【作者】 林晓钢；

【导师】 潘英俊；

【作者基本信息】 重庆大学 ， 光学工程， 2008， 博士

【摘要】 细胞是生命科学研究的基石,一切生命的关键问题都要到细胞中去寻找。细胞分裂可说是生命起源最基本也最重要的过程,细胞周期则是细胞分裂全部生理过程的综合体现,普遍存在于高等生物中。大量研究表明,细胞周期调控异常是肿瘤发生的主要机制,所以细胞周期理论对肿瘤的预防和临床诊治具有重要的指导意义。随着光学与生命科学的交叉,对细胞的光信息研究工作成为了生命科学的一个重要领域,当然光学技术和细胞研究的结合不可避免会产生各种各样的问题,利用光谱技术对细胞周期进行研究则是其中一个较为突出的问题。总结起来,从国内外学者的研究中,可以看出对于细胞在细胞周期中的光谱研究主要存在以下问题:①研究波段单一。几乎所有的研究机构和研究者均是在从红外光谱角度入手对细胞在细胞周期过程中红外波段的光谱变化进行研究,而没有充分利用到现有的丰富的光谱技术和手段以对细胞周期进行更为全面的研究。②分析方法复杂。生物细胞在红外波段有丰富的信息,是因为几乎生物细胞内所有分子在红外波段均有光谱体现,这就使得生物细胞的红外光谱相互混叠,难以获取有用的信息。这也使得对细胞内特定分子的光谱跟踪变得十分的困难。③很多与细胞周期光谱研究的关键问题没有明确的说明和解决。如进行细胞周期光谱研究的样品获取问题,进行细胞周期光谱研究的细胞样品应该是一种什么样的同步化水平,细胞周期光谱模型的建立方法等等这些问题均未得到解决。本文则是针对现有问题,利用紫外-可见光光谱分析技术和荧光光谱技术操作简便、分析快速等优点提取活体肿瘤细胞在细胞周期不同时相的光谱特征,在以下几个方面开展工作:①设计并实施HeLa细胞同步化处理方案根据细胞同步化原理及处理方法,提出了宫颈癌细胞HeLa细胞株进行细胞同步化的处理方案以及进行光谱实验所需要达到的细胞同步化水平,以获取满足实验要求的同步化于细胞周期各时相(G1期,S期,G2期和M期)的HeLa细胞样品。②设计实验以获取HeLa细胞在细胞周期各时相的紫外—可见吸收光谱设计了同步化后处于细胞周期各时相的HeLa细胞的紫外-可见光吸收光谱测量方案,进行HeLa细胞样品的紫外-可见光吸收光谱测量(190nm～800nm)。吸收光谱测量结果说明各时相的光谱变化基本反映了HeLa细胞周期变化的生色团和助色团变化过程,光谱中在204nm和260nm附近有两个吸收峰,其中204nm附近的吸收峰是由HeLa细胞内的芳香族氨基酸造成,260nm处的吸收峰则是蛋白质和核酸综合吸收的结果。这些变化说明HeLa细胞生长过程中紫外吸收光谱的变化反映了芳香族氨基酸、嘌呤和嘧啶衍生物与HeLa细胞生长过程的密切关系。③设计实验以获取HeLa细胞在细胞周期各时相的自体荧光光谱设计了对同步化后处于细胞周期各时相的HeLa细胞进行了自体荧光光谱测量的测量方案。以290nm为激发光获得了各时相细胞样品的荧光光谱,测量结果表示,各时相细胞样品荧光光谱峰值位置均位于360nm和680nm处,这两处荧光峰的强度随样品的不同而发生相应的变化,这说明细胞内产生荧光的物质含量随细胞的生长变化,从而导致荧光强度的变化。其光谱曲线反映了HeLa细胞细胞周期变化过程中氨基酸和卟啉衍生物含量的变化趋势。④建立HeLa细胞的光谱模型根据HeLa细胞紫外-可见光吸收光谱测量结果,采用生物系统光谱模型的建模方法,在种群水平上分别建立了描述HeLa细胞周期变化的一元线性回归光谱模型和多元线性回归光谱模型,并对模型进行了评价,证明了两个模型能够很好的反映HeLa细胞周期变化过程中的紫外吸收光谱变化。HeLa细胞细胞周期自体荧光光谱特征峰的强度变化趋势不适合采用线性回归模型对其进行描述,故在未对其作进一步研究。最后通过实验样本对模型进行验证,一元线性回归模型和多元线性回归模型对实验样本所处细胞周期时相判断的正确率在98%以上,验证结果说明本文建立的HeLa细胞的紫外吸收光谱模型能够很好的描述HeLa细胞周期变化过程中其特征峰吸光度的变化。更多还原

【Abstract】 Cell is the cornerstone of research in life sciences. All the key issues of life have to look for answers in cells. Cell division can be said to the most basic and most important process in origin of life. Cell cycle embodies all the physiological process in cell division, and it exists in higher creatures. A large number of studies have shown that abnormal regulation and control of cell cycle is the main cause of tumor, therefore the cell cycle theory gives directions to cancer prevention and clinical diagnosis and treatment of tumor.With the interdisciplinary of life sciences and optics, the study of cells’optical information has become a important area in life sciences, of course, the combination of optical technology and cell research will inevitably produce various problems, especially the cell cycle study using spectrum technology. On the whole, from the studies of domestic and foreign scholars, there are some questions about the spectrum study on cells which are in cell cycle.①Single optical band. Almost all the research institutes and researchers did studies on the changes of cell cycle using infrared spectroscopy. They did not make full use of the existing spectrum technologies to give the cell cycle a comprehensive study.②Complex analysis methods. Biological cells have a wealth of information in the infrared band, because almost all the elements of biological cells have light absorption in the infrared band, which leads to the infrared spectrum aliasing. And the track of specific molecules in cells becomes very difficult.③No clear explanations and resolutions for some key issues in the spectrum study on cells which are in cell cycle, such as the access to samples, what kinds of synchronization level about the samples, methods of building cell cycle spectrum model, parameter extraction and so on.This article aimed at the existing problems, using ultraviolet and visible spectroscopy and fluorescence spectroscopy, extracted the spectrum characteristics of living tumor cells at different phases. The followings are our research work:①Design and implement the synchronization processing plan about HeLa cells According to the principles of cell synchronization, the synchronization processing method about HeLa cells has been put forward, as well as the level of cell synchronization on the following experiments to obtain the HeLa cell samples which meet the need of the test requirement in the cell cycle Phase (G1 phase, S phase, G2 and M phase) .②Do experiments to obtain ultraviolet and visible (UV) absorption spectrum of HeLa cells in different cell cycle phases.Design the synchronization processing plan on ultraviolet and visible absorption spectrum about HeLa cells in different cell cycle phases. Then give ultraviolet and visible light absorption spectrum measurement on HeLa cell samples (190nm ~ 1100nm).The result shows that the spectrum of cell simple in different phases reflects the changing processes of chromophore and auxochrome with the changing HeLa cell cycle. Around 204nm and 260nm, the spectrum has two peaks. The former is caused by the aromatic amino acids, the latter by comprehensive absorption of protein and nucleic acid. These changes show that in the process of HeLa cells’growth ,UV absorption spectrum reflects the close relationship among the aromatic amino acids, purine and pyrimidine derivatives and HeLa cell growth process.③Do experiments to obtain the auto-fluorescence spectrum about HeLa cells in different cell cycle phasesDesign the experiment plan on auto-fluorescence spectrum of HeLa cells in different cell cycle phases. We got the autofluorescence spectrum of the samples exciting by 290nm light. The results indicated that the fluorescence spectrum has two peaks at 360nm and 680nm, the intensity of these two peaks changes in different samples. It shows that the materials producing fluorescence changes with the cell growth, resulting in the changes of fluorescence intensity. Its spectral curve reflects the trend of amino acid and porphyrin changing in HeLa cell cycle.④Build HeLa cell spectral modelAccording to the ultraviolet and visible light absorption spectra measurements about HeLa cells, using the method of biological spetrum modeling, the linear regression model spectrum and multiple linear regression model have been put forward evaluated .These two models both can have a very good response to changes in the HeLa cell cycle in the process of UV absorption spectrum. There will not be further studies about auto-fluorescence spectrum due to the changes of characteristic peak indensity can not be described by linear regression model.Finally, the correct rate of regression model and multiple linear regression model achieved 98%, which determine the simple cell at which phase in cell cycle. It shows that the UV absorption spectrum model can describe the absorbance changes of characteristic peak in cell cycle of HeLa cells.更多还原