激光光镊拉曼光谱在活体中的应用
摘要
拉曼光谱技术具有非侵入性、低破坏性、不用试剂和高度自动化等优点。在医学诊断中具有很大的应用潜力。激光光镊拉曼技术(Laser tweezers Raman spectroscopy, LTRS),是将光学囚禁(optical trapping)与拉曼光谱分析(Raman spectroscopy)结合并应用于悬浮细胞或细胞器研究的一项新的生物光子技术。该技术克服了普通显微拉曼无法克服的缺点,进一步提高了探测的灵敏度、精确度,使得研究溶液中的单个活细胞的生理生化过程成为可能,是研究单个细胞的生命过程和医学诊断的有用工具。目前拉曼光镊广泛被应用单个细胞分析上。
糖尿病是一种患病率高且并发症多的慢性终身性疾病,但有50%～70%病人并无明显的自觉症状,其慢性并发症在不知不觉中逐渐发生和发展,所以糖尿病有“隐形杀手”之称。由于糖尿病常伴有多种并发症如组织坏死、感染等,成为继肿瘤、心脑血管疾病之后的第三大非遗传性疾病,严重威胁着人类的健康和生命,因此有效地防治并发症对于降低糖尿病的死亡率非常重要。目前,血糖检测是糖尿病检测的重要目标之一,检测血糖的方法主要是从体内抽血通过生化检测进行分析,进行离体实验,没有在体内真正的生理条件下进行,频繁的采血既耗费时间,又增加了病人的痛苦和经济负担。由于葡萄糖在组织液和血液中的含量低且变化范围小,组织中大量的水,葡萄糖的吸光系数也远远小于水的吸光系数,血糖浓度变化导致的有效信号非常微弱,所以拉曼光谱进行血糖无创检测的精确度和可靠性方面存在一定的难处。基于上述原因本文试图从处于活体糖尿病小鼠血管中的白细胞和红细胞的拉曼光谱入手,来检测和筛查糖尿病患者。本文利用激光光镊拉曼系统(LTRS)对活体糖尿病小鼠中单个白细胞的拉曼光谱进行了研究分析。利用拉曼光镊并结合多元统计分析方法无损地对活体糖尿病小鼠中的白细胞进行了研究,分别检测了糖尿病和正常小鼠活体中的白细胞拉曼光谱,利用主成分分析(PCA)建立拉曼光谱诊断多元统计算法模型。结果表明：1、利用LTR确实可以获得活体内白细胞的拉曼光谱；患糖尿病小鼠和正常小鼠白细胞拉曼光谱差别明显,且实验存在较好的重现性。利用PCA统计分析方法得到诊断特异性和灵敏度达到了98%。2、在活体糖尿病小鼠白细胞中出现较高的蛋白质的特征峰1302cm-1这表明活体糖尿病白细胞中的蛋白质浓度比正常状态高。3、糖尿病小鼠内的白细胞与正常相比,DNA磷酸骨架基团强度和蛋白质酰胺强度升高,表明DNA双螺旋结构、蛋白质主链和氢键体系发生变化,二级构象改变。血氧供应不足是组织变性坏死等并发症最主要的病理生理基础。红细胞是氧气供应的
主要载体,红细胞结构和功能异常则是氧供应不足的最直接原因。由于成熟红细胞需要改变自身形状才能通过比自己直径小的微血管,因此,良好的红细胞变形能力是维持微循环物质与氧气的最重要保证。研究表明,糖尿病、高血压等[4-5]多种疾病红细胞变形能力下降,引起微循环灌注障碍和局部组织缺血缺氧,是糖尿病并发症极易发生的主要原因。近年来,红细胞变形能力作为从血液流变学角度探讨糖尿病微血管病变之发生机制、疗效原理和预防措施的一项客观指标或重要参数日益受到人们的重视。但是以往红细胞的研究过程繁琐往往是在体外进行的,这就必然会改变其生存环境,不能客观地反映其实际情况,原位无损的研究就显得尤为必要。本文利用无损的方法来研究糖尿病小鼠的原位红细胞成分及其在不同状态的成分变化,这个方法据我们所知尚未见报道。同样利用LTRS系统并结合多元统计分析方法对糖尿病小鼠中的红细胞进行了研究。得出结论如下：1.此系统的确获得了活体内红细胞的拉曼光谱;患糖尿病小鼠和正常小鼠红细胞拉曼光谱差别明显,且实验存在较好的重现性。利用PCA统计分析方法得到诊断特异性和灵敏度达到了95%。2.糖尿病小鼠内的红细胞与正常相比,血红蛋白、苯基丙氨酸强度升高,表明发生了血红蛋白糖基化,代谢过程异常。3.糖尿病小鼠红细胞中拉曼峰I1635/I1550的比值小于正常红细胞的比值,说明糖尿病小鼠红细胞携氧能力下降;I1126/I1080的比值大于正常红细胞此峰的比值,结果指出糖尿病红细胞膜的流动性比正常红细胞下降了。以上结论证明拉曼光镊技术是实时研究细胞生理、生化变化的快捷而有效的工具,有望成为在分子水平上对各种活细胞的检测、诊断的先进工具,具有非常广阔的前景。
The advantage of Raman spectroscopy is a non-invasive, low-destructive, do not have the reagents and a high degree of automation.it was greatly applied in medical diagnosis. Laser tweezers Raman spectroscopy is a new biological photonic technology that is combination of optical trapping and Raman spectroscopy. This technique has been used to study single living suspension cells. With this system, high-quality and sensity Raman spectra were obtained from single optically trapped biological cells in suspension. Raman spectroscopy is a promising tool for studying living process of the single cell and medicine diagnosis. Currently, Raman spectroscopy was applied in analysis of single cell.
Diabetes is a high prevalence of complications of chronic disease, but there are50%to70%of patients do not fell obvious symptoms. It was occurd unconsciously and developed gradually.so diabetes was called "invisible killer". Because diabetes is often accompanied by a variety of complications such as tissue necrosis, myocardial, it waas the third largest non hereditary diseases from cancer, cardiovascular and cerebrovascular diseases. Diabetes threatens seriously the health and life of human being, and therefore effectively prevent complications reduce the mortality rate of diabetes is very important. At present, detection of blood glucose is one of the important goals, the method of testing blood glucose is that blood from the body were analyzed by biochemical tests.In vitro experiments is not true in vivo under physiological conditions;frequent blood sampling is spending too much time and increasing the patient's suffering and economic burden. However, because the low glucose content and small range in the tissue fluid and blood, a lot of water in the organization, the glucose absorptivity is far less than the water absorptivity and changes in blood glucose concentration cause effective weak signal. The accuracy and reliability of Raman spectroscopy of the measurement blood glucose non-invasive, there is a certain difficulty. Raman spectra of normal WBC and DM WBC in vivo were studied by laser tweezers combined with Multivariate statistic analysis method.Raman spectrum and Multivariate statistic model was found by the principal components analysis (PCA).the result of Raman spectrum suggested:1.DM WBC and normal WBC could be differentiated with PCA. Diagnostic specificity and sensitivity are come to98%.2.The protein vibration bands at1302cm-1in DM WBC in vivo rat showed that the concent of protein in DM WBC are higher than normal WBC.3.The Raman intensities at O-P-O of DNA and Amide Bands of DM cell are significantly higher than normal cell,which showed that the changes in comformation of O-P-O、the backbone and hydrogen bonds of protein and two comformation of protein.
The lack of oxygen supply is the most important pathophysiological basis of tissue necrosis and other complications. blood cells is the main carrier of oxygen supply, abnormal structure and function of blood cell is the most direct cause of the lack of oxygen supply. Since the mature red blood cells change their shape could through the microvascular.therefore, a good red blood cell deformability is the most important guarantee to maintain the the microcirculation material with oxygen. Studies have shown that the decreasing of blood cell deformability cause microcirculation disorders and local tissue ischemia and hypoxia in diabetes and a variety of diseases, it is the main reason for diabetes complications can happen easily. In recent years, the blood cell deformability as the pathogenesis of diabetic microvascular disease from the perspective of hemorheology, an objective index of efficacy of principle and preventive measures important parameters has been increasing attentied. But the research process of blood cells in vitro in the past often is trouble, which is bound to changing their living environment that can not objectively reflect the actual situation.it is especially necessary in situ non-destructive research.Using non-destructive method to study the in situ blood cell composition of the diabetic mice and its composition changes in different states, this method has not been reported as far as we know. Raman spectra of normal RBC and DM RBC in vivo were studied by laser tweezers combined with Multivariate statistic analysis method.Raman spectrum and Multivariate statistic model was found by the principal components analysis (PCA).the result of Raman spectrum suggested.1.1.DM RBC and normal RBC could be differentiated with PCA. Diagnostic specificity and sensitivity are come to98%.2. The Raman intensities at ferrohemoglobin and phenylalanine of DM cell are significantly higher than normal cell,which showed that glycosylation ferrohemoglobin, metabolism abnormal.3.7he ratios of showirg the Oxgen-carring capacity I1635/I1550in DM were less than those in normal. It is that the Oxgen-carring capacity of RBC in DM was lower than that in normal subjects;7he ratios of showirg the fluldity I1126/I1080in normal subjects were less than those in DM. It is that the fluidity of erythroeyte membrane in DM was lower than that in normal subjects. The above conclusions showed that Raman optical tweezers technology in a quick and effective tool that studies of cell physiological and biochemical changes. It is expected to become the advanced tools that detection and diagnosis of all kinds of living cells at the molecular level.
糖尿病是一种患病率高且并发症多的慢性终身性疾病,但有50%～70%病人并无明显的自觉症状,其慢性并发症在不知不觉中逐渐发生和发展,所以糖尿病有“隐形杀手”之称。由于糖尿病常伴有多种并发症如组织坏死、感染等,成为继肿瘤、心脑血管疾病之后的第三大非遗传性疾病,严重威胁着人类的健康和生命,因此有效地防治并发症对于降低糖尿病的死亡率非常重要。目前,血糖检测是糖尿病检测的重要目标之一,检测血糖的方法主要是从体内抽血通过生化检测进行分析,进行离体实验,没有在体内真正的生理条件下进行,频繁的采血既耗费时间,又增加了病人的痛苦和经济负担。由于葡萄糖在组织液和血液中的含量低且变化范围小,组织中大量的水,葡萄糖的吸光系数也远远小于水的吸光系数,血糖浓度变化导致的有效信号非常微弱,所以拉曼光谱进行血糖无创检测的精确度和可靠性方面存在一定的难处。基于上述原因本文试图从处于活体糖尿病小鼠血管中的白细胞和红细胞的拉曼光谱入手,来检测和筛查糖尿病患者。本文利用激光光镊拉曼系统(LTRS)对活体糖尿病小鼠中单个白细胞的拉曼光谱进行了研究分析。利用拉曼光镊并结合多元统计分析方法无损地对活体糖尿病小鼠中的白细胞进行了研究,分别检测了糖尿病和正常小鼠活体中的白细胞拉曼光谱,利用主成分分析(PCA)建立拉曼光谱诊断多元统计算法模型。结果表明：1、利用LTR确实可以获得活体内白细胞的拉曼光谱；患糖尿病小鼠和正常小鼠白细胞拉曼光谱差别明显,且实验存在较好的重现性。利用PCA统计分析方法得到诊断特异性和灵敏度达到了98%。2、在活体糖尿病小鼠白细胞中出现较高的蛋白质的特征峰1302cm-1这表明活体糖尿病白细胞中的蛋白质浓度比正常状态高。3、糖尿病小鼠内的白细胞与正常相比,DNA磷酸骨架基团强度和蛋白质酰胺强度升高,表明DNA双螺旋结构、蛋白质主链和氢键体系发生变化,二级构象改变。血氧供应不足是组织变性坏死等并发症最主要的病理生理基础。红细胞是氧气供应的
主要载体,红细胞结构和功能异常则是氧供应不足的最直接原因。由于成熟红细胞需要改变自身形状才能通过比自己直径小的微血管,因此,良好的红细胞变形能力是维持微循环物质与氧气的最重要保证。研究表明,糖尿病、高血压等[4-5]多种疾病红细胞变形能力下降,引起微循环灌注障碍和局部组织缺血缺氧,是糖尿病并发症极易发生的主要原因。近年来,红细胞变形能力作为从血液流变学角度探讨糖尿病微血管病变之发生机制、疗效原理和预防措施的一项客观指标或重要参数日益受到人们的重视。但是以往红细胞的研究过程繁琐往往是在体外进行的,这就必然会改变其生存环境,不能客观地反映其实际情况,原位无损的研究就显得尤为必要。本文利用无损的方法来研究糖尿病小鼠的原位红细胞成分及其在不同状态的成分变化,这个方法据我们所知尚未见报道。同样利用LTRS系统并结合多元统计分析方法对糖尿病小鼠中的红细胞进行了研究。得出结论如下：1.此系统的确获得了活体内红细胞的拉曼光谱;患糖尿病小鼠和正常小鼠红细胞拉曼光谱差别明显,且实验存在较好的重现性。利用PCA统计分析方法得到诊断特异性和灵敏度达到了95%。2.糖尿病小鼠内的红细胞与正常相比,血红蛋白、苯基丙氨酸强度升高,表明发生了血红蛋白糖基化,代谢过程异常。3.糖尿病小鼠红细胞中拉曼峰I1635/I1550的比值小于正常红细胞的比值,说明糖尿病小鼠红细胞携氧能力下降;I1126/I1080的比值大于正常红细胞此峰的比值,结果指出糖尿病红细胞膜的流动性比正常红细胞下降了。以上结论证明拉曼光镊技术是实时研究细胞生理、生化变化的快捷而有效的工具,有望成为在分子水平上对各种活细胞的检测、诊断的先进工具,具有非常广阔的前景。
The advantage of Raman spectroscopy is a non-invasive, low-destructive, do not have the reagents and a high degree of automation.it was greatly applied in medical diagnosis. Laser tweezers Raman spectroscopy is a new biological photonic technology that is combination of optical trapping and Raman spectroscopy. This technique has been used to study single living suspension cells. With this system, high-quality and sensity Raman spectra were obtained from single optically trapped biological cells in suspension. Raman spectroscopy is a promising tool for studying living process of the single cell and medicine diagnosis. Currently, Raman spectroscopy was applied in analysis of single cell.
Diabetes is a high prevalence of complications of chronic disease, but there are50%to70%of patients do not fell obvious symptoms. It was occurd unconsciously and developed gradually.so diabetes was called "invisible killer". Because diabetes is often accompanied by a variety of complications such as tissue necrosis, myocardial, it waas the third largest non hereditary diseases from cancer, cardiovascular and cerebrovascular diseases. Diabetes threatens seriously the health and life of human being, and therefore effectively prevent complications reduce the mortality rate of diabetes is very important. At present, detection of blood glucose is one of the important goals, the method of testing blood glucose is that blood from the body were analyzed by biochemical tests.In vitro experiments is not true in vivo under physiological conditions;frequent blood sampling is spending too much time and increasing the patient's suffering and economic burden. However, because the low glucose content and small range in the tissue fluid and blood, a lot of water in the organization, the glucose absorptivity is far less than the water absorptivity and changes in blood glucose concentration cause effective weak signal. The accuracy and reliability of Raman spectroscopy of the measurement blood glucose non-invasive, there is a certain difficulty. Raman spectra of normal WBC and DM WBC in vivo were studied by laser tweezers combined with Multivariate statistic analysis method.Raman spectrum and Multivariate statistic model was found by the principal components analysis (PCA).the result of Raman spectrum suggested:1.DM WBC and normal WBC could be differentiated with PCA. Diagnostic specificity and sensitivity are come to98%.2.The protein vibration bands at1302cm-1in DM WBC in vivo rat showed that the concent of protein in DM WBC are higher than normal WBC.3.The Raman intensities at O-P-O of DNA and Amide Bands of DM cell are significantly higher than normal cell,which showed that the changes in comformation of O-P-O、the backbone and hydrogen bonds of protein and two comformation of protein.
The lack of oxygen supply is the most important pathophysiological basis of tissue necrosis and other complications. blood cells is the main carrier of oxygen supply, abnormal structure and function of blood cell is the most direct cause of the lack of oxygen supply. Since the mature red blood cells change their shape could through the microvascular.therefore, a good red blood cell deformability is the most important guarantee to maintain the the microcirculation material with oxygen. Studies have shown that the decreasing of blood cell deformability cause microcirculation disorders and local tissue ischemia and hypoxia in diabetes and a variety of diseases, it is the main reason for diabetes complications can happen easily. In recent years, the blood cell deformability as the pathogenesis of diabetic microvascular disease from the perspective of hemorheology, an objective index of efficacy of principle and preventive measures important parameters has been increasing attentied. But the research process of blood cells in vitro in the past often is trouble, which is bound to changing their living environment that can not objectively reflect the actual situation.it is especially necessary in situ non-destructive research.Using non-destructive method to study the in situ blood cell composition of the diabetic mice and its composition changes in different states, this method has not been reported as far as we know. Raman spectra of normal RBC and DM RBC in vivo were studied by laser tweezers combined with Multivariate statistic analysis method.Raman spectrum and Multivariate statistic model was found by the principal components analysis (PCA).the result of Raman spectrum suggested.1.1.DM RBC and normal RBC could be differentiated with PCA. Diagnostic specificity and sensitivity are come to98%.2. The Raman intensities at ferrohemoglobin and phenylalanine of DM cell are significantly higher than normal cell,which showed that glycosylation ferrohemoglobin, metabolism abnormal.3.7he ratios of showirg the Oxgen-carring capacity I1635/I1550in DM were less than those in normal. It is that the Oxgen-carring capacity of RBC in DM was lower than that in normal subjects;7he ratios of showirg the fluldity I1126/I1080in normal subjects were less than those in DM. It is that the fluidity of erythroeyte membrane in DM was lower than that in normal subjects. The above conclusions showed that Raman optical tweezers technology in a quick and effective tool that studies of cell physiological and biochemical changes. It is expected to become the advanced tools that detection and diagnosis of all kinds of living cells at the molecular level.
引文
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