臺灣博碩士論文加值系統

根據過去實驗已經證實了Grail（Gene related to anergy in lymphocyte）蛋白與p53蛋白間相互結合的關係。但Grail蛋白在細胞中所扮演的功能及其與p53間的交互影響目前仍不十分清楚。所以我們建立表達不同量Grail蛋白的細胞株，使用Actinomycin D和 UV來處理細胞藉以提高胞內p53蛋白之含量及造成細胞損傷效應與及在不同Grail表達情中進行對於細胞死亡及細胞週期的變異分析。
我們發現當使用Actinomycin D和 UV來處理細胞時，過度表現Grail蛋白在這兩種刺激下都會抑制細胞走向死亡及促進細胞G1期的停滯。然而在p53蛋白方面，我們也發現過度表達Grail蛋白在細胞遭受壓力後，會抑制p53蛋白的表達。令我們驚訝的是這四株C2C12/shGrail細胞處在Actinomycin D和 UV的壓力下，亦會明顯地抑制細胞走向死亡，且此抑制效果比過度表達Grail蛋白更強，然而這四種細胞株均仍表達了大量的p53蛋白。
因此我們建立了持續降解p53訊息RNAs的細胞株和持續降解p53訊息RNAs並同時過度表達Grail蛋白的細胞株，從而得知Actinomycin D和 UV造成細胞的死亡與p53蛋白有極其密切的關係，並且發現Grail蛋白在細胞遭受Actinomycin D和 UV壓力之下，對於細胞所產生的保護效應，是與p53蛋白有相互依存的關連性。
由於p53蛋白在細胞中的功能主要是造成細胞死亡和G1時期的停滯，我們也發現四種持續降解Grail訊息RNAs的細胞株在受到Actinomycin D和 UV的刺激之下有很高G1時期的停滯與大量的p21蛋白表達，所以我們進而建立了四種持續降解p21訊息RNAs的細胞株及持續降解p21訊息RNAs同時過度表達Grail蛋白的細胞株，在處理Actinomycin D和 UV之後發現不能表達p21蛋白確實會造成較多的死亡，而如果再加之與Grail蛋白的表達則會抑制因不能表達p21所造成的死亡。所以Grail蛋白的抑制死亡效用是不依賴著p21蛋白而是依賴著p53蛋白。至於不能表達Grail蛋白時為何大量的p53蛋白不對細胞造成死亡卻引發G1期停滯時，確切分子機制還需再進一步研究探討。

The interaction between Grail（Gene related to anergy in lymphocyte）and p53 can be detected via in vitro and in vivo experiments but the celluar functions of Grail still remain elusive. To gain more information on the physiological function of Grail , we created a Grail knock-down and a Grail- overexpression C2C12 stable cell lines.In response to Actinomycin D and UV , over-expression Grail could reduce apoptosis , increase the number of cells accumulated in G1 phase, and reduce p53 protein accumulation.
We found that , in response to Actinomycin D and UV , C2C12/shGrail is resistant to apoptosis with high cellular level of p53 accumulation.
To further explore the function of Grail in the context of p53 , we generated four p53 knock-down C2C12 cell lines in the background of endogenous and overexpressing Grail. We found that , in these C2C12 , the apoptosis induced by Actinomycin D and UV are p53-dependent,and Grail did not further reduce the apoptosis in C2C12 lacking the expression of p53.
Because the main cellular functions of p53 are to induce apoptosis and cell cycle G1 arrest , we generated eight p21 knock-down C2C12 cell lines in the background of endogenous and overexpressing Grail. We found that Grail significantly inhibites the damage-induced apoptosis in C2C12 lacking the expression of p21.Thus , the anti-apoptotic function of Grail is not required p21 but it is strictly p53-dependent.
From the results of Grail knock-down C2C12 , we hypothesize that the high level of p53 accumulation after stresses is nonfunctional indicating that Grail might play an essential role in damage-induced apoptotic signaling of p53. The exact molecular mechanism by which Grail affects p53’s function needs to be further characterized in the future.

圖1、利用有效濃度5μg/ml的puromycin殺死不含puromycin抗藥
性的C2C12所需時間。…………………………………………46
圖2、選殖反轉錄病毒及建議穩定表達的C2C12細胞株流程。……47
圖3、利用西方墨點法檢視C2C12細胞在遭受不同的DNA damage
reagent時，內生性p53蛋白的表現。………………………48
圖4、利用西方墨點法和免疫螢光染色去偵測Grail蛋白的表現。.49
圖5、Grail接合至pQCXIP載體的位置………………………………50
圖6、C2C12/pQCXIP、C2C12/pQCXIP-Grial及四個C2C12/pQCXIP
-Grial subclone的免疫螢光照片。……………………………51
圖7、六個C2C12/pQCXIP-Grial subclone的免疫螢光照片。……52
圖8、在未受刺激時，Grail蛋白不會影響細胞週期的分布。………53
圖9、利用流式細胞儀和西方墨點法檢視C2C12/pQCXIP、C2C12/
pQCXIP-Grial細胞株處理Actinomycin D 30 nM 及UV 200
（J/m2） 48小時後，細胞週期的分佈和p53和Grail蛋白的變化。
……………………………………………………………………54
圖10、Grail的cDNA上四個不同shRNAs所認識的位置。…………55
圖11、測試四個C2C12/shGrail的效用。……………………………56
圖12、未受刺激時，這四個C2C12/shGrail細胞株不會影響細胞週
期的分布。………………………………………………………57
圖13、C2C12/pQCXIP及四個不同C2C12/shGrail細胞株處理
Actinomycin D 30 nM 48小時後，利用流式細胞儀檢視細胞
週期的變化。……………………………………………………58
圖14、.C2C12/pQCXIP及四個不同C2C12/shGrail細胞株處理UV 200
（J/m2）48小時後，利用流式細胞儀檢視細胞週期的變化。
…………………………………………………………………59
圖15、C2C12/pQCXIP、C2C12/pQCXIP-Grial及四個不同C2C12/
shGrail細胞株處理Actinomycin D 30 nM 48小時後，利用
流式細胞儀檢視細胞週期的變化。……………………………60
圖16、C2C12/pQCXIP、C2C12/pQCXIP-Grial及四個不同C2C12/
shGrail細胞株處理UV 200（J/m2） 48小時後，利用流式細
胞儀檢視細胞週期的變化。……………………………………61
圖17、利用流式係細胞儀檢視C2C12/pQCXIP、C2C12/shmp53-12、
C2C12/shmp53 -346、C2C12/shmp53-12/Grail和C2C12/
shmp53-346/ Grail處理Actinomycin D 30 nM 48小時後，
其細胞週期的變化。…………………………………………62
圖18、利用流式係細胞儀檢視C2C12/pQCXIP、C2C12/shmp53-12、
C2C12/shmp53-346、C2C12/shmp53-12/Grail和C2C12/
shmp53-346/ Grail處理UV 200（J/m2） 48小時後，其細胞
週期的變化。…………………………………………………63
圖19、利用西方墨點法檢視C2C12/pQCXIP、C2C12/pQCXIP- C2C12
/shGrail 554、722、1036及1111和C2C12/shmp53-12、
C2C12/shmp53 -346、C2C12/shmp53-12/Grail和C2C12/
shmp53-346/ Grail處理Actinomycin D 30 nM 48小時後，
其p53、Grail及p21蛋白的變化。…………………………64
圖20、利用西方墨點法檢視C2C12/pQCXIP、C2C12/pQCXIP- Grial、
C2C12/ shGrail 554、722、1036及1111和C2C12/shmp53-12、
C2C12/shmp53-346、C2C12/shmp53-12/Grail和C2C12/shmp53
-346/ Grail處理UV 200（J/m2） 48 48小時後，其p53、Grail
及p21蛋白的變化。……………………………………………65
圖21、p21的cDNA上五個不同shRNA所認識的位置。………………66
圖22、將C2C12/shp21-42583、C2C12/shp21-42585、C2C12/shp21-
54901、C2C12/shp21-54902處理Actinomycin D 30 nM 48小
時後，利用流式細胞儀檢視細胞週期的變化。………………67
圖23、將C2C12/shp21-42583、C2C12/shp21-42585、C2C12/shp21-
54901、C2C12/shp21-54902處理Actinomycin D 30 nM 48小
時後，利用西方墨點法檢視p53、Grailr及p21蛋白的變化。
…………………………………………………………………68
圖24、將C2C12/shp21-42583、C2C12/shp21-42585、C2C12/shp21-
54901、C2C12/shp21-54902處理UV 200（J/m2） 48小時後，
利用流式細胞儀檢視細胞週期的變化。………………………69
圖25、將C2C12/shp21-42583、C2C12/shp21-42585、C2C12/shp21-
54901、C2C12/sh21p-54902、C2C12/shp21-42583/P-Grail、
C2C12/shp21-42585/ P-Grail、C2C12/shp21-54901
/P-Grail、 C2C12/sh21p-54902/P-Grail這幾種細胞株處
理Actinomycin D 30 nM 48小時後，使用流式細胞儀檢
視其細胞週期的變化。………………………………………70
圖26、將C2C12/shp21-42583、C2C12/shp21-42585、C2C12/shp21-
54901、C2C12/sh21p-54902、C2C12/shp21-42583/P-Grail、
C2C12/shp21-42585/ P-Grail、C2C12/shp21-54901/ P-Grail
、C2C12/sh21p-54902/P-Grail這幾種細胞株處理UV 200（J/m2）
48小時後，使用流式細胞儀檢視其細胞週期的變化。……………71

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