探討過氧化體生合成對於線蟲成蟲壽命與癌細胞存活的影響

The study of the down-regulation effect of the peroxisomal biogenesis factor on the lifespan of midlife-stage Caenorhabditis elegans and the survival of the cancer cell

邱韋凱(Wei-Kai Chiu)

指導教授 ： 蔡榮宗

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00060

摘要

(1)近年來人口老化已是近數十年來全球的趨勢，而有關延遲老化和抵抗衰老的研究和生物產業的發展也在近年蓬勃發展，目前有許多研究探討生物體老化之相關的分子機制，而造成生物體老化的因素之成面包含了生物體所接觸外在的環境壓力因子(例如:毒素、放射線)，也有細胞內生性的分子機制因子(例如:氧化物傷害、細胞激素)，會去促使生物體走向老化死亡。而近年來許多有關老化的研究常利用生命週期短的生物模型來進行實驗與分析，先前文獻顯示氧化代謝物和外在壓力都會影響線蟲的壽命。過氧化體為人體重要胞器之一，有許多的過氧化氫酶和氧化酶能參與脂肪酸或膽固醇的代謝，其中過氧化體生合成基因扮演極重要的角色，過氧化體功能若缺失會造成神經退化症(例如: 腎上腺腦白質退化症、阿茲海默症) 。因此，我們利用線蟲動物模型以RNAi干擾的實驗方式來研究過氧化體生合成基因對於壽命的影響，結果顯示過氧化體生合成基因(prx-3、prx-5、prx-13、prx-19)表現被抑制時，線蟲成蟲的壽命會變短，另一方面結果也顯示當prx-19受到RNAi干擾時線蟲之細胞凋亡相關基因(egl-1、ced-3、ced-4、ced-9)表現量會有增加的表現，顯示過氧化體生合成基因的正常表現對於線蟲成蟲壽命是重要。
(2)根據衛生署民國101年統計全國死亡人數，惡性腫瘤於國人十大死因之首，近年來在全球也是人類醫療中最為重大的疾病之一，癌症的生長過程可能透過外在環境因素和生物體內分子機制因素來誘導促進引起細胞組織的癌化，許多文獻指出過氧化體所產生的活性氧化物ROS可能造成促進癌細胞的生長，也有一些文獻指出過氧化體增生活化受體PPAR會在不同癌細胞中有不同的表現量，有的顯示利用過氧化體增生活化受體PPAR之抑制劑能抑制癌細胞生長，有的則顯示過氧化體PPAR被活化時能抑制癌細胞的生長。因PPAR可能透過增加過氧化體數量或調控其他基因來影響癌細胞的生長，因此無法得知過氧化體在癌症存活中真正扮演的角色，所以本研究想直接透過抑制過氧化體生合成基因的表現，來分析過氧化體對於癌細胞存活的重要性。另一個支持我們做這些研究的理由是本研究第一部分有關過氧化體生合成基因和線蟲成蟲壽命之影響實驗中看到當線蟲過氧化體生合成基因被抑制表現時壽命會變短，結果也顯示當線蟲過氧化體生合成基因prx-19的表現被抑制時，會促進線蟲相關細胞凋亡基因的表現，顯示過氧化體的生合成會影響老化及細胞凋亡。因此本研究第二部份利用RNAi干擾方式抑制腦癌細胞GBM8401之過氧化體生合成基因PEX19，接著利用細胞存活實驗顯示腦癌細胞GBM8401和肝癌細胞Huh7生長會受到抑制，而腦癌細胞GBM8401特別顯著，而非癌症之人類胚腎細胞HEK293和HEK293T的生長能力並未受到抑制，利用流式細胞儀分析細胞週期，結果顯示腦癌細胞GBM8401的sub-G1期會增加，Western Blot的結果也顯示當PEX19的蛋白抑制時會促進細胞凋亡相關蛋白的表現，也會使訊息傳遞中磷酸化ERK的量會有增加的情形，此外也分析細胞移動能力，結果顯示在PEX19被抑制時腦癌細胞GBM8401移動能力會下降，在裸鼠動物實驗中結果也顯示當PEX19被抑制時，會抑制腫瘤生長能力，因此實驗結果證實癌細胞之過氧化體生合成基因表現受到抑制時會抑制其生長及移動的能力。本研究結果顯示指出一個新的治療癌症的策略，亦即透過降低過氧化體生合成基因的表現將可達成治療癌症的目標。

關鍵字

過氧化體 ； 線蟲 ； 壽命 ； 腦癌 ； 細胞凋亡

並列摘要

(1)In recent years, the aging population in recent decades has been a global trend, and such delay aging and anti-aging research and development of biotechnology industry is also booming in recent years, caused by the aging of the organism factors into the surface in contact with the organism contains the external environment stress factors (eg: toxins, radiation), there is also the molecular mechanisms of cellular endogenous factors (eg: oxides injury, cytokines), will go to promote aging of the organism to die. In recent years many of the aging studies often use a short life cycle model for biological experiments and analysis, previous literature shows that oxidative metabolites and external pressure will affect the Caenorhabditis elegans life. Peroxisome organelle is one of the important human,there are many catalase and oxidase to participate in the metabolism of fatty acids or cholesterol,which peroxisome biogenesis genes play a very important role, Peroxisome function if lose will cause neurodegenerative disorders (eg: ALD, AD). Therefore, we use RNAi interference experiments to study ways of peroxisome biogenesis genes for the life of an animal model of Caenorhabditis elegans. The results showed that peroxisome biogenesis genes (prx-3, prx-5, prx-13, prx-19) expression is suppressed, adult worms become shorter life span, The other hand, the results also show that when the prx-19 C. elegans by RNAi interference of apoptosis-related genes(egl-1、ced-3、ced-4、ced-9) expression have increased performance, show peroxisome biogenesis genes normally expressed is important for the longevity of Caenorhabditis elegans.
(2)According to the Department of Health of the Republic of 101 deaths nationwide statistics, cancer of the first leading cause of death in people. In recent years, the world is also the most significant human health in one of the diseases, During the growth of cancer by external environmental factors and possible biological factors to induce in vivo molecular mechanisms to promote causes cancerous cells and tissues, many studies indicate Peroxisome reactive oxygen generated ROS may promote the growth of cancer cells caused by, there are also some study that peroxisome proliferator activated receptor PPAR in different cancer cells have different expression, some shows use of peroxisome proliferator activated receptor PPAR inhibitors can inhibit the growth of cancer cells, some displays are peroxisome PPAR activation can inhibit cancer cell growth. PPAR may be due to the body by increasing the number of peroxide or regulate other genes to affect cancer cell growth, Peroxisome therefore no way of knowing the real cancer survivors in the role, so this study is to directly through inhibition of peroxisome biogenesis gene expression, to analyze the peroxisome importance for the survival of cancer cells for cancer effects.In the first part of this study peroxisome biogenesis genes and longevity of C. elegans experiment to see if the effect of nematode peroxisome biogenesis gene expression was inhibited when life becomes shorter, The results also show that when the nematode peroxisome biogenesis gene prx-19 are downregulation, thereby promoting apoptosis-related gene expression in C. elegans ,Thus the second part of the study methods using RNAi interference suppression of brain cancer GBM8401 Peroxisome biogenesis genes PEX19, Then using cell survival experiments showed brain cancer GBM8401 and Huh7 hepatoma cell growth will be inhibited, and brain cancer GBM8401 particularly significant, rather than the cancer cells in the human embryonic kidney HEK293 and HEK293T not been inhibited the growth of capacity, Cell cycle experimental results show that the the sub-G1 phase of brain cancer GBM8401 will increase,and when PEX19 protein inhibition promotes apoptosis-related protein expression, phosphorylated ERK also have increased. The other hand, cell migration experiments showed brain cancer GBM8401 mobility will decline. In nude animal experiments also showed that when PEX19 is suppressed, inhibited tumor growth capacity,, therefore, experiments confirmed that cancer cells from peroxisome biogenesis gene expression is suppressed will inhibit their ability to grow. The results showed that the treatment of cancer of a new strategy, that is by reducing the peroxisome biogenesis gene expression will be able to reach the goal of cancer treatment.

並列關鍵字

Peroxisome ； C. elegans ； Lifespan ； Glioblastoma ； Apoptosis

參考文獻

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