偵測癌症中扮演一個生物標記的腳色；並且，Noxa的突變或缺乏功能性沒有普遍盛行於癌症細胞中。因此，希望藉著這篇論文裡提到利用干擾型核醣核酸的方法所得到的實驗結果，能提供一些資訊，有助於胃腺癌病人的基因治療研究。

Noxa encodes a BH3 domain-only protein of the Bcl-2 family. Ectopically expressed Noxa affects apoptosis in various cells, such as neural and myloma cells. Noxa in human gastric adenocarcinoma AGS cells was not yet fully studied. Besides, the exact molecular mechanisms of Noxa (p53-dependent or p53-independent) activating apoptosis exhibit diversity from cell types and stresses. Thus, the frame of different Noxa expression background of AGS cells was conducted for further molecular mechanism studies on Noxa in this study.
Five pLKO.1 plasmids (No. 1~5), each inserted a special designed 21-nucleotides fragment of Noxa-siRNA gene, and one pLKO.1-Luc plasmid containing Noxa-siRNA mismatch sequences were prepared from corresponding E. coli DH5α strains which were originally purchased from National RNAi Core Facility (NRC) in Academia Sinica. Two marker genes, functioned to against antibiotics, Amp and puromycin, are also located on these lentivirus-based plasmids. Corresponding Noxa siRNA-targeting sequences No. 2~ 5 were confirmed by sequencing. Plasmids (No. 3 and No. 4) containing two different target sequences near the ORF regions of the Noxa gene and plasmid pLKO.1-luc were transfected to AGS cells, respectively (Fig. 1 and 2), using the puromycin selection. In addition, the p53 siRNA containing AGS cells were established by means of transfecting p53 siRNA inserted pLKO-AS1 transfection successfully (Fig. 9-10). Seventy percent reduction of p53 expression in p53 siRNA transfected AGS cells was found in comparison with the mock cells (Fig. 11). Therefore, the different Noxa expression affected by SA-, X irradiated-, or cisplatin-induced apoptosis in P53 knockdown AGS cells now are ready for further investigation.

The cytotoxic effects on cell survival and apoptosis in p53 wild type AGS cells with different Noxa statuses after treatment with IR, sodium arsenite (SA) or cisplatin were examined. Noxa siRNA transfection has no significant influence on the survival rate of X-irradiated AGS cells. However, a significant decrease on apoptosis was detected in Noxa knockdown AGS cells after 5 Gy X-irradiation (Fig. 6). Also, the polyploidy population exhibited obviously only in mock AGS cells after X-irradiation. The reduction of Noxa expression was investigated to decrease the X-ray induced apoptosis in AGS cells. After treating with 3 or 5 μM SA for 24 hours, the AGS cells with Noxa siRNA had about 20 % increase in survival rate and 10 % decreases in apoptosis in comparison with mock AGS cells (Fig. 7). Noxa was found to modulate SA-induced apoptosis in AGS cells. On the other hand, the effects of cisplatin were opposite to those of SA on AGS cells with different Noxa statuses. Noxa siRNA transfected AGS cells had 10 % lesser in survival rate and 10 % more in apoptosis after 2 or 4 μM cisplatin treatments for 24 hours in comparison with mock AGS cells (Fig. 8). It is possible that the reduction of Noxa expression affected the interaction between the members in Bcl-2 family and promoted apoptosis after cisplatin treatment in Noxa knockdown AGS cells.
The p53 knockdown AGS cells had been constructed in this study for examing the effects of both P53 and Noxa proteins on cell apoptosis and survival. Noxa protein has not been confirmed to be a biomarker for detecting cancer. Also, mutations or deletions of Noxa are not always detected in cancer cells. Therefore, my data using siRNA methods in this study supported the possible clinical usage for wild type Noxa gene therapy for Human Gastric Adenocarcinoma (AGS) patients.

中文摘要 1
Abstract 3
Motives 5
Introduction 7
1. Gastric cancer 7
2. Apoptosis and Bcl-2 family 7
3. Noxa, the BH3-only protein of Bcl-2 family 9
4. RNA interference 11
5. Noxa and X-ray irradiation (Radiation therapy) 12
6. Arsenic compounds in cancer therapy 14
7. Chemotherapy drug-cisplatin and gastric cancer 16
Materials and Methods 17
Materials 17
1. Drugs and antibodies 17
2. Cell lines 17
3. Plasmids 18
4. Selection marker- Puromycin 19
Methods 20
1. Cell culture conditions 20
2. X-ray irradiation or drug treatment 20
3. Reverse transcription (RT) – PCR 21
4. Sulforhodamine B (SRB) cell viability assay 22
5. Flow cytometric analysis 23
6. Dual-parameter flow cytometric analysis 23
7. Plasmid construction and transfection 24
8. Western blot analysis 26
Results 27
1. Selection of Noxa siRNA- transfected cells 27
2. The characteristics of transfected AGS cells 30
3. Effects of X-irradiation on Noxa siRNA 4-transfected AGS cells 31
4. Effects of sodium arsenite (SA) on Noxa siRNA 4- transfected AGS cells 32
5. Effects of cisplatin on Noxa siRNA 4-transfected AGS cells 33
6. Construction of RNA interference system (p53 siRNA for example) 34
Discussion 37
1. RNA interference for Noxa 37
2. X-irradiation effects on cellular apoptosis 39
3. Sodium arsenite effects on cellular apoptosis 39
4. Cisplatin effects on cellular apoptosis 41
5. The system for RNA interference 42
References 44
Figures 50
Appendix 1 71
Appendix 2 72
Appendix 3 73
Appendix 4 74
Appendix 5 75

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