肝细胞及其与药物相互作用的拉曼光谱研究
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摘要
拉曼光谱是一种振动光谱,能够快速并且无损伤的探测生物体系。应用拉曼光谱研究细胞体系有极大的优势,细胞不需要标记或固定等特殊处理,并且可以在生理环境状态下进行拉曼测试。本论文应用拉曼光谱技术探测了正常肝细胞(HL-7702)和肿瘤肝细胞(BEL-7402)的拉曼光谱特性并且对正常肝细胞和肿瘤肝细胞进行了判别区分;探讨了抗癌药物与肝细胞的相互作用,并且应用表面增强拉曼光谱对肿瘤肝细胞进行了探测。具体内容如下:
(1)应用显微拉曼光谱对单个正常肝细胞和肿瘤肝细胞进行了测量,结果显示细胞内不同位点的拉曼光谱区别较大,这说明了细胞内化学组成和结构的区别。并且应用T-检验,主成分分析(PCA)和判别分析(LDA)对正常肝细胞和肿瘤肝细胞进行了统计分析。T-检验结果表明位于785 cm~(-1)和1004 cm~(-1)的两个拉曼峰在两个细胞线中差别最大,是判别区分正常肝细胞和肿瘤肝细胞的重要参数。PCA-LDA分析显示利用拉曼光谱对正常肝细胞和肿瘤肝细胞的区分准确率可以达到100%。
(2)利用拉曼光谱对抗癌药物阿霉素与两种细胞线的相互作用进行了分析。研究了药物与肿瘤肝细胞的作用机制和对正常肝细胞的毒副作用。发现位于1313和1585 cm~(-1)归属为鸟嘌呤碱基的拉曼峰发生明显改变,表明阿霉素可能是通过与DNA鸟嘌呤碱基的N7位置形成氢键而对肝细胞产生毒杀作用,
(3)A54(AGKGTPSLETTP)是一种能够特异性结合肝癌细胞的多肽,利用拉曼光谱研究了阿霉素与多肽A54复合后对肿瘤肝细胞的毒杀效果和对正常肝细胞的毒副作用情况。实验结果表明A54能够提高阿霉素对肿瘤细胞的毒性并降低对正常肝细胞的毒副作用。
(4)应用表面增强拉曼散射效应使得对单个活的肿瘤肝细胞进行超灵敏拉曼测量成为可能。利用金溶胶(35 nm)作为活性衬底,进入到细胞而使活细胞内的化学成分在相对较短的时间(1 s每点)内产生拉曼增强信号。表面增强拉曼光谱成像显示在细胞内不同的位点拉曼光谱不同,反映了细胞内化学结构的差异。
Raman spectroscopy, based on vibrational spectroscopy, is a rapid and nondestructive technique for studying biological system. Raman spectroscopy has the advantages for studying the cells in vitro, namely that no labels and fixation are required. Furthermore, Raman measurements on cells can be performed in physiological- like conditions. In this thesis, we applied Raman spectroscopy to study the character of the hepatocytes and discriminate the normal (HL-7702) and malignant (BEL-7402) hepatocytes, investigate the interactions between the antitumor drugs and hepatocytes, and explore the malignant hepatocytes by surface enhanced Raman spectroscopy (SERS). The details are the followings:
(1) Micro-Raman spectroscopy was used to study the characters of single normal and malignant hepatocyte. The study indicates the Raman spectra are different for the different point in the cells, which suggests that the chemical component and structure are different in the different positions of cells. In addition, statistical analysis, including of T-test, principal component analysis (PCA), and linear discriminant analysis (LDA), was performed on the Raman spectra of malignant and normal hepatocytes. The T-test-LDA results show the bands at 785 cm~(-1) and 1004 cm~(-1) are significantly different between the malignant and normal hepatocytes and are the most crucial in distinguishing between the malignant and normal hepatocytes. The best results with the differentiation accuracy of 100% can be obtained with PCA-LDA.
(2) Raman spectroscopy was used to explore the interactions between doxorubicin (DOX) and hepatocytes in vitro. And the interaction mechanism of DOX with malignant hepatocytes and side-effect of DOX to the normal hepatocytes were studied. The changes in the Raman bands for guanine (1313 and 1585 cm~(-1)) of DNA suggest that DOX may interact with DNA by the hydrogen bond to the N7 position of guanine to kill the cells.
(3) The peptide A54 (AGKGTPSLETTP), a kind of hepatocarcinoma-binding peptide, was coupled with chemical toxin doxorubicin. We investigate the killing effect of A54-DOX to malignant hepatocytes and side-effect to normal hepatocytes by Raman spectroscopy. The results indicate that A54 can improve the cytotoxicity of DOX to malignant hepatocytes and decrease the side-effect of DOX to normal hepatocytes.
(4) Ultrasensitive Raman measurements in single living malignant hepatocyte are possible through exploiting the effect of surface-enhanced Raman scattering. Colloidal gold particles (35 nm in size) that are deposited inside cells as "SERS-active nanostructures" result in strongly enhanced Raman signals of the native chemical constituents of the malignant hepatocytes in relatively short collection times (1 second for one mapping point). SERS mapping over a cell shows different Raman spectra at different places, reflecting the very inhomogeneous chemical constitution of the cells.
(1)应用显微拉曼光谱对单个正常肝细胞和肿瘤肝细胞进行了测量,结果显示细胞内不同位点的拉曼光谱区别较大,这说明了细胞内化学组成和结构的区别。并且应用T-检验,主成分分析(PCA)和判别分析(LDA)对正常肝细胞和肿瘤肝细胞进行了统计分析。T-检验结果表明位于785 cm~(-1)和1004 cm~(-1)的两个拉曼峰在两个细胞线中差别最大,是判别区分正常肝细胞和肿瘤肝细胞的重要参数。PCA-LDA分析显示利用拉曼光谱对正常肝细胞和肿瘤肝细胞的区分准确率可以达到100%。
(2)利用拉曼光谱对抗癌药物阿霉素与两种细胞线的相互作用进行了分析。研究了药物与肿瘤肝细胞的作用机制和对正常肝细胞的毒副作用。发现位于1313和1585 cm~(-1)归属为鸟嘌呤碱基的拉曼峰发生明显改变,表明阿霉素可能是通过与DNA鸟嘌呤碱基的N7位置形成氢键而对肝细胞产生毒杀作用,
(3)A54(AGKGTPSLETTP)是一种能够特异性结合肝癌细胞的多肽,利用拉曼光谱研究了阿霉素与多肽A54复合后对肿瘤肝细胞的毒杀效果和对正常肝细胞的毒副作用情况。实验结果表明A54能够提高阿霉素对肿瘤细胞的毒性并降低对正常肝细胞的毒副作用。
(4)应用表面增强拉曼散射效应使得对单个活的肿瘤肝细胞进行超灵敏拉曼测量成为可能。利用金溶胶(35 nm)作为活性衬底,进入到细胞而使活细胞内的化学成分在相对较短的时间(1 s每点)内产生拉曼增强信号。表面增强拉曼光谱成像显示在细胞内不同的位点拉曼光谱不同,反映了细胞内化学结构的差异。
Raman spectroscopy, based on vibrational spectroscopy, is a rapid and nondestructive technique for studying biological system. Raman spectroscopy has the advantages for studying the cells in vitro, namely that no labels and fixation are required. Furthermore, Raman measurements on cells can be performed in physiological- like conditions. In this thesis, we applied Raman spectroscopy to study the character of the hepatocytes and discriminate the normal (HL-7702) and malignant (BEL-7402) hepatocytes, investigate the interactions between the antitumor drugs and hepatocytes, and explore the malignant hepatocytes by surface enhanced Raman spectroscopy (SERS). The details are the followings:
(1) Micro-Raman spectroscopy was used to study the characters of single normal and malignant hepatocyte. The study indicates the Raman spectra are different for the different point in the cells, which suggests that the chemical component and structure are different in the different positions of cells. In addition, statistical analysis, including of T-test, principal component analysis (PCA), and linear discriminant analysis (LDA), was performed on the Raman spectra of malignant and normal hepatocytes. The T-test-LDA results show the bands at 785 cm~(-1) and 1004 cm~(-1) are significantly different between the malignant and normal hepatocytes and are the most crucial in distinguishing between the malignant and normal hepatocytes. The best results with the differentiation accuracy of 100% can be obtained with PCA-LDA.
(2) Raman spectroscopy was used to explore the interactions between doxorubicin (DOX) and hepatocytes in vitro. And the interaction mechanism of DOX with malignant hepatocytes and side-effect of DOX to the normal hepatocytes were studied. The changes in the Raman bands for guanine (1313 and 1585 cm~(-1)) of DNA suggest that DOX may interact with DNA by the hydrogen bond to the N7 position of guanine to kill the cells.
(3) The peptide A54 (AGKGTPSLETTP), a kind of hepatocarcinoma-binding peptide, was coupled with chemical toxin doxorubicin. We investigate the killing effect of A54-DOX to malignant hepatocytes and side-effect to normal hepatocytes by Raman spectroscopy. The results indicate that A54 can improve the cytotoxicity of DOX to malignant hepatocytes and decrease the side-effect of DOX to normal hepatocytes.
(4) Ultrasensitive Raman measurements in single living malignant hepatocyte are possible through exploiting the effect of surface-enhanced Raman scattering. Colloidal gold particles (35 nm in size) that are deposited inside cells as "SERS-active nanostructures" result in strongly enhanced Raman signals of the native chemical constituents of the malignant hepatocytes in relatively short collection times (1 second for one mapping point). SERS mapping over a cell shows different Raman spectra at different places, reflecting the very inhomogeneous chemical constitution of the cells.
引文
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