【摘要】： 環(huán)境中的多種理化因素,包括紫外線、離子輻射和香煙煙霧等,都會對機體產(chǎn)生多種形式的損傷。特別是對于遺傳信息載體-DNA的損傷,會引起DNA結(jié)構(gòu)的改變和遺傳物質(zhì)的損失,即產(chǎn)生所謂的遺傳毒性。近年來,隨著這一領(lǐng)域的研究逐漸深入,人們對細胞內(nèi)DNA損傷產(chǎn)生及響應(yīng)機制已經(jīng)有了一個初步的了解。但由于我們所知有限,這一領(lǐng)域還存在著大量懸而未決的科學(xué)問題,通過對這些問題的解答,將使我們在DNA損傷方面的認知得到進一步加深。 熱休克是否引起DNA損傷一直存在爭議,我們實驗室前期發(fā)現(xiàn)熱休克不能誘導(dǎo)DNA雙鏈斷裂的特異性指標-γH2AX焦點的產(chǎn)生,但其它實驗室卻得到相反的結(jié)果。我們針對這一問題開展了進一步的研究,結(jié)果發(fā)現(xiàn)不同的熱休克處理方式對產(chǎn)生的細胞毒性包括遺傳毒性也各不相同,而不同的細胞系對熱休克處理的響應(yīng)也各不相同。45℃熱休克引起細胞不同程度的死亡。在遺傳毒性方面,五種熱休克處理方式誘導(dǎo)細胞內(nèi)γH2AX焦點產(chǎn)生的能力也各不相同,只有金屬浴不論在42℃還是45℃下,FL細胞或是CHL細胞中都能夠誘導(dǎo)γH2AX焦點的產(chǎn)生。兩種細胞的表現(xiàn)方式也有所不同,CHL細胞對熱休克處理的響應(yīng)比FL細胞更加靈敏。而且我們發(fā)現(xiàn)γH2AX焦點產(chǎn)生與細胞存活率并無必然聯(lián)系,因此γH2AX焦點不能作為熱休克引起細胞死亡的判斷標準。 順鉑是一種臨床常用的抗癌藥物,主要用于生殖系統(tǒng)癌癥和頭頸部癌癥的治療,但其作用機制仍未明確。系統(tǒng)生物學(xué)的方法能夠為我們提供一個高通量的、系統(tǒng)全面的研究平臺。近年來,已經(jīng)有多個研究小組利用蛋白質(zhì)組學(xué)的方法對順鉑的細胞損傷機制進行了研究,但作為一種以DNA損傷為主的藥物,引起的主要應(yīng)答反應(yīng)集中在細胞核內(nèi),因此我們利用蛋白質(zhì)組學(xué)的方法對核蛋白組進行了表達譜分析,并最終篩選到了19個順鉑處理后引起表達量發(fā)生改變的蛋白質(zhì)。這些蛋白包括核纖層蛋白、mRNA表達調(diào)控蛋白、細胞周期相關(guān)蛋白和生物大分子合成相關(guān)蛋白等,功能涉及細胞基本代謝和生理過程的多個方面。我們對其中部分蛋白進行了表達驗證,并通過對Fas基因兩種變異剪切產(chǎn)物的表達量檢測,證實順鉑處理確實引起了HeLa細胞中變異剪切現(xiàn)象的發(fā)生。 納米技術(shù)是一種開發(fā)應(yīng)用納米材料的新型技術(shù),納米材料因其物理結(jié)構(gòu)上的特殊性,在應(yīng)用方面有著極大的優(yōu)勢,但它是否會對機體產(chǎn)生毒害作用尚未明確。量子點作為一種熒光納米材料,廣泛應(yīng)用于細胞成像等生物醫(yī)學(xué)領(lǐng)域。細胞存活率檢測結(jié)果顯示PEG包被的CdSe/ZnS核/殼量子點對細胞基本不產(chǎn)生毒害作用。我們同時對量子點的細胞毒性進行了蛋白質(zhì)組學(xué)分析,共找到了21個差異表達的蛋白質(zhì)點,占蛋白總數(shù)的0.5%。其中5個蛋白質(zhì)點得到質(zhì)譜鑒定,包括兩個鋅指蛋白、兩個角蛋白和一個過氧化物酶。鑒于量子點處理后的HSF細胞中只有0.5%的蛋白質(zhì)表達量受到顯著影響,結(jié)合細胞存活率的檢測結(jié)果,可以認為這種量子點的細胞毒性較小,是一種比較安全的納米材料。
[Abstract]:Many physical and chemical factors in the environment, including ultraviolet radiation, ion radiation and cigarette smoke, can damage the organism in many forms. In particular, damage to the DNA vector-DNA can cause a change in DNA structure and loss of genetic material, that is, to produce so-called genotoxicity. In recent years, as the research in this field is in-depth, there has been a preliminary understanding of the mechanism of DNA damage generation and response in cells. But because of our limited knowledge, there is still a lot of outstanding scientific problems in this area, and through answering these questions, we will further deepen our knowledge of DNA damage. Whether heat shock causes DNA damage has always been controversial. We found that heat shock could not induce DNA double-strand break specific index in the early stage of our laboratory. As a result of this study, we conducted a further study of this problem, and the results showed that different heat shock treatments had different genotoxicity, and the response of different cell lines to heat shock treatment was different. In terms of genetic toxicity, the ability of five types of heat shock treatment to induce the focus of H2AX in cells is different, and only the metal bath can induce H2AX coke in FL cells or CHL cells, whether at 42 & deg; C or 45 & deg; C The effect of CHL cells on heat shock treatment was higher than that of FL cells. It was even more sensitive. And we found that the focus of H2AX was not linked to the survival of cells, so that the focus of H2AX was unable to cause cell death as heat shock. Judgment standard. Cisplatin is a commonly used anti-cancer drug, mainly used in the treatment of reproductive system cancer and head and neck cancer, but Its mechanism of action remains unclear. The system biology approach can provide us with a high throughput, In recent years, a number of research groups have used proteomics methods to study the mechanism of cell damage in cisplatin, but as a drug based on DNA damage, the main response is The reaction was concentrated in the nucleus, so we used the method of proteomic analysis to analyze the nuclear protein group, and finally screened the expression of 19 cisplatin. The protein comprises nuclear fiber layer protein, mRNA expression regulatory protein, cell cycle related protein and biological macromolecule synthesis related protein and the like, and the function relates to cell basic metabolism and In many aspects of the physiological process, we have demonstrated the expression of some of these proteins, and confirmed that cisplatin treatment did give rise to HeLa cells by detecting the expression of two variants of Fas gene. The occurrence of variation shear phenomenon. Nanotechnology is a new type of technology for the development of nano-materials. Nano-materials have great advantages in application because of its particularity in their physical structure, but whether it and the quantum dot is used as a fluorescent nanometer material, and is widely used as a fluorescent nanometer material. Applied in the biomedical field of cell imaging and the like, and the cell survival rate detection results show that the PEG-coated CdSe/ ZnS core/ shell quantity At the same time, the cytotoxicity of quantum dots was analyzed by proteomic analysis, and 21 differentially expressed eggs were found. The white particles accounted for 0.5% of the total protein. Among them, 5 protein spots were identified by mass spectrometry, including two proteins. Two keratin and one peroxidase. In view of the significant effect of only 0.5% of the protein expression in the HSF cells treated with quantum dots, the cytotoxicity of the quantum dots can be considered to be less toxic than the detection results of cell survival.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R363

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