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標題: Cdk5/p35蛋白在維生素甲酸誘導之HeLa細胞株凋亡中扮演的角色
The roles of Cdk5/p35 proteins in retinoic acid-induced HeLa cell apoptosis
作者: 郭宏碩
Kuo, Hung-Shou
關鍵字: RA;維生素甲酸;Cdk5;p35;p53;HeLa cell;cell cycle;apoptosis;子宮頸癌;細胞週期;細胞凋亡
出版社: 生命科學系所
Cdk5 (Cyclin dependent kinase 5)是Ser/Thr激酶,屬於Cdks成員,但卻不具有調控細胞週期的功能。Cdk5本身不具有活性,必須有活化蛋白如p35和p39的存在才會具有磷酸化下游蛋白的活性。在這之中,p35容易被截切成p25以及p10兩個片段,而p25的半生期較長、結構較為穩定,因此具有過度活化Cdk5蛋白活性的能力。在攝護腺癌細胞中,已有文獻證明由藥物所引起的細胞凋亡,與Cdk5/p35 (p25)有關。p53存在於細胞核內,其相關生理功能包含有停滯細胞週期(cell cycle arrest)、細胞分化(cell differentiation)、細胞衰老(cell senescence)以及細胞凋亡(apoptosis),最常被提到的便是導致細胞凋亡,在癌症生物學裡,更有腫瘤抑制者(tumor suppressor)的稱號。維生素甲酸(Retinoic acid,以下簡稱為RA)屬於維生素甲類化合物,因最具生物活性而被廣泛研究。在前人的研究中發現,RA對於癌症細胞的影響除了可以抑制其生長外,更甚者可以導致細胞進行凋亡。
在我們的研究裡發現在加入RA之後,子宮頸癌HeLa細胞株的型態上有所變化,除了可以利用MTT分析以及trypan blue染色發現到細胞存活率有降低的情形外,以流式細胞儀分析細胞週期的結果,Sub G1期的比例有增加,代表細胞在進行細胞凋亡以及停止生長。以免疫螢光染色的結果可以發現,Cdk5的分佈位置會與p35及p53大致相同外,在加入RA後,會有螢光強度增加的情形,同時也發現到核內有較大量累積的情形。在p53的下游蛋白如p21、p27及caspase-3也可以發現加入RA處理後會有類似現象。以西方轉漬法可以發現RA會誘導Cdk5與其相關蛋白的表現量增加,並可以發現到PARP有被截切成較小片段的現象。根據以上結果,我們認為在RA所誘導的HeLa 細胞株細胞凋亡過程中,Cdk5也參與在其中。

Cdk5 is a Ser/Thr protein kinase, belong to Cdks. However, unlike other Cdks, Cdk5 has no function during cell cycle. Cdk5 has no activity just binding with its activator p35 or p39, and could phosphorylate downstream protein. p35 could be cut two fragments--p10 and p25. However, p25 has longer half-life and more stable structure than p35, so p25 can over-express Cdk5 protein. Accroding to reports, Cdk5/p35 involved in drugs-induced cell apoptosis. p53 exists in nuclear, its biological function contained cell cycle arrest, cell differentiation, cell scenesce and cell apoptosis. Of cancer biology, p53 usually reported as tumor suppressor because it can inhibit cell growth. Retinoic acid (RA) belongs to Vitamin A compounds, because of its most biological function, be studied in various area. Studies showed RA not only inhibited cancer cell growth, but also induced cancer cell apoptosis.
In our study showed that morphology of cervical cancer HeLa cells changed by treated with RA. By MTT assay and trypan blue staining, cell viability decreased; flow cytometry assay also showed ratio of Sub G1 phase increased, it meant cell was apoptosis and stopped growth. From immunostaining, we found distribution of Cdk5 was almost the same as p35 and p53, and when treated with RA, their expression were increased, especially in nuclear. Down stream protein of p53, as p21, p27 and caspase-3 also showed this distribution. Western-blotting showed RA could induced Cdk5 and Cdk5-related protein expression increased, and PARP could cut to smaller fragment. In conclusion, we believe that Cdk5 involved with RA induced HeLa cell apoptosis.
Appears in Collections:生命科學系所

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