Rho small GTPases的訊息傳遞對細胞老化之影響

Rho small GTPases in cellular senescence

劉佳宜(Chia-Yi Liu)

指導教授 ： 張智芬

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01033

摘要

正常未經過癌化的真核細胞在細胞培養的過程中，其具有一定的分裂壽命，當細胞分裂次數到達極限時，細胞不再分裂，此現象稱為細胞老化。當細胞老化後，其外型會由細長立體轉為攤開扁平變大。本篇論文即是針對這個顯著的細胞老化特徵去探討三個調節細胞骨架的蛋白：RhoA、Cdc42、與Rac1在細胞老化中所扮演的角色。
本研究中，我們以一株正常人類肺部纖維母細胞(IMR-90)作為研究模型，在先比較老化細胞與年輕細胞的細胞骨架後，我們發現，無論在一般培養未同步化或是在同步血清刺激下，細胞骨架中的一個主要蛋白，肌纖蛋白(actin)在老細胞中皆較無動態性，這樣的結果，僅僅造成老化細胞較慢的游移速度，然而卻沒有破壞老化細胞的游移能力。
進一步探討RhoA、Cdc42、與Rac1在老化過程中所受到的調節，我們發現，這三個蛋白的表現量在未經同步化的細胞中，並不受老化過程的影響，然而，它們的活性在老化細胞中卻明顯降低，因此，我們利用重組腺病毒的系統，在老化細胞中表現活化態的RhoA (V14)、Cdc42 (V12)、及Rac1 (V12)，結果發現，提高任一個蛋白的活性皆可以回復老化細胞DNA複製的能力，也造成老化細胞細胞骨架的改變，然而，在這樣的調節下，一個與老化相關的標記(SA-β-Gal)卻沒有被減弱，因此，我們認為在老化過程中RhoA、Cdc42、與Rac1所受到的調節，應該參與部份的角色，即使如此，在老化細胞中提高這幾個蛋白的活性卻無法回復所有的老化特徵。

關鍵字

細胞老化

並列摘要

Normal human fibroblasts cannot divide indefinitely in culture. The nature of this finite replicative life span of cells restricts cell division by a process known as cellular or replicative senescence. When cells enter the senescent state, their cell shape will change from filamentous to flat. In this thesis, I investigated the role of three cytoskeleton regulatory proteins, RhoA, Cdc42, and Rac1 in cellular senescence.
In this research, I used normal human lung fibroblasts, IMR-90, as a study model. In the experiment of F-actin stain, I found that senescent cells displayed a less actin dynamics than young cells did either in an asynchronous state or a serum-stimulated state. However, the migration ability still existed but with a slower rate in senescent cells.
To further investigate the regulation of RhoA, Cdc42, and Rac1 in cellular senescence, I found that the expression of these three proteins in asynchronous cells was senescence-independent. However, the activities of RhoA, Cdc42, and Rac1 were decreased during cellular senescence. Hence, I used recombinant adenoviral systems to express constitutively active forms of RhoA (V14), Cdc42 (V12), and Rac1 (V12) in senescent cells. We found that expression of RhoA (V14), Cdc42 (V12), or Rac1 (V12) in senescent cells could restore the DNA synthesis capacity and result in cytoskeleton changes. However, such a regulation could not repress the expression of a senescent marker, SA-β-Gal. Accordingly, we proposed that age-dependent change in regulation of RhoA, Cdc42, and Rac1 may take a part in the process of cellular senescence; however, upregulation of these RhoGTPases could not reverse all senescent phenotypes.

並列關鍵字

Rho ； cellular senescence

參考文獻

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