激光光镊拉曼光谱在活体中的应用

发布时间：2018-09-10 05:51

【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。
[Abstract]:Raman spectroscopy has the advantages of non-invasive, low destructive, reagent-free and highly automated. It has great potential in medical diagnosis. Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with Raman spectroscopy and is applied to medical diagnosis. Suspension cell or organelle research is a new biophoton technology. This technology overcomes the shortcomings that ordinary micro-Raman can not overcome, further improves the sensitivity and accuracy of detection, makes it possible to study the physiological and biochemical processes of a single living cell in solution, is the study of the life process of a single cell and medical diagnosis. Currently, Raman optical tweezers are widely used in single cell analysis.
Diabetes mellitus is a chronic life-long disease with high morbidity and many complications, but 50%-70% of the patients do not have obvious symptoms, and its chronic complications gradually occur and develop unconsciously. Therefore, diabetes mellitus is known as "invisible killer". Because diabetes is often accompanied by a variety of complications such as tissue necrosis, infection, and so on, it becomes secondary swelling. Tumor, the third largest non-hereditary disease after cardiovascular and cerebrovascular diseases, is a serious threat to human health and life. Therefore, effective prevention and treatment of complications is very important to reduce the mortality of diabetes mellitus. Frequent blood collection not only consumes time, but also increases the patient's pain and economic burden. Because the glucose content in tissue blood and blood is low and the change range is small, there is a large amount of water in the tissue, and the absorption coefficient of glucose is far less than that of water absorption system. Because the effective signal caused by the change of blood glucose concentration is very weak, the accuracy and reliability of non-invasive detection of blood glucose by Raman spectroscopy are difficult. Raman spectroscopy of single leukocyte in diabetic mice in vivo was studied by laser tweezers Raman spectroscopy (LTRS). Leucocytes in diabetic mice in vivo were nondestructively studied by Raman tweezers and multivariate statistical analysis. Leucocytes in diabetic mice and normal mice were detected respectively. The results show that: 1. Raman spectra of leukocytes in vivo can be obtained by using LTR; the Raman spectra of leukocytes in diabetic mice and normal mice are obviously different, and the experiment has good reproducibility. The diagnostic specificity and sensitivity were 98%. 2. The characteristic peak of high protein in the leukocytes of diabetic mice in vivo was 1302 cm-1, indicating that the protein concentration in the leukocytes of diabetic mice in vivo was higher than that of normal mice. 3. Compared with normal leukocytes of diabetic mice, the strength of DNA phosphorylated skeleton group and the strength of protein amide were increased. It indicates that DNA double helix structure, protein main chain and hydrogen bond system have changed, secondary conformation has changed. Insufficient oxygen supply is the most important pathophysiological basis of complications such as tissue degeneration and necrosis.
The main carrier, the abnormal structure and function of red blood cells, is the most direct cause of oxygen shortage.Because mature red blood cells need to change their shape to pass through the smaller-diameter microvessels, good red blood cell deformability is the most important guarantee for maintaining microcirculation substances and oxygen.Studies have shown that diabetes, hypertension and so on [4-5]. [Decreased erythrocyte deformability, microcirculation perfusion disturbance and local tissue ischemia and hypoxia are the main causes of diabetic complications. In recent years, erythrocyte deformability has been an objective indicator of the pathogenesis, therapeutic principle and preventive measures of diabetic microangiopathy from the perspective of hemorheology. However, in the past, red blood cells were often studied in vitro, which would inevitably change their living environment and could not objectively reflect their actual situation. Therefore, in situ non-destructive research is particularly necessary. The same LTRS system and multivariate statistical analysis were used to study erythrocytes in diabetic mice. The results were as follows: 1. The Raman spectra of erythrocytes in vivo were obtained by this system; diabetic mice and normal mice were obtained. The diagnostic specificity and sensitivity were 95% by PCA statistical analysis. 2. Compared with normal mice, the intensity of hemoglobin and phenylalanine increased, indicating that hemoglobin was glycosylated and the metabolic process was abnormal. The ratio of I1635/I1550 in erythrocyte of diabetic mice was lower than that of normal erythrocyte, indicating that the oxygen carrying capacity of erythrocyte of diabetic mice was decreased; the ratio of I1126/I1080 was higher than that of normal erythrocyte, indicating that the fluidity of erythrocyte membrane of diabetic mice was lower than that of normal erythrocyte. It is a fast and effective tool for real-time study of cell physiology and biochemical changes. It is expected to become an advanced tool for the detection and diagnosis of various living cells at the molecular level.
【学位授予单位】：广西师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.51;O433.4

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