激光光鑷?yán)庾V在活體中的應(yīng)用
發(fā)布時(shí)間：2018-09-10 05:51
【摘要】：拉曼光譜技術(shù)具有非侵入性、低破壞性、不用試劑和高度自動(dòng)化等優(yōu)點(diǎn)。在醫(yī)學(xué)診斷中具有很大的應(yīng)用潛力。激光光鑷?yán)夹g(shù)(Laser tweezers Raman spectroscopy, LTRS),是將光學(xué)囚禁(optical trapping)與拉曼光譜分析(Raman spectroscopy)結(jié)合并應(yīng)用于懸浮細(xì)胞或細(xì)胞器研究的一項(xiàng)新的生物光子技術(shù)。該技術(shù)克服了普通顯微拉曼無(wú)法克服的缺點(diǎn),進(jìn)一步提高了探測(cè)的靈敏度、精確度,使得研究溶液中的單個(gè)活細(xì)胞的生理生化過(guò)程成為可能,是研究單個(gè)細(xì)胞的生命過(guò)程和醫(yī)學(xué)診斷的有用工具。目前拉曼光鑷廣泛被應(yīng)用單個(gè)細(xì)胞分析上。 糖尿病是一種患病率高且并發(fā)癥多的慢性終身性疾病,但有50%～70%病人并無(wú)明顯的自覺(jué)癥狀,其慢性并發(fā)癥在不知不覺(jué)中逐漸發(fā)生和發(fā)展,所以糖尿病有“隱形殺手”之稱(chēng)。由于糖尿病常伴有多種并發(fā)癥如組織壞死、感染等,成為繼腫瘤、心腦血管疾病之后的第三大非遺傳性疾病,嚴(yán)重威脅著人類(lèi)的健康和生命,因此有效地防治并發(fā)癥對(duì)于降低糖尿病的死亡率非常重要。目前,血糖檢測(cè)是糖尿病檢測(cè)的重要目標(biāo)之一,檢測(cè)血糖的方法主要是從體內(nèi)抽血通過(guò)生化檢測(cè)進(jìn)行分析,進(jìn)行離體實(shí)驗(yàn),沒(méi)有在體內(nèi)真正的生理?xiàng)l件下進(jìn)行,頻繁的采血既耗費(fèi)時(shí)間,又增加了病人的痛苦和經(jīng)濟(jì)負(fù)擔(dān)。由于葡萄糖在組織液和血液中的含量低且變化范圍小,組織中大量的水,葡萄糖的吸光系數(shù)也遠(yuǎn)遠(yuǎn)小于水的吸光系數(shù),血糖濃度變化導(dǎo)致的有效信號(hào)非常微弱,所以拉曼光譜進(jìn)行血糖無(wú)創(chuàng)檢測(cè)的精確度和可靠性方面存在一定的難處�；谏鲜鲈虮疚脑噲D從處于活體糖尿病小鼠血管中的白細(xì)胞和紅細(xì)胞的拉曼光譜入手,來(lái)檢測(cè)和篩查糖尿病患者。本文利用激光光鑷?yán)到y(tǒng)(LTRS)對(duì)活體糖尿病小鼠中單個(gè)白細(xì)胞的拉曼光譜進(jìn)行了研究分析。利用拉曼光鑷并結(jié)合多元統(tǒng)計(jì)分析方法無(wú)損地對(duì)活體糖尿病小鼠中的白細(xì)胞進(jìn)行了研究,分別檢測(cè)了糖尿病和正常小鼠活體中的白細(xì)胞拉曼光譜,利用主成分分析(PCA)建立拉曼光譜診斷多元統(tǒng)計(jì)算法模型。結(jié)果表明：1、利用LTR確實(shí)可以獲得活體內(nèi)白細(xì)胞的拉曼光譜；患糖尿病小鼠和正常小鼠白細(xì)胞拉曼光譜差別明顯,且實(shí)驗(yàn)存在較好的重現(xiàn)性。利用PCA統(tǒng)計(jì)分析方法得到診斷特異性和靈敏度達(dá)到了98%。2、在活體糖尿病小鼠白細(xì)胞中出現(xiàn)較高的蛋白質(zhì)的特征峰1302cm-1這表明活體糖尿病白細(xì)胞中的蛋白質(zhì)濃度比正常狀態(tài)高。3、糖尿病小鼠內(nèi)的白細(xì)胞與正常相比,DNA磷酸骨架基團(tuán)強(qiáng)度和蛋白質(zhì)酰胺強(qiáng)度升高,表明DNA雙螺旋結(jié)構(gòu)、蛋白質(zhì)主鏈和氫鍵體系發(fā)生變化,二級(jí)構(gòu)象改變。血氧供應(yīng)不足是組織變性壞死等并發(fā)癥最主要的病理生理基礎(chǔ)。紅細(xì)胞是氧氣供應(yīng)的 主要載體,紅細(xì)胞結(jié)構(gòu)和功能異常則是氧供應(yīng)不足的最直接原因。由于成熟紅細(xì)胞需要改變自身形狀才能通過(guò)比自己直徑小的微血管,因此,良好的紅細(xì)胞變形能力是維持微循環(huán)物質(zhì)與氧氣的最重要保證。研究表明,糖尿病、高血壓等[4-5]多種疾病紅細(xì)胞變形能力下降,引起微循環(huán)灌注障礙和局部組織缺血缺氧,是糖尿病并發(fā)癥極易發(fā)生的主要原因。近年來(lái),紅細(xì)胞變形能力作為從血液流變學(xué)角度探討糖尿病微血管病變之發(fā)生機(jī)制、療效原理和預(yù)防措施的一項(xiàng)客觀指標(biāo)或重要參數(shù)日益受到人們的重視。但是以往紅細(xì)胞的研究過(guò)程繁瑣往往是在體外進(jìn)行的,這就必然會(huì)改變其生存環(huán)境,不能客觀地反映其實(shí)際情況,原位無(wú)損的研究就顯得尤為必要。本文利用無(wú)損的方法來(lái)研究糖尿病小鼠的原位紅細(xì)胞成分及其在不同狀態(tài)的成分變化,這個(gè)方法據(jù)我們所知尚未見(jiàn)報(bào)道。同樣利用LTRS系統(tǒng)并結(jié)合多元統(tǒng)計(jì)分析方法對(duì)糖尿病小鼠中的紅細(xì)胞進(jìn)行了研究。得出結(jié)論如下：1.此系統(tǒng)的確獲得了活體內(nèi)紅細(xì)胞的拉曼光譜;患糖尿病小鼠和正常小鼠紅細(xì)胞拉曼光譜差別明顯,且實(shí)驗(yàn)存在較好的重現(xiàn)性。利用PCA統(tǒng)計(jì)分析方法得到診斷特異性和靈敏度達(dá)到了95%。2.糖尿病小鼠內(nèi)的紅細(xì)胞與正常相比,血紅蛋白、苯基丙氨酸強(qiáng)度升高,表明發(fā)生了血紅蛋白糖基化,代謝過(guò)程異常。3.糖尿病小鼠紅細(xì)胞中拉曼峰I1635/I1550的比值小于正常紅細(xì)胞的比值,說(shuō)明糖尿病小鼠紅細(xì)胞攜氧能力下降;I1126/I1080的比值大于正常紅細(xì)胞此峰的比值,結(jié)果指出糖尿病紅細(xì)胞膜的流動(dòng)性比正常紅細(xì)胞下降了。以上結(jié)論證明拉曼光鑷技術(shù)是實(shí)時(shí)研究細(xì)胞生理、生化變化的快捷而有效的工具,有望成為在分子水平上對(duì)各種活細(xì)胞的檢測(cè)、診斷的先進(jìn)工具,具有非常廣闊的前景。
[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.
【學(xué)位授予單位】：廣西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R318.51;O433.4
本文編號(hào)：2233542
[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.
【學(xué)位授予單位】：廣西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R318.51;O433.4
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