【作者】 康立丽；

【导师】 黄耀熊；

【作者基本信息】 暨南大学 ， 生物医学工程， 2008， 博士

【摘要】 随着生命科学探究的逐步深入,对生物体内单细胞的分子水平信息的探测成为生物医学领域一个重要的研究方向。由于拉曼光谱提供的细胞分子水平信息可用以研究其生理活动以及疾病等方面的分子机制与过程,成为用于生物样品研究特别是对活态单细胞进行原位、无扰、实时或近实时研究的有力工具之一。显微激光共焦拉曼散射技术相对于其它技术有比较突出的优点,例如可在无需对被测样品作标记及制备的情况下提供物质的分子组成和结构等样品内在的信息,并可对各种样品包括活细胞进行三维扫描测定每一局域的分子组成与分布。本文采用法国Horbia JY公司生产的倒置激光共焦显微拉曼散射仪（配置514.5nm激发光）,进行基于该技术系统的用于各种生物样品,尤其是活细胞的有关测定技术的研究,并将其应用于红细胞、淋巴细胞进行单细胞水平的拉曼光谱实验研究。本文的主要工作包括:显微激光共焦拉曼散射仪的系统性能参数快速测定和校正方法研究;针对活细胞进行不同扫描模式（点扫描、线扫描与二维扫描）的扫描参数优化研究及扫描结果分析、处理技术研究;在此基础上对活态红细胞和淋巴细胞进行了应用研究。包括不同胞龄红细胞、不同生命状态红细胞和添加唾液酸老龄红细胞拉曼光谱及胞内血红蛋白的分布和携氧能力的测定;900MHz电磁辐射及254nm紫外辐射作用下的淋巴细胞拉曼光谱的测定。根据本文的研究,得到以下结论:（1）为了确保所得样品的拉曼光谱的质量、可信度及提高可比性,需要在实验前进行系统性能参数快速测试和校正工作。快速测试及校正的项目主要包括波数精度、系统稳定性、激光焦点位置和照射到样品的激光功率密度、激光光斑尺寸等。对于需要共焦条件的扫描,还要进行系统共焦性能的测试。（2）进行活态细胞的激光共焦拉曼散射光谱研究时,为了实现原位、基本无扰的测定,需要对不同的实验对象进行扫描参数优化研究。需优化的参数主要包括照射到样品的激光功率密度、曝光积分时间、扫描间隔、共焦孔径等。在扫描参数优化的条件下才可得到细胞样品在其自然生命状态下的拉曼光谱。（3）为了正确并全面地反映拉曼光谱丰富的指纹特性,需要对样品的拉曼光谱进行一系列处理,如宇宙射线消除、谱线的分类、谱线平均和谱线的平滑等。特别是对谱线的分类处理可有效地使对分子构象敏感的谱线所包含的关键信息得以正确解读,凸显拉曼光谱的指纹特性优点。（4）对谱线进行分类、平均处理可更好地表现活态细胞丰富的指纹谱,获得活态红细胞拉曼光谱的多样性信息。且根据氧合标记带的复杂变化,可将红细胞拉曼光谱分为T态、M1、M2、M3、M4和R态,从而便于准确地反映活细胞血红蛋白氧合状态及有关变化过程。此外,结合对在514nm激发波长下属于共振谱线的v₄谱线进行分析,可实现对活细胞氧合状态变化相关的血红蛋白变构效应进行定性及半定量研究。（5）轻龄红细胞、老龄红细胞及添加唾液酸的老龄红细胞的拉曼光谱差异主要为谱线强度的差异、血红蛋白分布的差异、珠蛋白信息的差异及携氧能力的差异。具体为:a)老龄红细胞的拉曼光谱谱线强度较高、信号平滑;轻龄红细胞的拉曼信号较弱,谱线较粗糙:添加唾液酸老龄红细胞的拉曼谱线强度介于轻、老龄红细胞之间。b)轻龄红细胞和添加唾液酸老龄红细胞的胞内血红蛋白分布较为均匀,而老龄红细胞的血红蛋白在细胞膜处易形成高强度拉曼信号,提示老龄红细胞血红蛋白与细胞膜的结合位点发生了交联、聚集。c)老龄红细胞的拉曼光谱体现的珠蛋白信息比轻龄红细胞的明显。老龄红细胞血红蛋白的酪氨酸更容易暴露、其无规卷曲结构增加、有序结构降低:添加唾液酸后的老龄红细胞血红蛋白的酪氨酸处于埋藏状态,而且蛋白的有序结构增加、无序结构减少;轻龄红细胞的拉曼光谱珠蛋白信号较弱。d)老龄红细胞的血红蛋白从T态到R态的跃迁速率比轻龄红细胞的慢,添加唾液酸老龄红细胞的的跃迁速率介于轻、老龄红细胞之间。这表明轻龄红细胞有更好的携氧能力,添加唾液酸的老龄红细胞的携氧能力有所改善。e)添加唾液酸的老龄红细胞的拉曼信号强度、胞内血红蛋白分布、从T态到R态跃迁速率等均有改善。这说明唾液酸不仅对细胞膜起作用,也对细胞内老化的血红蛋白的功能恢复起作用。（6）正常生理环境条件下（PH=7.4）的红细胞的拉曼信号强度较弱,荧光背景较弱;而偏离生理环境PH越大（酸性增加或碱性增加）,红细胞的拉曼光谱信号则越强,其荧光背景也明显增加。酸性条件下还出现755cm^-1谱线的增强。说明血红蛋白处于偏离生理环境PH值的环境下（例如一些病态情形）,分子构象发生变化,影响其功能。（7）低温制备的畸形红细胞的拉曼光谱显示细胞畸形处的拉曼信号异常增高,提示畸变处有血红蛋白的聚集、变性。（8）在900MHz电磁辐射（功率密度5mW/cm²）作用下的淋巴细胞拉曼光谱研究结果表明:短时间的电磁辐射作用（≤20min）不会对离体的淋巴细胞产生明显影响,只会使蛋白物质发生轻微变化,DNA也没有显著变化;辐射时间达到40min后,蛋白质和DNA等的特征谱线出现了变化,表现为疏水氨基酸的暴露、碱基的损伤等;辐射时间达到60min后,蛋白和DNA的拉曼光谱变化更加显著,提示蛋白有序结构的减少、无序结构的增加,DNA出现无序和单链的损伤等;（9）淋巴细胞在功率密度为10W/cm²的254nm紫外线辐射作用时间≤5min时,主要出现微弱的蛋白无序结构的增加、有序结构的减少;辐射时间≥10min时,不仅有蛋白无序结构的增加、有序结构的减少,而且出现一些疏水氨基酸的暴露使蛋白构象改变;一些与碱基有关的谱线由于“增色效应”出现强度增加,表明碱基-碱基堆积作用受到破坏,并可能出现嘧啶二聚体等紫外线作用光产物;辐射时间≥15min时,蛋白无序结构的增加、有序结构的减少的情形更加明显,同样出现疏水氨基酸的暴露;碱基的增色效应更加明显。（10）紫外辐射作用淋巴细胞的拉曼光谱显示出比RF电磁波作用的淋巴细胞更明显的多样性。形成多样性的原因是淋巴细胞拉曼光谱中容易出现包含1156cm^-1和1520cm^-1的谱线（类胡萝卜素物质的拉曼特征谱线）。出现明显的类胡萝卜素物质的拉曼特征谱线的细胞表现的损伤迹象较少。证明淋巴细胞在紫外辐射作用过程中,其细胞质内高尔基体释放类胡萝卜素用于机体的保护,消除自由基、实现抗氧化的作用。随着紫外辐射时间的增加,细胞内类胡萝卜素的含量下降,细胞损伤明显。本论文创新点主要在于:（1）设计了系统性能参数快速测试、校正方法和程序;（2）针对活态红细胞进行了系统的扫描参数优化研究以及数据后处理等多种扫描技术（点扫描技术、线扫描技术和二维扫描技术）的研究。并利用上述扫描技术进行了红细胞和淋巴细胞的实验研究。（3）针对生物样品拉曼光谱的复杂性,为体现样品丰富的指纹谱,在参数优化的基础上提出应对样品的拉曼光谱进行分类再平均的处理方法。（4）利用分类再平均的方法和短曝光积分时间等扫描参数优化的结果首次提出将血红蛋白的拉曼谱线细分为T、M1、M2、M3、M4和R态（提示血红蛋白的氧含量依次增加）;（5）利用活态红细胞拉曼光谱的多样性和v₄谱线的变化对血红蛋白的变构效应进行了定性及半定量的探讨:（6）首次利用激光共焦拉曼光谱散射技术研究了轻、老龄及添加唾液酸老龄红细胞的多种扫描结果差异。设计了简单易行的血红蛋白T态到R态的跃迁速率比较实验,首次实现了对活细胞内有关分子结构动态变化的测定研究,得到了轻、老龄及添加唾液酸老龄红细胞的跃迁速率差异。并对唾液酸对老龄红细胞内血红蛋白的作用机制进行了探讨;（7）首次利用激光共焦拉曼光谱散射技术研究了不同PH值下的红细胞及低温制备的畸形红细胞;证明了血红蛋白会在畸形处发生聚集;（8）首次利用激光共焦拉曼光谱散射技术对900MHz电磁辐射和254nm紫外线辐射作用下的淋巴细胞进行了研究。揭示了超过40分钟的辐射,5mW/cm² 900 MHz电磁辐射会引起淋巴细胞酪氨酸的暴露、巯基基团的激活、蛋白有序结构的减少和无序结构的增加及其碱基的损伤;10W/m²的紫外辐射作用时间达到10min后,淋巴细胞会发生酪氨酸、色氨酸等疏水氨基酸的暴露,α螺旋、β折叠的减少和无规卷曲的增加;且在RF电磁辐射、紫外辐射条件作用下,淋巴细胞会释放类胡萝卜素用于机体保护的过程。并对紫外辐射作用下淋巴细胞拉曼光谱的多样性及细胞内胡萝卜素的变化进行研究。总之,本文发展的活态单细胞显微激光共焦拉曼散射光谱扫描技术,特别是线扫描、二维扫描方法以及分类再平均的谱线处理方法为活态细胞研究提供了有益参考,该技术可广泛应用于其他单细胞的测试。更多还原

【Abstract】 The detection of molecular information of a living cell level has become a major research hot point with the development of life science.The molecular information provided by Raman spectrum can be used for the study of physiological of living creatures,the mechanism and process of diseases.So Raman scatter technique is a powerful tool for biological sample,especially for in situs,noninvasive,and real time or near real time measurement on living single cell.The confocal Raman micro-spectroscopy technique has many advantages over other techniques in doing so.For example,it can provide the information about the molecular component and structure of different samples without special preparation and any labeling,and it can detect the micro-region in living cells with 3D scanning.In this thesis,an inverted confocal Raman scatter spectroscopy system produced by Horbia JY（equipped with 514.5nm laser） was used to study living cells.The work we have done includes:（1） Fast test and calibration of performance parameters for the confocal Raman spectroscopy system in measuring living cells.（2） The techniques for living cells measurement including point scan, line mapping and 2D mapping and the technique of measuring the variation of the molecular conformation with time;（3） The optimization of scan parameters for living cells with different scan modes.The analysis and processing technique of Raman spectrum.（4） Confocal Raman micro-spectroscopy on erythrocytes and lymphocytes based on the work mentioned above.The measurements include:（a） The Raman spectra of erythrocytes with different cell age and states of intracellular Hb.（b） The variation of the Raman spectra of lymphocytes under the radiation of 900MHz electromagnetic filed and 254nm UV exposure respectively.According to the study and the experiment results,we can come to the following conclusions:（1） The fast test and calibration of performance parameters are vital to obtain high quality,reliability and comparable ability of Raman spectra.The wave riuinber precision,the stability:of system,the position of laser spot and the laser power density etc.should be calibrated.For confocal scanning,the confocal performance testing is also needed.（2） In order to meet the requirement of performing in situs,noninvasive measurement,the scanning parameters should be optimized for different samples. The optimized parameters mainly included the;laser power density at sample, exposure time,scan step,pinhole etc.The Raman spectrum of cells in vivo can be achieved with optimization of scan parameters.（3） In order to obtain high quality fingerprint spectrum,a series of process should be taken,such as the deletion of cosmic ray,classification,average and smooth of Raman spectra,etc.The classification of Raman spectra is significant in enhancing the bands,which are sensitive to the structure of molecule to highlight the fingerprint spectrum.（4） The diversity of Raman spectra of erythrocytes had been achieved by our developed method.The Raman spectra of erythrocytes can be divided into T,M1, M2,M3,M4 and R states according to the oxidation band and O₂ level marker band.The different states of Hb’s Raman spectra can show the changing of its structure.Moreover,we qualitatively and semi quantitatively analyzed the allosteric effect of Hb using the v4 band（514.5nm excitation line） in living erythrocytes.（5） Differences in the signal intensity,molecule distribution,globin structure and the ability of carrying O₂ are shown among the young,old and old with added salic acid（old+SA） erythrocytes.（a） For old erythrocytes,its Raman spectrum is very strong and smooth; however,the Raman spectrum of young erythrocytes is relatively weak and coarse. And the result of "old+SA" is between that of young and old erythrocytes.（b） The distribution of Hb in old erythrocytes is not uniform,and exhibits high signal at the membrane of cells.The distributions of Hb in young and "old+SA" erythrocytes are more uniform.The results suggest that crosslink and aggregation of Hb around the cell membrane appeared in old erythrocytes.（c） The Raman spectrum of old erythrocytes shows obvious information of globin.The exposure of tyrosine（Tyr）,the increase of unordered coil structure and decrease of ordered structure happen easily in old erythrocytes.However,in "old+SA" erythrocytes,the Tyr becomes embedded,the ordered structure increases and the unordered structure decreases.The signal of globin in young erythrocytes is very weak.（d） The transition speed of the Hb from T（tense） state to R（relaxed） state in old erythrocytes is slower than that of young ones,and the results of "old+SA" is intervenient.The T to R transition results shows that young erythrocytes have better ability of carrying oxygen than that of old ones.SA improves the ability of carrying oxygen in old erythrocytes.（e） SA not only decreases the intensity of Hb’s Raman spectrum in old erythrocytes,but also improves the uniformity of the Hb distribution and the T to R transition speed in old ones,which indicates the SA has effect on the function of Hb in aging erythrocytes.（6） The intensity and background of the Raman signal of erythrocytes under the normal condition（PH=7.4） are weaker than that of erythrocytes under the acidic （PH＜7.0） or alkaline（PH＞7.6） condition.Greater the deviation from normal condition,higher the intensity and background counts.The intensity of the band at 755cm^-1 increases with the decrease of PH value in acidic situation.This suggests the structure and function of Hb will change if the PH value is deviant（for example,sickness）.（7） The Raman spectra of distorted erythrocytes exhibits dramatical enhance at the distortion which suggests the aggregation and denaturation of Hb.（8） The results of lymphocytes under 900MHz electromagnetic field shows there are no obvious change in their Raman spectra,only appeared slight change in protein when the exposure time is short（less than 20min）.However,if the exposure time were up to 40min,the characteristic bands of Raman spectra in protein and DNA would change.For example,the hydrophobic amino acid appeared exposed,the base damage etc.When the exposure time is up to 60min, there would be more changes in the Raman spectra of protein and DNA,such as the decrease of ordered structure,the increase of unordered structure and damage in single chain.（9） When the time of 254nm UV（power density was 10W/cm²） exposure to lymphocytes is equal to or less than 5min,there are slight increase in the unordered structure and slight decrease in the ordered structure in protein;When the exposure time is longer than 10min,not only the situation mentioned above would developed further,but also the hydrophobic amino acid would be exposed and the intensity of some bands increase for the reason of "hyperchromic effect".The results indicated the base-base effect was damaged and the CPD may be appeared as the photoproduct of UV.When the exposure time is up to 15min,the changes mentioned above become more obvious.（10） More divers Raman spectra for the lymphocytes under UV exposure than those under RF radiation based on analysis of the bands at 1156cm^-1 and 1520cm^-1. The two bands were assigned to the carotenoids,when the bands of carotenoids appeared,the cells is slightly damaged,indicating that the lymphocytes have released carotenoids at Gall bodies to protect themselves and eliminated the free radical as the antioxygen agent.With the UV exposure time increasing,the amount of carotenoids in cell decreases and the damage become obvious.Our innovations in this work are as follow:（1） Designing a fast method and sequence of the test and calibration of Raman confocal micro-spectroscopy.（2） Optimization of the scanning parameters and data process for point scan,line scan and 2D scan modes.And apply the technique on erythrocytes and lymphocytes measurements.（3） Have developed the classification and average method to incarnate the abundant fingerprint spectrum.（4） Classify the Raman spectrum of Hb into 6 states:T,M1,M2,M3,M4 and R based on the classification and average method and parameters’ optimization.（5） Have performed qualitative and semi-quantity analysis of alloestric effect of Hb in living erythrocytes based on the change of v₄ band in Raman spectrum of erythrocytes.（6） Report the Raman spectrum of young,old and old+SA erythrocytes first time. Design an easy and practical method to do the T to R state transition experiment and realize dynamic test on the variation of the molecular structure in living cells.（7） Report the Raman spectra of erythrocytes at different PH value and the distortion erythrocytes first time.（8） Study the effects of 900MHz electromagnetic field and 254nm UV on the lymphocytes with the confocal Raman micro-spectroscopy,and report the diversity of Raman spectrum of lymphocytes and the carotenoids changes after the radiations.In summary,the confocal Raman micro-spectroscopy we developed for single living cell measurements,especially the line scan,2D scan methods,classification and average method can provide helpful tool in living cell studies,and they are expected to have a variety of application on the measurements of different single living cells.更多还原