单个活态细胞的显微激光共焦拉曼光谱扫描技术及应用研究
|
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v_4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/cm~2)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/cm~2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v_4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/cm~2 900 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/m~2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。
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 O_2 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 O_2 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/cm~2) 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 v_4 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.
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v_4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/cm~2)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/cm~2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v_4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/cm~2 900 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/m~2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。
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 O_2 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 O_2 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/cm~2) 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 v_4 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.
引文
1.Carey P.R.,Biochemical Applications of Raman and Resonance Raman Spectroscopies.Academic Press,1982,第二版,6-132.
2.Michael J.P Analytical application of Raman spectroscopy.Blackwell Science,2005,第二版,1-282.
3.许以明.拉曼光谱及其在结构生物学中的应用.北京:化学工业出版社,2005,第1版.11-32.
4.Doom S.K.,O'Connell M.J.,Zheng LX.,T Y.T.,Huang Sh.M and Liu J.Raman Spectral Imaging of a Carbon Nanotube Intramolecular Junction.Physical View Letters.PRL94,2005:016802-1-4.
5.Confocal Raman Microscopy - Imaging in the Pharmaceutical Industry.WITEC Report.
6.Jouko Vyorykka,Confocal Raman microscopy in chemical and physical characterization of coated and printed papers.Thesis,Technische University,2004.
7.Ling J.Direct Raman Imaging Techniques for Studying the Subcellular Distribution of a Drug.Applied Optics.2002,41(28):6006-6017.
8.Feofanov A.V.,Grichine A.I.,Shitova L.A.,Karmakova T.A.,Yakubovskaya R.I.Confocal Raman Microspectroscopy and Imaging Study of Theraphthal in Living Cancer Cells.Biophys.J,2000,78(1):499-512.
9.Charonov S.,Chourpa I.,Valisa P.and Manfait M.Confocal Raman microscopic imaging of biological samples.Proc.SPIE.1997,2980:210-216.
10.Igor N.,Igor C.and Mivhel M.Applications of Raman and surface-enhanced Raman scattering spectroscopy in medicine.J.Raman Spectrosc.1994,25:13-23.
11.Reinhard S.S.Structure and dynamics of biomolecules probed by Raman spectroscopy.J.Raman Spec.2005,36:276-278.
12.Wong P.T.,Wong P.K.,Caputo T.A.,Godwin T.A.and Rigas B.Infrared spectroscopy of exfoliated human cervical cells:Evidence of extensive structural changes during carcinogenesis.Proc.Natl.Acad.Sci.1991,8(24):10988-10992.
13.Frank C.L.,Redd D.C.B.,Gansler T.S.AND Mccreery R.L.Characterization of human breast specimens with near-IR Raman spectroscopy.Anal Chem.1994,66:319-326.
14.Anita M.,Rebecca R.Raman spectroscopy for the detection id cancers and precancers.J. Biomed.Opt.1996,1:31-70.
15.Stewart S.,Shea D.A.,Tamowski C.P.,Morris M.D.,Wang D.,Franceschi R.,Lin D.L.and Keller E.Trends in early mineralization of murine calvarial osteoblastic cultures:a Raman microscopic study.J.Raman Spectrosc.2002,33:536-543.
16.Schaeberle M.D.,Kalasinsky V.F.,Luke J.L.Levin I.W.and Treado P.J.Raman chemical imaging:Histopathologe of indusions in human broart tissue.Anal Chem.1996,68:1829-1833.
17.许以明,徐国瑞,杨先春和张志义.三种卟啉类药物对DNA光敏损伤的比较.辐射研究与辐射工艺学报,1989,7(4):60-62.
18.Chenery D.and Bowring H.Infrared and Raman spectroscopic imaging in biosciences.Spectroscopy Europe.2003,15(4):8-14.
19.Puppels G.J.,De Mul F.F.M.,Otto C.Greve J.Studying single living cells and chromosomes by confocal Raman microspectroscopy.Nature.1990,347:301-303.
20.Puppels G.J.,Bakker Schut T.C.,Sijtsema N.M.,Grond M.,Maraboeuf F.,de Grauw C.G.,Figdor C.G.,and Greve J.Development application of Raman microspectroscopic and Raman imaging techniques for cell biological studies.Mole.Struc.1995,347:477-473.
21.Caspers P.J.,Lucassen G.W.and Puppels G.J.Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin.Biophysical J.2003,85:572-582.
22.Puppels G.J.,Garritsen H.S.P.,Kummer J.A.and Greve J.Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy.Cytometry.1993,14(3):251-256.
23.Puppels G.J.,Garritsen H.S.P.,Segers-Nollten G.M.J.,de Mul F.F.M.and Greve J.Raman microspectroscopic approach to the study of human granulocytes.Biophys.J.1991,60:1046-1056.
24.Puppels G.J.,Bakker Schut T.C.,Sijtsema N.M.,Grond M,Maraboeuf F.,de Grauw C.G.,Figdor C.G.and Greve J.Development and application of Raman microspectroscopic and Raman imaging techniques for cell biological studies.J.of Molecular Structure.1995,347:477-484.
25.Rosch P.,Harz M.,Schmitt M.and Popp J.Raman spectroscopic identification of single yeast cells.J.Raman Spectrosc.2005,36:377-379.
26.Salmaso B.L.N.,Puppels G.J.,Caspers P.J.,Floris R.,Wever R.,Greve J.Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human cosinophilic granulocytes.Biophysical J.1994,67(1):436-446.
27.Huang Y.S.,Karashima T.,Yamamoto M.,Ogura T.,Hamaguchi H.Raman spectroscopic signature of life in a living yeast cell.J.Raman Spectrosc.2004,35:525-526.
28.Sijtsema N.M.,Wouters S.D.,De Grauw C.J.,Otto C.,and Greve J.Confocal direct imaging Raman microscope:design and applications in biology.Appl.Spectrosc.1998,52:348-355.
29.Sijtsema N.M.Towards chemical imaging of living cells:design and application of a confocal Raman Microscope.[Thesis]Chap.1,University of Twente,1997
30.Wood B.R.,Caspers P.,Puppels G.J.,Pandiancherds S.and McNaughton D.Resonance Raman spectroscopy of red blood cells using near-infrared laser excitation.Anal.Bioanal Chem.2007,387(5),1691-1703.
31.Wood B.R.,Hammer L.,Davis L.and McNaughton D.Raman microspectroscopy and imaging provides insights into heme aggregation and denaturation within human erythrocytes.J.Biomed.Opt.2005,10(1):014005.
32.Wood B.R.,McNaughton D.Raman excitation wavelength investigation of single red blood cells in vivo.J.Raman Spec.2002,33:517-523.
33.Wood B.R.,McNaughton D.Micro-Raman Characterization of High- and Low-Spin Heme Moieties within Single Living Erythrocytes.Biospectroscopy.2002,67:259-262.
34.Cherney D.P.,Conboy J.C.and Harris J.M.Optical-trapping Raman microscopy detection of single unilamellar lipid vesicles.Anal.Chem.2003,75(23):6621-6628.
35.Creely C.M.,Singh G.P.and Petrov D.Dual wavelength optical tweezers for confocal Raman spectroscopy.Opt.Commun.2005,245:465-470.
36.Creely C.M.,Volpe G.,Singh G.P.Raman imaging on floating cells.Optics Express.2005,13(16):6105-6110.
37.Ramser K.,Logg K.,Goksor M.,Enger J.,Kall M.and Hanstrop D.Resonance Raman spectroscopy of optically trapped functional erythrocytes.J.Biomed.Opt.2004,9(3):593-600.
38.于敏华,包晓群和王卫东.脑脊液激光喇曼光谱及荧光光谱的试验观察.激光杂志,1996, 17(4):207-209.
39.杨祥良,徐辉碧,韩秀娴和孙奕.正常人胎儿晶体与老年性白内障晶体的激光喇曼光谱研究.激光技术,1995,19(6):382-384.
40.赵元黎,吕晶,申培红,梁二军,姚淑霞和葛向红.乳腺肿瘤组织的拉曼光谱研究.激光与红外.2004,34(6):502-504.
41.闫循领,董瑞新,王秋国,陈双峰,张宗旺,张学俊和张雷.乳腺癌病人单个细胞的Raman光谱.光谱学与光谱分析.2005,25(1):58-61.
42.张京伟,沈爱国,魏云,王小华,胡继明和叶勇.胃癌和胃正常黏膜拉曼光谱检测.生物医学工程学杂志.,2004,21(6):910-912.
43.吕刚,宫衍香,马传涛,和李长富.红细胞的拉曼光谱研究.德州学院学报.2005,21(6):23-25.
44.赵元黎,吕晶,葛向红,姚淑霞和梁二军.乳腺癌周边组织的拉曼光谱研究.光谱学与光谱分析.2006,26(7):1267-1271.
45.http://isl.lanl.gov/raman.html
46.Shelnutt J.A.,Rousseau D.L.,Friedman J.M.and Simon S.R.Protein-heme interaction in hemoglobin:Evidence from Raman difference spectroscopy.Natl.Acad.Sct.1979,76(9):4409-4413.
47.Brunner H.,Mayer A.and Sussner H.Resonance Raman scattering on the haem group of oxy- and deoxyhaemoglobin.J Mol.Biol.1972,70:153-156.
48.Brunner H.and Sussner H.Resonance Raman scattering on haemoglobin.Biochim.Biophys.Acta.1973,310:20-31.
49.Wood B.R.,Tait B.and McNaughton D.Micro-Raman characterization of the R to T state transition of haemoblobin within a single living erythrocyte.BBA.2001,1539:58-70.
50.Ramser K.,Bjerneld E.J.,Fant C.and Kall M.Raman imaging and spectroscopy of single functional erythrocytes:a feasibility study.Proc.SPIE.2002,4614:20-27.
51.Bulkin B.J.Raman spectroscopic study of human erythrocyte membranes.Biochimica et Biophysica Acta.1972,274:649.
52.Xu Y.M.,Zhao H.X.and Zhang ZH.Y.Raman spectroscopic study of microcosmic and photosensitive damage on the liposomes of the mixed phospholipids sensitized by hypocrellin and its derivatives.Photochem.Photobiol.B:Biology.1998,43:41-46.
53.Albrecht A.C.and Hurley M.C.On the Dependence of Vibrational Raman Intensity on the Wavelength of Incident Light.J.Chem.Phys.1971,55(9):4438-4443.
54.程光熙,拉曼布里渊散射,北京:科学出版社,2003,第一版,714-727.
55.Confocal Raman Microscopy.Raman Products Technical note,No.1350.
56.Zhang J.SH.,Sun CH.D.and Lu D.Sets effect on Zn.Acta Photonica Sinica.1999,28(6):551-554.
57.任斌 田中群 表面增强拉曼光谱的研究进展.现代仪器,2004,5:1-8,13.
58.Fleischmann M.,Hendra P.J.and McQuillan A.J.Raman spectra of pyridine adsorbed ata silver electrode.Chem.Phys.Lett.1974,26:163-166.
59.Moskovits M.Surface enhanced Raman Spectroscopy.Rev.Mod.Phys.1985,57(3):783-826.
60.Otto A.,Mrozek I.,Grabhorn H.and Akemann W.Surface enhanced Raman scattering.J.Phys.:Condensed Matter.1992,4:1143-1212.
61.Puppels G.J.,Olminkhof JH,Segers-Nolten G.M.,Otto C.,de Mul F.F.Greve J.Laser irradiation and Raman spectroscopy of single living cells and chromosomes:sample degradation occurs with 514.5nm but not with 660nm laser light.Exp.Cell.Res.1991,195:361-367.
62.Ramser K.,Bjerneld E.J.,Fant C.,Kaell M.Importance of substrate and photo-induced effects in Raman spectroscopy of single functional erythrocytes.J.BioMed.Opt.2003,8(2):173-178.
63.Hu S.ZH,Smith K.M.,Spiro T.G.Assignment of Protoheme Resonance Raman Spectrum by Heme Labeling in Myoblobin.J.Am.Chem.Soc.1996,118:12638-12646.
64.Tantra R.,McCabe A.,Bailey M.et al.Comparable Raman Spectroscopy Across Different Instruments and Excitation Wavelengths.NPL REPORTDQL-AS012,2004.
65.Horiba JY公司质量控制手册
66.降雨强,李昌勇,周源,张临杰,肖连团,贾锁堂,马存根和周国生.低强度激光血管内照射治疗的机理初探.激光生物学报.10(3):209-211,2001.
67.陈敏,骆清铭.弱激光的生物学效应及对红细胞变形性的改善作用.激光生物学报.11(1):55-57,2002.
68.http://en.wikipedia.org/wiki/Red_blood_cell
69.姚泰,生理学,北京:人民卫生出版社,2001,第三版.
70.Perutz M F.Molecular anatomy,physiology,and pathology of hemoglobin.In:Molecular basis of blood Diseases.Philadelphia,W B Saunders Co,1987,127
71.龙桂芳,张俊武.血红蛋白与血红蛋白病.广西:广西科学技术出版社.2003年,第一版.
72.张之南,李蓉生.红细胞疾病-基础与临床.北京:科学出版社.2000年,第一版.
73.Stryer L.Biochemistry.,New York,W.H.Freeman and Company,3rd.ed.1988.
74.Guido Guidotti.Studies on the chemistry of hemoglobin Ⅲ The interactions of the aβsubunits of hemoglobin.The journal of Biological Chemistry.1967.242(16):3694-3703
75.Guido Guidotti.Studies on the chemistry of hemoglobin Ⅳ The mechanism of reaction with ligands.The journal of Biological Chemistry.1967,242(16):3704-3712
76.Streaks T.C.and Spiro T.G.Hemoglobin:Resonance Raman Spectra.Biochim.Biophys.Acta.1972,263:830-833.
77.Strekas T.C.and Spiro T.G.Cytochrome c:Resonance Raman Spectra.Biochim.Biophys.Acta.1972,278:188-192.
78.Spiro T.G.and Strekas T.C.Resonance Raman scattering of heme proteins.Effect of oxidation and spin/state.Am.Chem.Soc.1974,96(2):338-345.
79.Tones Filho I.P.,Terner J.,Pittman R.N.,Somera L.G.and Wa(?)d K.R.Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy.Am.J.Phys.Heart.Circ.Phys.2005,289:488-495.
80.Aono,S.,Kato T.,Matsuki M.,Nakajima H.,Ohta T.,Uchida T.And Kitagawa T.Resonance Raman and Ligand Binding Studies of the Oxygen-sensing Signal Transducer Protein HemAT from Bacillus subtilis.J.Biol.Chem.2002,277(16):13528-13538.
81.Bruzzese F.J.,Dix J.A.,Rava R.P.and Cerny L.C.Resonance Raman spectroscopy of chemically modified haemoglobins.Biomater Artif.Cells Artif.Organs.1990,18:143-156.
82.Zhao X.J.,Spiro T.G.Ultraviolet resonance Raman spectroscopy of Hemoglobin with 200and 212nm excitation:H-bonds of Tyrosines and Prolines.J.Raman spectroscopy.1998,29:49-55.
83.Engler N.,Ostermann A.,Gassmann A.,et al.Protein Dynamics in an Intermediate State of Myoglobin:Optical Absorption,Resonance Raman Spectroscopy,and X-ray Structure Analysis.Biophys.J.2000,78(4):2081-2092.
84.De Paula J.C.Raman spectroscopy of metalloporphyrins,http://www.haverford.edu/chem
85.Monod J.,Wyman J.and Changeux J.P.On the nature of allosteric transitions:a plausible model.J.Mol.Biol.1965,12:88-118.
86.Koshland D.E.,Nemethy G.and Filmer D.Comparison of experimental binding data and theoretical models in proteins containing subunits.Biochemistry.1966,5:365-385.
87.Baldwin J.M.Structure and function of haemoglobin.Prog.Biophys.Mol.Biol.1975,29:225-320.
88.Ackers G.K.,Dalession P.M.,Lew G.H.,Daugherty M.A.and Holt J.M.Single residue modification of only one dimmer within the hemoglobin tetramer reveals autonomous dimmer function.Proc.Natl.Acad.Sci.2002,99(15):9777-9782.
89.Rhoda E.H.,Margaret J.L.,Gediminas V.A.V.,Shuocai H.,Joel M.F.and Ronald L.N.Conformational changes in oxyhemoglobin C(Gluβ6 →Lys) detected by spectroscopic probing.J.Bio.Chem.1996,271(1):372-375.
90.Mihailescu M.R.and Russu I.M.A signature of the T → R transition in human hemoglobin.PNAS.2001,98(7):3773-3777.
91.萨出尔夫,罗辽复和李前忠.血红蛋白分子氧合过程的构象变化特性.内蒙古大学学报,2003,34(6):648-652.
92.Xu Ch.Y.,Tobi D.and Bahar I.Allostedc Changes in Protein Structure Computed by Simple Mechanical Model:Hemoglobin T←→R2 Transition,J.Mol.Biol.2003,333:153-168.
93.Xu CH-Y,Tobi D.and Bahar I.Allosteric changes in protein structure computed by a simple mechanical model:Hemoglobin.J.Mol.Biol.2003,333:153-168.
94.Shigenori N.,Masako N.,Yasuhisa M.,Takashi Y.and Teizo K.Quaternary structure of intermediately ligated huaman hemoglobin A and influence from strong allosteric effectors:Resonance Raman investigation.Biophys.J.2005
95.Nagatomo S.,Nagai M.,Mizutani Y.,Yonetani T.And Kitagawa T.Quaternary Structures of Intermediately Ligated Human Hemoglobin A and Influences from Strong Allosteric Effectors:Resonance Raman,Biophy.J.2005,89:1203-1213.
96.Danon D.and Marikovshy Y.Difference of chafe electrophoresis at surface erythrocytes jeunes and agees et al.Compt.Rend Acad.Sci.1961,253:1271-1272.
97. Lutz H.U., Stammler P., Fasher S., Ingold M. and Fehr J. Density separation of human red blood cells on self forming percoll Gradients: Correlation with cell age. Biochem Biophys Acta. 1992,116:1-10.
98. Kocsis A., Biro E., Racz E., Kiss A. and Kappelmayer J. Mean corpuscular volume is not a reliable marker of red cell age in case of anisocytosis. Annu. Hematol. 1998, 77(4): 149-151.
99. Simpson L.O. and O'Neill D.J. Red cell shape changes in the blood of people 60years of age and older imply a role for blood rheology in the aging process. Derontology. 2003,49(5): 310-315.
100. Sutera S.P., Gardner R.A., Boylan C.W., Carroll G.L, Chang K.C., Marvel J.S., Kilo C., Gonen B. and Williamson J.R. Age related changes in deformability of human erythrocytes. Blood, 1985,65:275-282.
101. Waugh R.E., Narla M., Jackson C.W., Mueller T.J., Suzuki T. and Dale G.L. Rheologic properties of senescent erythrocytes: Loss of surface area and volume with red blood cell age. Blood. 1992, 79:1351-1358.
102. Canham, P.B. Difference in Geometry of Young and Old Human Erythrocytes Explained by a Filtering Mechanism. Circ Res. 1969,25:39-45.
103. Rettig M.P., Low P.S., Gimm J.A., Mohandas N., Wang J.ZH. and Christian J.A. Evaluation of biochemical changes during in vivo erythrocyte senescence in the dog. Blood. 1999,93: 376-384.
104. Knowles D.W., Chasis JA, Evans EA, Mohandas N. Cooperative action between band 3 and glycophorin A in human erythrocytes: Immobilization of band 3 induced by antibodies to glycophorin A. Biophys J. 1994; 66:1726-1732.
105. Hiroshi I., Effects of abnormal Hb on red cell membranes. Rinsho. Byori. 1999, 47(3): 232-237.
106. Ando K., Beppu M, Kikugawa K, Nagai R. and Horiuchi S. Membrane proteins of human erythrocytes are modified by advanced glycation end products during aging in the circulation. Biochem Biophys Res. Commun. 1999, 258(1): 123-127.
107. Kiefer C.R., Trainor J.F. Mckenney J.B., Valeri C.R. and Snyder L.M. Hemoglobin-spectrin complexes: interference with spectrin tetramer assembly as a mechanism for compartmentalization of Band 1 and Band 2 Complexes.Blood.1995,86(1):366-371.
108.程正江 人带3蛋白结构和功能研究进展.国外医学生理、病理科学与临床分册,2000,20(5):377-380.
109.Salhany J.M.,Cordes K.A.and Sloan R.L.Characterization of the pH dependence of hemoglobin binding to band 3:evidence for a pH-dependent conformational change within the hemoglobin-band 3 complex.Biochim Biophys Acta.1998,1372(1):107-113.
110.余国宇,田兴亚.衰老红细胞与带3蛋白.昆明医学院学报,2004,2:110-113.
111.Rauenbuehler P.,Cordes K.A.and Salhany J.M.Identification of the hemoglobin binding sites on the inner surface of the erythrocytes membrane.Biochim.Biophys.Acta.1982,692:361-370.
112.陈兴尧.红细胞表面电荷对其结构与功能参数的影响(硕士学位论文).暨南大学,2006.
113.Chen X.Y.,Huang Y.X.,Liu W.J.,Yuan Zh.J.Membrane surface charge and morphological & mechanical properties of young and old erythrocyte.Current Applied Physics.2007,7(1):94-96.
114.郑新景.量子点对细胞表面电荷的标记(硕士学位论文) 暨南大学,2007.
115.刘文静 新型快速荧光动态图像摄录与处理分析系统及其对活细胞的应用研究(博士学位论文) 暨南大学,2006.
116.Nathan Sharon,Halina Lis.LECTINS.Kluwer Academic Publishers,2003,Second Edition..
117.Bulai T.,Bratosin D.,Pins A.,Montreuil J.Zanetta J.P.Diversity of the human erythroeyte membrane sialic acids in relation with blood groups.FEBS Letters,2003;534:185-189.
118.Brooks S.A.,Dwek M.V.and Schumacher U.Functional & Molecular Glycobiology.BIOS Scientific Publishers Limited,2001.
119.Lutz H.U.and Fehr J.Total sialic acid content of glycophofins during senescence of human red blood cells.J.Bio.Chem.1979,254(22):11177-11180.
120.C.Traving,R.Schauer.Structure,function and metabolism of sialic acids.Cell.Mol.Life Sci.1998;54:1330-1349.
121.Bennett E.,Urcan M.S.,Tinkle S.S.,Koszowski A.G..and Levinson S.R.Contribution of sialic acid to the voltage dependence of sodium channel gating. A possible electrostatic mechanism. J Gen. Physiol. 1997; 109 (3): 327-343.
122. Ufret-Vinenty CA, Baro DJ, Santana LE Differential contribution of sialic acid to the function of repolarizing K+ currents in ventricular myocytes. Am. J. Physiol Cell Physiol. 2002; 281 (2): 464-474.
123. Thampoe JI, Furukawa K, Vallve E and Lloyd K.O. Sialyltransferase levels and ganglioside expression in melanoma and other cullured human cancer cells. Cancer Res. 1989,49: 6258-6264.
124. Kanai A., Sutherland D.R., Fibach E., Matta K.L., Hindernburg A., Brockhausen I., Kuhns W., Taub R.N., van den Eijnden D.H. and Baker M.A. Human leukemic myelobasts and myeloblastoid cells contain the enzyme cytidine 5-monophosphate-N-acetylneuraminic acid: Gal β 1-3 GalNAC α(2,3)-sialyltransferase. Cancer Res. 1990; 50: 5003-5007.
125. Percoll Methodology and Applications, Handbooks from Amersham Biosciences, 18-1115-69.
126. Erslev AJ and Beutler E. Production and destruction of erythrocytes. William Hematology, fifth edition, McGrew-Hill, Inc. New York, 1995,425-440.
127. Kosztolanyi G. and Jobst K. Electrokinetic analysis of the fetal erythrocyte membrane age trypsin digestion. Pediatric Research. 1980,14:138-141.
128. Edwards M. and Rigas D.A. Electrolyte- liable increase of oxygen affinity during in vivo aging of hemoglobin. J. Clinic. Investi. 1967,46(10): 1579-1588.
129. Baskurt O.K., Tugral E., Neu B. and Meiselman H.J. Particle electrophoresis as a tool to understand the aggregation behavior of red blood cells. Electrophoresis. 2002; 23 (13): 2103-2109.
130. Miles J.D. and Demetrios A.G. Electrolyte-Habile increase of oxygen affinity during in vivo aging of Hemoglobin. J. Clinic. Investi. 1967,46(10):1579-1588.
131. Iwona C., Yoji S. and Norihiko T. Changes of RBC aggregation in oxygenation-deoxygenation: pH dependency and cell morphology. Am J Physiol Heart Circ Physiol. 2003, 284: 2335-2342.
132. Condon M.R., Kim J.E., Deitch E.A., Machiedo G.W., Appearance of an erythrocyte population with deceased deformability and hemoglobin content following sepsis. American Journal of Phsiology.2003,284(4):2177-2184.
133.张秀琳,谢益宽.膜表面电荷学说及唾液酸对Na+、K+通道门控特性的影响.生理科学进展,2005,35(2):167-169.
134.姚成灿,姚平,黄耀熊.环境温度对人红细胞膜形态和表面电荷的即时影响.暨南大学学报(自然科学版),2005(2):689-693.
135.姚成灿,黄耀熊,李校坤,阮萍.PH值对红细胞膜力学特性和胞内蛋白结构和功能的影响 科学通报.2003,48(10):1050-1053.
136.Frans L.A.W.,Bregt R.S.,Yvonne A.M.G.,Hemoglobin loss from erythrocytes in vivo results from spleen-facilitated vesiculation.Blood.2003,101:747-751.
137.吴正洁.温度对人红细胞及血红蛋白结构功能的影响(硕士学位论文) 暨南大学,2007.
138.http://info.med.hc360.com/html/001/005/002/23802.htm
139.Mashevich M,Folkman D,Kesar A.Barbul A.,Korenstein R.,Jerby E.and Avivi L.Exposure of human peripheral blood lymphocytes to electromagnetic fields associated with celluar phones leads to choromosomal instability.Bioelectromagnetics.2003,24(2):82-90.
140.Caraj-VrhovacV,Fucic A,HorvatD.The correlation between the frequency of micronuclei and specific chromosome aberrations in human lymphocytes exposed to microwave radiation in vitro.Mutat Res.1992,281(3):181-186.
141.马菲,熊鸿燕,张耀和杨丽华.高强度电磁辐射对长期暴露人群血液成分的损伤效应研究.疾病控制杂志.2005,9(5):437-440.
142.Ramanauskaite R.B.,Segers-Nolten I.G.M.J.,Grauw K.J.,Sijtsema N.M.,van der Maas L.,Greve J.,Otto C.and Figfor C.G..Carotenoid levels in human lymphocytes measured by Raman micro spectroscopy.Pure & Appl.Chem.1997,69(10):2131-2134.
143.姚成灿,姚平,唐国汉,郑青,李校坤和黄耀熊.射频电磁波与环境温度对单个活态红细胞膜力学性质的影响.中国职业医学.2005,32(3):11-13.
144.姚成灿.900MHz电磁波和有关理化因素对人红细胞的即时作用(博士学位论文).暨南大学,2004.
145.刘齐海.900MHz电磁波对淋巴细胞的作用(硕士学位论文).暨南大学,2006.
146.丁振华,范建中.紫外辐射生物学与医学.北京:人民军医出版社,2000:16-21.
147.Erfurth S.C.and Peticolas W.L.Melting and premelting phenomenon in DNA by laser Raman scattering.Biopolymer.1975,14:247-264.
148.Ojeda F.,Guarda M.I.,Lovengreen C.,Hidalgo M.A.,Folch H.,Hartel S.and Maldonado C.Ultraviolet exposure of thymcytes:selective inhibition of apoptosis.Int.J.Radiat.Biol.2004,80(6):445-450.
149.Zoltan N.,Attlia B.,Gyorgyi R.Eva P.,Dobozy A.And Kemeny L.Efficacy of different UV-emitting light sources in the induction of T-cell apoptosis.Photochemistry and photobiology.2004,79(5):434-439.
150.Morison W.L.,Parrish J.A.,Bloch K.J.and Krugler J.I.In vivo effect of UV-B on lymphocytes function.Britsh J.Dermatology.1979,101(5):513.
151.孙鼎文.紫外辐射(一).紫外辐射的主要效应及紫外辐射源.现代计量测试.1999,15-16.
152.周华和朱惠刚.紫外辐射对免疫功能的影响.上海环境科学.1996,15(6):37-39.
153.何慧和孙晓青.紫外线照射照射诱导人外周血淋巴细胞凋亡的研究.中华理疗杂志.2000,23(1):24-26.
154.余萍 和 李京培 紫外线照射对人外周血细胞凋亡及吞噬功能的观察.安徽医科大学学报.2002,37(5):377-379.
155.Spielberg H.,June C.H.,Blair O.C.,Nystrom-Rosander C.,Cereb N.and Deeg H.J.UV irradiation of lymphocytes triggers an increase in intracellular Ca2+ and prevents lectin-stimulated Ca2+ mobilization:evidence for UV- and nifedipine-sensitive Ca2+channels.Exp.Hematol.1991,19(8):742-748.
156.Berliner J.,Himes S.W.,Aoki C.T.and No A.The sites of unscheduled DNA synthesis within irradiated human lymphocytes.Radiation Research.1975,63:544-552.
157.Schieven G.L.,Kirihara J.M.,Gilloland L.K.,Uckun F.M.and Ledbetter J.A.Ultraviolet radiation rapidly induces tyrosine phosphorylation and calcium signaling in lymphocytes.MBC.1993,4(5):523-530.
158.Goettsch W.,Garssen J.,Hurks H.M.H.,Gruijl F.R.de,Loveren H.van.Effects of in vitro UV-B exposure on the immune system:with special emphasis on T cell activity.RIVM Report 850017002,1993.
159.许以明,张志义,赵克俭,张仲伦.高能质子辐射作用的分子激励-DNA空间结构微观损伤的拉曼光谱特征.中国科学(B辑).1991,12:1273-1278.
160.Xu Y.M.,Zhou ZH.X.,Yang H.Y.,Xu Y.and Zhang ZH.Y.Raman spectroscopic study of microscomic photodamage of the space structure of DNA sensitized by Yangzhou hematoporphyrin derivative and hotofrin Ⅱ.J.Photochem Photobio B:Biology.1999,52:30-34.
161.Morrion H.Bioorganic Photochemistry.Vol 1.New York:John Wiley and Sons,1990.
162.Wang S.Y.Photochemistry and photobiology of Nucleic Acids.Vol 1 and 2.New York:Awadcmic Press,1976.
163.Oison J.A.Molecular action of cartenoids in:Canfield LM(ed) Carotenoids in Human health.Annuls of New York the Axademy of Sciences.1993,31(691):156-166.
164.Edge R.and Truscott T.G Carotenoids - Free radical interactions.Spectrum,2000,3:12-20.
165.Hughes D.A.Effects of carotenoids on human immune function.Pro.Nutr.Soc.1999,58:713-718.
166.Herraiz L.A.,Hsieh W.C.,Parker R.S.,Swanson J.E.,Bendich A.and Roe D.A.Effect of UV exposure and β-carotene supplementation on delayed-type hypersensitivity response in healthy older men.J.American.College of Nutrition.1998,17(6):617-624.
167.宋雁.类胡萝卜素抗氧化和促氧化作用的影响因素.卫生研究.2003,32(4):417-419.
168.唐刘蕴泉.β-胡萝素应用的研究进展.肠内肠外营养.2005,12(2):121-123.
169.赵文恩,韩雅珊,戴蕴青 类胡萝卜素的生物学性能.生物学杂志.1998,15(3):1-3.
170.范晓岚,杨军,糜漫天和杨慧.β-胡萝素的抗氧化作用于疾病预防.中国公共卫生.2003,19(4):479-480.
171.张世平,张颖,栗学军,栗兰珍,张兴志和刘基芳.β胡萝素和维生素E抗辐射作用研究的临床应用.白求恩医科大学学报.2000:26(5):509-510.
172.李怡兰,乔珊珊,周蕾,姜淑清和李国星.β-胡萝素的抗辐射作用.环境与健康杂志.2004,21(5):300-302.
2.Michael J.P Analytical application of Raman spectroscopy.Blackwell Science,2005,第二版,1-282.
3.许以明.拉曼光谱及其在结构生物学中的应用.北京:化学工业出版社,2005,第1版.11-32.
4.Doom S.K.,O'Connell M.J.,Zheng LX.,T Y.T.,Huang Sh.M and Liu J.Raman Spectral Imaging of a Carbon Nanotube Intramolecular Junction.Physical View Letters.PRL94,2005:016802-1-4.
5.Confocal Raman Microscopy - Imaging in the Pharmaceutical Industry.WITEC Report.
6.Jouko Vyorykka,Confocal Raman microscopy in chemical and physical characterization of coated and printed papers.Thesis,Technische University,2004.
7.Ling J.Direct Raman Imaging Techniques for Studying the Subcellular Distribution of a Drug.Applied Optics.2002,41(28):6006-6017.
8.Feofanov A.V.,Grichine A.I.,Shitova L.A.,Karmakova T.A.,Yakubovskaya R.I.Confocal Raman Microspectroscopy and Imaging Study of Theraphthal in Living Cancer Cells.Biophys.J,2000,78(1):499-512.
9.Charonov S.,Chourpa I.,Valisa P.and Manfait M.Confocal Raman microscopic imaging of biological samples.Proc.SPIE.1997,2980:210-216.
10.Igor N.,Igor C.and Mivhel M.Applications of Raman and surface-enhanced Raman scattering spectroscopy in medicine.J.Raman Spectrosc.1994,25:13-23.
11.Reinhard S.S.Structure and dynamics of biomolecules probed by Raman spectroscopy.J.Raman Spec.2005,36:276-278.
12.Wong P.T.,Wong P.K.,Caputo T.A.,Godwin T.A.and Rigas B.Infrared spectroscopy of exfoliated human cervical cells:Evidence of extensive structural changes during carcinogenesis.Proc.Natl.Acad.Sci.1991,8(24):10988-10992.
13.Frank C.L.,Redd D.C.B.,Gansler T.S.AND Mccreery R.L.Characterization of human breast specimens with near-IR Raman spectroscopy.Anal Chem.1994,66:319-326.
14.Anita M.,Rebecca R.Raman spectroscopy for the detection id cancers and precancers.J. Biomed.Opt.1996,1:31-70.
15.Stewart S.,Shea D.A.,Tamowski C.P.,Morris M.D.,Wang D.,Franceschi R.,Lin D.L.and Keller E.Trends in early mineralization of murine calvarial osteoblastic cultures:a Raman microscopic study.J.Raman Spectrosc.2002,33:536-543.
16.Schaeberle M.D.,Kalasinsky V.F.,Luke J.L.Levin I.W.and Treado P.J.Raman chemical imaging:Histopathologe of indusions in human broart tissue.Anal Chem.1996,68:1829-1833.
17.许以明,徐国瑞,杨先春和张志义.三种卟啉类药物对DNA光敏损伤的比较.辐射研究与辐射工艺学报,1989,7(4):60-62.
18.Chenery D.and Bowring H.Infrared and Raman spectroscopic imaging in biosciences.Spectroscopy Europe.2003,15(4):8-14.
19.Puppels G.J.,De Mul F.F.M.,Otto C.Greve J.Studying single living cells and chromosomes by confocal Raman microspectroscopy.Nature.1990,347:301-303.
20.Puppels G.J.,Bakker Schut T.C.,Sijtsema N.M.,Grond M.,Maraboeuf F.,de Grauw C.G.,Figdor C.G.,and Greve J.Development application of Raman microspectroscopic and Raman imaging techniques for cell biological studies.Mole.Struc.1995,347:477-473.
21.Caspers P.J.,Lucassen G.W.and Puppels G.J.Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin.Biophysical J.2003,85:572-582.
22.Puppels G.J.,Garritsen H.S.P.,Kummer J.A.and Greve J.Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy.Cytometry.1993,14(3):251-256.
23.Puppels G.J.,Garritsen H.S.P.,Segers-Nollten G.M.J.,de Mul F.F.M.and Greve J.Raman microspectroscopic approach to the study of human granulocytes.Biophys.J.1991,60:1046-1056.
24.Puppels G.J.,Bakker Schut T.C.,Sijtsema N.M.,Grond M,Maraboeuf F.,de Grauw C.G.,Figdor C.G.and Greve J.Development and application of Raman microspectroscopic and Raman imaging techniques for cell biological studies.J.of Molecular Structure.1995,347:477-484.
25.Rosch P.,Harz M.,Schmitt M.and Popp J.Raman spectroscopic identification of single yeast cells.J.Raman Spectrosc.2005,36:377-379.
26.Salmaso B.L.N.,Puppels G.J.,Caspers P.J.,Floris R.,Wever R.,Greve J.Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human cosinophilic granulocytes.Biophysical J.1994,67(1):436-446.
27.Huang Y.S.,Karashima T.,Yamamoto M.,Ogura T.,Hamaguchi H.Raman spectroscopic signature of life in a living yeast cell.J.Raman Spectrosc.2004,35:525-526.
28.Sijtsema N.M.,Wouters S.D.,De Grauw C.J.,Otto C.,and Greve J.Confocal direct imaging Raman microscope:design and applications in biology.Appl.Spectrosc.1998,52:348-355.
29.Sijtsema N.M.Towards chemical imaging of living cells:design and application of a confocal Raman Microscope.[Thesis]Chap.1,University of Twente,1997
30.Wood B.R.,Caspers P.,Puppels G.J.,Pandiancherds S.and McNaughton D.Resonance Raman spectroscopy of red blood cells using near-infrared laser excitation.Anal.Bioanal Chem.2007,387(5),1691-1703.
31.Wood B.R.,Hammer L.,Davis L.and McNaughton D.Raman microspectroscopy and imaging provides insights into heme aggregation and denaturation within human erythrocytes.J.Biomed.Opt.2005,10(1):014005.
32.Wood B.R.,McNaughton D.Raman excitation wavelength investigation of single red blood cells in vivo.J.Raman Spec.2002,33:517-523.
33.Wood B.R.,McNaughton D.Micro-Raman Characterization of High- and Low-Spin Heme Moieties within Single Living Erythrocytes.Biospectroscopy.2002,67:259-262.
34.Cherney D.P.,Conboy J.C.and Harris J.M.Optical-trapping Raman microscopy detection of single unilamellar lipid vesicles.Anal.Chem.2003,75(23):6621-6628.
35.Creely C.M.,Singh G.P.and Petrov D.Dual wavelength optical tweezers for confocal Raman spectroscopy.Opt.Commun.2005,245:465-470.
36.Creely C.M.,Volpe G.,Singh G.P.Raman imaging on floating cells.Optics Express.2005,13(16):6105-6110.
37.Ramser K.,Logg K.,Goksor M.,Enger J.,Kall M.and Hanstrop D.Resonance Raman spectroscopy of optically trapped functional erythrocytes.J.Biomed.Opt.2004,9(3):593-600.
38.于敏华,包晓群和王卫东.脑脊液激光喇曼光谱及荧光光谱的试验观察.激光杂志,1996, 17(4):207-209.
39.杨祥良,徐辉碧,韩秀娴和孙奕.正常人胎儿晶体与老年性白内障晶体的激光喇曼光谱研究.激光技术,1995,19(6):382-384.
40.赵元黎,吕晶,申培红,梁二军,姚淑霞和葛向红.乳腺肿瘤组织的拉曼光谱研究.激光与红外.2004,34(6):502-504.
41.闫循领,董瑞新,王秋国,陈双峰,张宗旺,张学俊和张雷.乳腺癌病人单个细胞的Raman光谱.光谱学与光谱分析.2005,25(1):58-61.
42.张京伟,沈爱国,魏云,王小华,胡继明和叶勇.胃癌和胃正常黏膜拉曼光谱检测.生物医学工程学杂志.,2004,21(6):910-912.
43.吕刚,宫衍香,马传涛,和李长富.红细胞的拉曼光谱研究.德州学院学报.2005,21(6):23-25.
44.赵元黎,吕晶,葛向红,姚淑霞和梁二军.乳腺癌周边组织的拉曼光谱研究.光谱学与光谱分析.2006,26(7):1267-1271.
45.http://isl.lanl.gov/raman.html
46.Shelnutt J.A.,Rousseau D.L.,Friedman J.M.and Simon S.R.Protein-heme interaction in hemoglobin:Evidence from Raman difference spectroscopy.Natl.Acad.Sct.1979,76(9):4409-4413.
47.Brunner H.,Mayer A.and Sussner H.Resonance Raman scattering on the haem group of oxy- and deoxyhaemoglobin.J Mol.Biol.1972,70:153-156.
48.Brunner H.and Sussner H.Resonance Raman scattering on haemoglobin.Biochim.Biophys.Acta.1973,310:20-31.
49.Wood B.R.,Tait B.and McNaughton D.Micro-Raman characterization of the R to T state transition of haemoblobin within a single living erythrocyte.BBA.2001,1539:58-70.
50.Ramser K.,Bjerneld E.J.,Fant C.and Kall M.Raman imaging and spectroscopy of single functional erythrocytes:a feasibility study.Proc.SPIE.2002,4614:20-27.
51.Bulkin B.J.Raman spectroscopic study of human erythrocyte membranes.Biochimica et Biophysica Acta.1972,274:649.
52.Xu Y.M.,Zhao H.X.and Zhang ZH.Y.Raman spectroscopic study of microcosmic and photosensitive damage on the liposomes of the mixed phospholipids sensitized by hypocrellin and its derivatives.Photochem.Photobiol.B:Biology.1998,43:41-46.
53.Albrecht A.C.and Hurley M.C.On the Dependence of Vibrational Raman Intensity on the Wavelength of Incident Light.J.Chem.Phys.1971,55(9):4438-4443.
54.程光熙,拉曼布里渊散射,北京:科学出版社,2003,第一版,714-727.
55.Confocal Raman Microscopy.Raman Products Technical note,No.1350.
56.Zhang J.SH.,Sun CH.D.and Lu D.Sets effect on Zn.Acta Photonica Sinica.1999,28(6):551-554.
57.任斌 田中群 表面增强拉曼光谱的研究进展.现代仪器,2004,5:1-8,13.
58.Fleischmann M.,Hendra P.J.and McQuillan A.J.Raman spectra of pyridine adsorbed ata silver electrode.Chem.Phys.Lett.1974,26:163-166.
59.Moskovits M.Surface enhanced Raman Spectroscopy.Rev.Mod.Phys.1985,57(3):783-826.
60.Otto A.,Mrozek I.,Grabhorn H.and Akemann W.Surface enhanced Raman scattering.J.Phys.:Condensed Matter.1992,4:1143-1212.
61.Puppels G.J.,Olminkhof JH,Segers-Nolten G.M.,Otto C.,de Mul F.F.Greve J.Laser irradiation and Raman spectroscopy of single living cells and chromosomes:sample degradation occurs with 514.5nm but not with 660nm laser light.Exp.Cell.Res.1991,195:361-367.
62.Ramser K.,Bjerneld E.J.,Fant C.,Kaell M.Importance of substrate and photo-induced effects in Raman spectroscopy of single functional erythrocytes.J.BioMed.Opt.2003,8(2):173-178.
63.Hu S.ZH,Smith K.M.,Spiro T.G.Assignment of Protoheme Resonance Raman Spectrum by Heme Labeling in Myoblobin.J.Am.Chem.Soc.1996,118:12638-12646.
64.Tantra R.,McCabe A.,Bailey M.et al.Comparable Raman Spectroscopy Across Different Instruments and Excitation Wavelengths.NPL REPORTDQL-AS012,2004.
65.Horiba JY公司质量控制手册
66.降雨强,李昌勇,周源,张临杰,肖连团,贾锁堂,马存根和周国生.低强度激光血管内照射治疗的机理初探.激光生物学报.10(3):209-211,2001.
67.陈敏,骆清铭.弱激光的生物学效应及对红细胞变形性的改善作用.激光生物学报.11(1):55-57,2002.
68.http://en.wikipedia.org/wiki/Red_blood_cell
69.姚泰,生理学,北京:人民卫生出版社,2001,第三版.
70.Perutz M F.Molecular anatomy,physiology,and pathology of hemoglobin.In:Molecular basis of blood Diseases.Philadelphia,W B Saunders Co,1987,127
71.龙桂芳,张俊武.血红蛋白与血红蛋白病.广西:广西科学技术出版社.2003年,第一版.
72.张之南,李蓉生.红细胞疾病-基础与临床.北京:科学出版社.2000年,第一版.
73.Stryer L.Biochemistry.,New York,W.H.Freeman and Company,3rd.ed.1988.
74.Guido Guidotti.Studies on the chemistry of hemoglobin Ⅲ The interactions of the aβsubunits of hemoglobin.The journal of Biological Chemistry.1967.242(16):3694-3703
75.Guido Guidotti.Studies on the chemistry of hemoglobin Ⅳ The mechanism of reaction with ligands.The journal of Biological Chemistry.1967,242(16):3704-3712
76.Streaks T.C.and Spiro T.G.Hemoglobin:Resonance Raman Spectra.Biochim.Biophys.Acta.1972,263:830-833.
77.Strekas T.C.and Spiro T.G.Cytochrome c:Resonance Raman Spectra.Biochim.Biophys.Acta.1972,278:188-192.
78.Spiro T.G.and Strekas T.C.Resonance Raman scattering of heme proteins.Effect of oxidation and spin/state.Am.Chem.Soc.1974,96(2):338-345.
79.Tones Filho I.P.,Terner J.,Pittman R.N.,Somera L.G.and Wa(?)d K.R.Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy.Am.J.Phys.Heart.Circ.Phys.2005,289:488-495.
80.Aono,S.,Kato T.,Matsuki M.,Nakajima H.,Ohta T.,Uchida T.And Kitagawa T.Resonance Raman and Ligand Binding Studies of the Oxygen-sensing Signal Transducer Protein HemAT from Bacillus subtilis.J.Biol.Chem.2002,277(16):13528-13538.
81.Bruzzese F.J.,Dix J.A.,Rava R.P.and Cerny L.C.Resonance Raman spectroscopy of chemically modified haemoglobins.Biomater Artif.Cells Artif.Organs.1990,18:143-156.
82.Zhao X.J.,Spiro T.G.Ultraviolet resonance Raman spectroscopy of Hemoglobin with 200and 212nm excitation:H-bonds of Tyrosines and Prolines.J.Raman spectroscopy.1998,29:49-55.
83.Engler N.,Ostermann A.,Gassmann A.,et al.Protein Dynamics in an Intermediate State of Myoglobin:Optical Absorption,Resonance Raman Spectroscopy,and X-ray Structure Analysis.Biophys.J.2000,78(4):2081-2092.
84.De Paula J.C.Raman spectroscopy of metalloporphyrins,http://www.haverford.edu/chem
85.Monod J.,Wyman J.and Changeux J.P.On the nature of allosteric transitions:a plausible model.J.Mol.Biol.1965,12:88-118.
86.Koshland D.E.,Nemethy G.and Filmer D.Comparison of experimental binding data and theoretical models in proteins containing subunits.Biochemistry.1966,5:365-385.
87.Baldwin J.M.Structure and function of haemoglobin.Prog.Biophys.Mol.Biol.1975,29:225-320.
88.Ackers G.K.,Dalession P.M.,Lew G.H.,Daugherty M.A.and Holt J.M.Single residue modification of only one dimmer within the hemoglobin tetramer reveals autonomous dimmer function.Proc.Natl.Acad.Sci.2002,99(15):9777-9782.
89.Rhoda E.H.,Margaret J.L.,Gediminas V.A.V.,Shuocai H.,Joel M.F.and Ronald L.N.Conformational changes in oxyhemoglobin C(Gluβ6 →Lys) detected by spectroscopic probing.J.Bio.Chem.1996,271(1):372-375.
90.Mihailescu M.R.and Russu I.M.A signature of the T → R transition in human hemoglobin.PNAS.2001,98(7):3773-3777.
91.萨出尔夫,罗辽复和李前忠.血红蛋白分子氧合过程的构象变化特性.内蒙古大学学报,2003,34(6):648-652.
92.Xu Ch.Y.,Tobi D.and Bahar I.Allostedc Changes in Protein Structure Computed by Simple Mechanical Model:Hemoglobin T←→R2 Transition,J.Mol.Biol.2003,333:153-168.
93.Xu CH-Y,Tobi D.and Bahar I.Allosteric changes in protein structure computed by a simple mechanical model:Hemoglobin.J.Mol.Biol.2003,333:153-168.
94.Shigenori N.,Masako N.,Yasuhisa M.,Takashi Y.and Teizo K.Quaternary structure of intermediately ligated huaman hemoglobin A and influence from strong allosteric effectors:Resonance Raman investigation.Biophys.J.2005
95.Nagatomo S.,Nagai M.,Mizutani Y.,Yonetani T.And Kitagawa T.Quaternary Structures of Intermediately Ligated Human Hemoglobin A and Influences from Strong Allosteric Effectors:Resonance Raman,Biophy.J.2005,89:1203-1213.
96.Danon D.and Marikovshy Y.Difference of chafe electrophoresis at surface erythrocytes jeunes and agees et al.Compt.Rend Acad.Sci.1961,253:1271-1272.
97. Lutz H.U., Stammler P., Fasher S., Ingold M. and Fehr J. Density separation of human red blood cells on self forming percoll Gradients: Correlation with cell age. Biochem Biophys Acta. 1992,116:1-10.
98. Kocsis A., Biro E., Racz E., Kiss A. and Kappelmayer J. Mean corpuscular volume is not a reliable marker of red cell age in case of anisocytosis. Annu. Hematol. 1998, 77(4): 149-151.
99. Simpson L.O. and O'Neill D.J. Red cell shape changes in the blood of people 60years of age and older imply a role for blood rheology in the aging process. Derontology. 2003,49(5): 310-315.
100. Sutera S.P., Gardner R.A., Boylan C.W., Carroll G.L, Chang K.C., Marvel J.S., Kilo C., Gonen B. and Williamson J.R. Age related changes in deformability of human erythrocytes. Blood, 1985,65:275-282.
101. Waugh R.E., Narla M., Jackson C.W., Mueller T.J., Suzuki T. and Dale G.L. Rheologic properties of senescent erythrocytes: Loss of surface area and volume with red blood cell age. Blood. 1992, 79:1351-1358.
102. Canham, P.B. Difference in Geometry of Young and Old Human Erythrocytes Explained by a Filtering Mechanism. Circ Res. 1969,25:39-45.
103. Rettig M.P., Low P.S., Gimm J.A., Mohandas N., Wang J.ZH. and Christian J.A. Evaluation of biochemical changes during in vivo erythrocyte senescence in the dog. Blood. 1999,93: 376-384.
104. Knowles D.W., Chasis JA, Evans EA, Mohandas N. Cooperative action between band 3 and glycophorin A in human erythrocytes: Immobilization of band 3 induced by antibodies to glycophorin A. Biophys J. 1994; 66:1726-1732.
105. Hiroshi I., Effects of abnormal Hb on red cell membranes. Rinsho. Byori. 1999, 47(3): 232-237.
106. Ando K., Beppu M, Kikugawa K, Nagai R. and Horiuchi S. Membrane proteins of human erythrocytes are modified by advanced glycation end products during aging in the circulation. Biochem Biophys Res. Commun. 1999, 258(1): 123-127.
107. Kiefer C.R., Trainor J.F. Mckenney J.B., Valeri C.R. and Snyder L.M. Hemoglobin-spectrin complexes: interference with spectrin tetramer assembly as a mechanism for compartmentalization of Band 1 and Band 2 Complexes.Blood.1995,86(1):366-371.
108.程正江 人带3蛋白结构和功能研究进展.国外医学生理、病理科学与临床分册,2000,20(5):377-380.
109.Salhany J.M.,Cordes K.A.and Sloan R.L.Characterization of the pH dependence of hemoglobin binding to band 3:evidence for a pH-dependent conformational change within the hemoglobin-band 3 complex.Biochim Biophys Acta.1998,1372(1):107-113.
110.余国宇,田兴亚.衰老红细胞与带3蛋白.昆明医学院学报,2004,2:110-113.
111.Rauenbuehler P.,Cordes K.A.and Salhany J.M.Identification of the hemoglobin binding sites on the inner surface of the erythrocytes membrane.Biochim.Biophys.Acta.1982,692:361-370.
112.陈兴尧.红细胞表面电荷对其结构与功能参数的影响(硕士学位论文).暨南大学,2006.
113.Chen X.Y.,Huang Y.X.,Liu W.J.,Yuan Zh.J.Membrane surface charge and morphological & mechanical properties of young and old erythrocyte.Current Applied Physics.2007,7(1):94-96.
114.郑新景.量子点对细胞表面电荷的标记(硕士学位论文) 暨南大学,2007.
115.刘文静 新型快速荧光动态图像摄录与处理分析系统及其对活细胞的应用研究(博士学位论文) 暨南大学,2006.
116.Nathan Sharon,Halina Lis.LECTINS.Kluwer Academic Publishers,2003,Second Edition..
117.Bulai T.,Bratosin D.,Pins A.,Montreuil J.Zanetta J.P.Diversity of the human erythroeyte membrane sialic acids in relation with blood groups.FEBS Letters,2003;534:185-189.
118.Brooks S.A.,Dwek M.V.and Schumacher U.Functional & Molecular Glycobiology.BIOS Scientific Publishers Limited,2001.
119.Lutz H.U.and Fehr J.Total sialic acid content of glycophofins during senescence of human red blood cells.J.Bio.Chem.1979,254(22):11177-11180.
120.C.Traving,R.Schauer.Structure,function and metabolism of sialic acids.Cell.Mol.Life Sci.1998;54:1330-1349.
121.Bennett E.,Urcan M.S.,Tinkle S.S.,Koszowski A.G..and Levinson S.R.Contribution of sialic acid to the voltage dependence of sodium channel gating. A possible electrostatic mechanism. J Gen. Physiol. 1997; 109 (3): 327-343.
122. Ufret-Vinenty CA, Baro DJ, Santana LE Differential contribution of sialic acid to the function of repolarizing K+ currents in ventricular myocytes. Am. J. Physiol Cell Physiol. 2002; 281 (2): 464-474.
123. Thampoe JI, Furukawa K, Vallve E and Lloyd K.O. Sialyltransferase levels and ganglioside expression in melanoma and other cullured human cancer cells. Cancer Res. 1989,49: 6258-6264.
124. Kanai A., Sutherland D.R., Fibach E., Matta K.L., Hindernburg A., Brockhausen I., Kuhns W., Taub R.N., van den Eijnden D.H. and Baker M.A. Human leukemic myelobasts and myeloblastoid cells contain the enzyme cytidine 5-monophosphate-N-acetylneuraminic acid: Gal β 1-3 GalNAC α(2,3)-sialyltransferase. Cancer Res. 1990; 50: 5003-5007.
125. Percoll Methodology and Applications, Handbooks from Amersham Biosciences, 18-1115-69.
126. Erslev AJ and Beutler E. Production and destruction of erythrocytes. William Hematology, fifth edition, McGrew-Hill, Inc. New York, 1995,425-440.
127. Kosztolanyi G. and Jobst K. Electrokinetic analysis of the fetal erythrocyte membrane age trypsin digestion. Pediatric Research. 1980,14:138-141.
128. Edwards M. and Rigas D.A. Electrolyte- liable increase of oxygen affinity during in vivo aging of hemoglobin. J. Clinic. Investi. 1967,46(10): 1579-1588.
129. Baskurt O.K., Tugral E., Neu B. and Meiselman H.J. Particle electrophoresis as a tool to understand the aggregation behavior of red blood cells. Electrophoresis. 2002; 23 (13): 2103-2109.
130. Miles J.D. and Demetrios A.G. Electrolyte-Habile increase of oxygen affinity during in vivo aging of Hemoglobin. J. Clinic. Investi. 1967,46(10):1579-1588.
131. Iwona C., Yoji S. and Norihiko T. Changes of RBC aggregation in oxygenation-deoxygenation: pH dependency and cell morphology. Am J Physiol Heart Circ Physiol. 2003, 284: 2335-2342.
132. Condon M.R., Kim J.E., Deitch E.A., Machiedo G.W., Appearance of an erythrocyte population with deceased deformability and hemoglobin content following sepsis. American Journal of Phsiology.2003,284(4):2177-2184.
133.张秀琳,谢益宽.膜表面电荷学说及唾液酸对Na+、K+通道门控特性的影响.生理科学进展,2005,35(2):167-169.
134.姚成灿,姚平,黄耀熊.环境温度对人红细胞膜形态和表面电荷的即时影响.暨南大学学报(自然科学版),2005(2):689-693.
135.姚成灿,黄耀熊,李校坤,阮萍.PH值对红细胞膜力学特性和胞内蛋白结构和功能的影响 科学通报.2003,48(10):1050-1053.
136.Frans L.A.W.,Bregt R.S.,Yvonne A.M.G.,Hemoglobin loss from erythrocytes in vivo results from spleen-facilitated vesiculation.Blood.2003,101:747-751.
137.吴正洁.温度对人红细胞及血红蛋白结构功能的影响(硕士学位论文) 暨南大学,2007.
138.http://info.med.hc360.com/html/001/005/002/23802.htm
139.Mashevich M,Folkman D,Kesar A.Barbul A.,Korenstein R.,Jerby E.and Avivi L.Exposure of human peripheral blood lymphocytes to electromagnetic fields associated with celluar phones leads to choromosomal instability.Bioelectromagnetics.2003,24(2):82-90.
140.Caraj-VrhovacV,Fucic A,HorvatD.The correlation between the frequency of micronuclei and specific chromosome aberrations in human lymphocytes exposed to microwave radiation in vitro.Mutat Res.1992,281(3):181-186.
141.马菲,熊鸿燕,张耀和杨丽华.高强度电磁辐射对长期暴露人群血液成分的损伤效应研究.疾病控制杂志.2005,9(5):437-440.
142.Ramanauskaite R.B.,Segers-Nolten I.G.M.J.,Grauw K.J.,Sijtsema N.M.,van der Maas L.,Greve J.,Otto C.and Figfor C.G..Carotenoid levels in human lymphocytes measured by Raman micro spectroscopy.Pure & Appl.Chem.1997,69(10):2131-2134.
143.姚成灿,姚平,唐国汉,郑青,李校坤和黄耀熊.射频电磁波与环境温度对单个活态红细胞膜力学性质的影响.中国职业医学.2005,32(3):11-13.
144.姚成灿.900MHz电磁波和有关理化因素对人红细胞的即时作用(博士学位论文).暨南大学,2004.
145.刘齐海.900MHz电磁波对淋巴细胞的作用(硕士学位论文).暨南大学,2006.
146.丁振华,范建中.紫外辐射生物学与医学.北京:人民军医出版社,2000:16-21.
147.Erfurth S.C.and Peticolas W.L.Melting and premelting phenomenon in DNA by laser Raman scattering.Biopolymer.1975,14:247-264.
148.Ojeda F.,Guarda M.I.,Lovengreen C.,Hidalgo M.A.,Folch H.,Hartel S.and Maldonado C.Ultraviolet exposure of thymcytes:selective inhibition of apoptosis.Int.J.Radiat.Biol.2004,80(6):445-450.
149.Zoltan N.,Attlia B.,Gyorgyi R.Eva P.,Dobozy A.And Kemeny L.Efficacy of different UV-emitting light sources in the induction of T-cell apoptosis.Photochemistry and photobiology.2004,79(5):434-439.
150.Morison W.L.,Parrish J.A.,Bloch K.J.and Krugler J.I.In vivo effect of UV-B on lymphocytes function.Britsh J.Dermatology.1979,101(5):513.
151.孙鼎文.紫外辐射(一).紫外辐射的主要效应及紫外辐射源.现代计量测试.1999,15-16.
152.周华和朱惠刚.紫外辐射对免疫功能的影响.上海环境科学.1996,15(6):37-39.
153.何慧和孙晓青.紫外线照射照射诱导人外周血淋巴细胞凋亡的研究.中华理疗杂志.2000,23(1):24-26.
154.余萍 和 李京培 紫外线照射对人外周血细胞凋亡及吞噬功能的观察.安徽医科大学学报.2002,37(5):377-379.
155.Spielberg H.,June C.H.,Blair O.C.,Nystrom-Rosander C.,Cereb N.and Deeg H.J.UV irradiation of lymphocytes triggers an increase in intracellular Ca2+ and prevents lectin-stimulated Ca2+ mobilization:evidence for UV- and nifedipine-sensitive Ca2+channels.Exp.Hematol.1991,19(8):742-748.
156.Berliner J.,Himes S.W.,Aoki C.T.and No A.The sites of unscheduled DNA synthesis within irradiated human lymphocytes.Radiation Research.1975,63:544-552.
157.Schieven G.L.,Kirihara J.M.,Gilloland L.K.,Uckun F.M.and Ledbetter J.A.Ultraviolet radiation rapidly induces tyrosine phosphorylation and calcium signaling in lymphocytes.MBC.1993,4(5):523-530.
158.Goettsch W.,Garssen J.,Hurks H.M.H.,Gruijl F.R.de,Loveren H.van.Effects of in vitro UV-B exposure on the immune system:with special emphasis on T cell activity.RIVM Report 850017002,1993.
159.许以明,张志义,赵克俭,张仲伦.高能质子辐射作用的分子激励-DNA空间结构微观损伤的拉曼光谱特征.中国科学(B辑).1991,12:1273-1278.
160.Xu Y.M.,Zhou ZH.X.,Yang H.Y.,Xu Y.and Zhang ZH.Y.Raman spectroscopic study of microscomic photodamage of the space structure of DNA sensitized by Yangzhou hematoporphyrin derivative and hotofrin Ⅱ.J.Photochem Photobio B:Biology.1999,52:30-34.
161.Morrion H.Bioorganic Photochemistry.Vol 1.New York:John Wiley and Sons,1990.
162.Wang S.Y.Photochemistry and photobiology of Nucleic Acids.Vol 1 and 2.New York:Awadcmic Press,1976.
163.Oison J.A.Molecular action of cartenoids in:Canfield LM(ed) Carotenoids in Human health.Annuls of New York the Axademy of Sciences.1993,31(691):156-166.
164.Edge R.and Truscott T.G Carotenoids - Free radical interactions.Spectrum,2000,3:12-20.
165.Hughes D.A.Effects of carotenoids on human immune function.Pro.Nutr.Soc.1999,58:713-718.
166.Herraiz L.A.,Hsieh W.C.,Parker R.S.,Swanson J.E.,Bendich A.and Roe D.A.Effect of UV exposure and β-carotene supplementation on delayed-type hypersensitivity response in healthy older men.J.American.College of Nutrition.1998,17(6):617-624.
167.宋雁.类胡萝卜素抗氧化和促氧化作用的影响因素.卫生研究.2003,32(4):417-419.
168.唐刘蕴泉.β-胡萝素应用的研究进展.肠内肠外营养.2005,12(2):121-123.
169.赵文恩,韩雅珊,戴蕴青 类胡萝卜素的生物学性能.生物学杂志.1998,15(3):1-3.
170.范晓岚,杨军,糜漫天和杨慧.β-胡萝素的抗氧化作用于疾病预防.中国公共卫生.2003,19(4):479-480.
171.张世平,张颖,栗学军,栗兰珍,张兴志和刘基芳.β胡萝素和维生素E抗辐射作用研究的临床应用.白求恩医科大学学报.2000:26(5):509-510.
172.李怡兰,乔珊珊,周蕾,姜淑清和李国星.β-胡萝素的抗辐射作用.环境与健康杂志.2004,21(5):300-302.