靶向HSP60的化学合成多肽及其在凋亡细胞成像中的应用研究
发布时间：2018-09-03 12:43
【摘要】：目的：细胞凋亡在维持机体生长发育等正常生命活动中发挥着重要作用,也与许多疾病的发生、发展及治疗过程息息相关,比如心肌细胞、神经元细胞的过度凋亡分别是心力衰竭等心血管系统疾病、阿尔茨海默症等神经退行性疾病的重要诱因；而组织中过少的凋亡则可能导致癌症、自身免疫病等重大疾病的发生。更值得注意的是,许多药物都是基于诱导或抑制细胞凋亡而发挥其治疗作用。因此,实时监测凋亡的发生,特别是建立可视化方法,对于评价药物或治疗方案的疗效与毒副作用,以及深入研究和理解细胞凋亡机制、疾病的发生与发展的机理均具有重要意义。考虑到人工设计的小分子多肽具有易化学修饰、体内清除速率较快、以及性价比高等优点,本研究从一批人工设计的化学合成多肽序列中筛选了能与凋亡细胞高效结合的多肽分子,并通过多种检测手段在多种类型的细胞凋亡模型中考察其细胞凋亡成像能力,在药物诱导肿瘤凋亡和急性心肌损伤的动物模型中考察其体内凋亡成像能力。通过蛋白质相互作用研究方法探究并验证该多肽结合的靶蛋白。在细胞和动物水平初步评价多肽的安全性。方法：以肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导Jurkat细胞凋亡为模型,通过激光共聚焦显微镜观察多肽与传统凋亡试剂共染色情况,以验证多肽的凋亡结合能力；通过流式细胞术分析多肽作用浓度、作用时间以及细胞凋亡程度对其结合量的影响；通过流式细胞术考察多肽与不同凋亡时期标志物共染色情况,以分析多肽的凋亡时期特异性；通过Pull-down实验及MALDI-FTICR质谱分析对多肽结合靶蛋白进行鉴定,并通过与靶蛋白的荧光共定位分析以及westernblot方法对靶蛋白的鉴定结果进行验证。进一步,在不同凋亡诱导剂诱导的多种细胞凋亡模型中,通过流式细胞术、荧光显微镜、激光共聚焦显微镜、酶标仪等多种检测手段,考察多肽在细胞水平的凋亡成像能力。建立小鼠乳腺癌移植模型及急性心肌损伤模型,尾静脉注射FITC标记多肽,通过小动物活体成像分析考察多肽的体内凋亡成像能力；通过TUNEL检测法及Caspase-3活性检测进行组织凋亡分析,以对多肽标记区域进行凋亡验证。通过尾静脉注射FITC标记多肽,考察其在健康BALB/c小鼠中的体内分布代谢以及排出情况。通过CCK-8、Annexin V/PI双染色考察多肽对细胞活性及凋亡的影响；通过激光共聚焦显微镜、酶标仪等检测多肽对细胞固有功能如细胞骨架、细胞粘附能力的影响,以评价多肽的体外安全性。在健康BALB/c小鼠模型中,尾静脉注射多肽,通过组织切片的HE染色观察多肽主要蓄积器官的形态学变化,以初步评价多肽的体内安全性。结果：(1)筛选出的水溶性多肽序列P17在可与多种凋亡细胞株结合,包括多种人类白血病细胞(Jurkat、THP-1、K-562、 U937)、人类实体瘤细胞(Hela、MDA-MB-231、DU4475)、人类血管内皮细胞(HUVEC)、小鼠原代心肌细胞、大鼠心肌细胞(H9c2)等。(2)P17在凋亡细胞上的信号比对照组高出10倍以上；结合迅速,孵育15分钟后结合即可达到峰值；结合稳定,信号强度可维持4小时以上：并可通过流式细胞术、普通荧光显微镜、激光共聚焦显微镜、酶标仪等多种方法进行观测。(3)与不同凋亡时期标志物共染色结果显示,P17倾向于与晚期凋亡的细胞结合。(4)经质谱分析鉴定,Pull-down实验中P17捕获的靶蛋白为热休克蛋白60(HSP60)。荧光共定位及westernblot结果证实,P17可与HSP60特异性结合,同时HSP60在细胞凋亡后表达量显著增加。(5)在小鼠乳腺癌移植瘤模型中,通过尾静脉注射FITC标记的P17探针,可在小动物活体成像仪上观测到小鼠肿瘤组织内由化疗药诱导的凋亡区域或自发凋亡区域,并可在冰冻组织切片上清楚观测到P17结合的凋亡区域。(6)在阿霉素诱导的急性心肌损伤模型中,尾静脉注射P17后1-9小时内,阿霉素处理小鼠的全身及心脏内可见明显荧光信号,外周血中荧光信号强度平稳下降；健康小鼠的荧光信号主要集中在膀胱区域,且心脏内无明显荧光信号,外周血中荧光强度在注射后1小时达到峰值,随后快速降低。(7)对健康BALB/c小鼠尾静脉注射P17,1小时后肝、肾两个主要代谢器官的荧光信号强度达到峰值,随后下降,并于注射后6小时恢复到生理盐水对照组水平。(8)CCK-8及Annexin V/PI双染色结果显示,P17对细胞的增殖活性没有影响,也不会引起细胞凋亡。同时,P17对细胞正常功能如细胞骨架、细胞粘附能力也没有明显影响。(9)对健康BALB/c小鼠尾静脉注射P17后24小时,肝、肾组织细胞结构完整,没有发生变性肿胀、炎性细胞浸润、充血等异常病理改变。结论：P17通过与细胞内HSP60特异性结合而高效标记凋亡细胞或发生凋亡的组织,并具备较快的体内清除速率,有望发展成一种可用于外及体内无创凋亡成像的新型凋亡探针,并展现出在评价药物毒副作用、评价药物疗效等方面潜在的应用价值。同时,P17揭示了HSP60与晚期凋亡之间的潜在联系,为进一步明确HSP60在凋亡中的具体作用提供了新思路。
[Abstract]:OBJECTIVE: Apoptosis plays an important role in maintaining normal life activities such as growth and development, and is also closely related to the occurrence, development and treatment of many diseases, such as cardiomyocytes, neuronal excessive apoptosis is the heart failure and other cardiovascular diseases, Alzheimer's disease and other neurodegenerative diseases. More importantly, many drugs are based on inducing or inhibiting apoptosis. Therefore, real-time monitoring of the occurrence of apoptosis, especially the establishment of visual methods, can be used to evaluate drugs or treatment options. It is of great significance to study the mechanism of apoptosis and to understand the mechanism of the occurrence and development of diseases. Considering the advantages of easy chemical modification, rapid in vivo clearance, and high cost-effectiveness of the synthetic peptides, a series of synthetic peptides were designed. Polypeptide molecules that can bind to apoptotic cells efficiently were screened, and the imaging ability of apoptotic cells was investigated in various apoptotic models by various detection methods. The imaging ability of apoptotic cells in vivo was investigated in animal models of drug-induced tumor apoptosis and acute myocardial injury. To explore and validate the target protein binding to the peptide. To evaluate the safety of the peptide at cellular and animal levels. Methods: The apoptosis of Jurkat cells induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was used as a model. The co-staining of the peptide with traditional apoptotic reagents was observed by laser confocal microscopy to verify the apoptotic node of the peptide. The binding capacity of peptides was analyzed by flow cytometry; the binding capacity of peptides was determined by concentration, time and degree of apoptosis; the co-staining of peptides with different apoptotic markers was investigated by flow cytometry to analyze the specificity of apoptotic phase of peptides; the effect of peptides was analyzed by Pull-down test and MALDI-FTICR mass spectrometry. The target proteins were identified by fluorescence co-localization analysis and Western blot. Furthermore, in the apoptosis models induced by different apoptosis inducers, the hands were examined by flow cytometry, fluorescence microscopy, laser confocal microscopy, enzyme labeling and other methods. To establish a mouse model of breast cancer transplantation and acute myocardial injury, FITC-labeled peptides were injected into tail vein, and the imaging ability of apoptosis in vivo was examined by imaging analysis of small animals; the tissue apoptosis was analyzed by TUNEL and Caspase-3 activity assay, in order to get more than one. The distribution, metabolism and excretion of FITC-labeled peptides in healthy BALB/c mice were investigated by tail vein injection. The effects of peptides on cell activity and apoptosis were investigated by CCK-8 and Annexin V/PI double staining. The intrinsic work of peptides on cells was detected by laser confocal microscopy and enzyme labeling apparatus. In a healthy BALB/c mouse model, polypeptides were injected into the tail vein to observe the morphological changes of the main accumulation organs of polypeptides by HE staining in tissue sections, so as to preliminarily evaluate the in vivo safety of polypeptides. It can bind to a variety of apoptotic cell lines, including a variety of human leukemia cells (Jurkat, THP-1, K-562, U937), human solid tumor cells (Hela, MDA-MB-231, DU4475), human vascular endothelial cells (HUVEC), mouse primary cardiomyocytes, rat cardiomyocytes (H9c2), etc. (2) P17 signal on apoptotic cells was more than 10 times higher than the control group. It can be observed by flow cytometry, common fluorescence microscopy, laser confocal microscopy, enzyme labeling and other methods. (3) Co-staining with different apoptotic markers showed that P17 tended to be associated with late apoptosis. The results of fluorescence co-localization and Western blot confirmed that P17 could specifically bind to HSP60, and the expression of HSP60 increased significantly after apoptosis. (5) FITC labeling was injected into tail vein in mouse breast cancer xenograft model. The P17 probe can be used to observe the apoptotic region or spontaneous apoptotic region induced by chemotherapeutics in tumor tissue of mice in vivo, and the P17-binding apoptotic region can be clearly observed in frozen tissue sections. (6) In the model of adriamycin-induced acute myocardial injury, 1-9 hours after injection of P17, adriamycin was injected into tail vein. The fluorescence signal intensity in the whole body and heart of the treated mice decreased steadily, and the fluorescence signal intensity in the peripheral blood of the healthy mice was mainly concentrated in the bladder region, and there was no obvious fluorescence signal in the heart. The fluorescence intensity in the peripheral blood reached the peak one hour after injection, and then decreased rapidly. The fluorescence signal intensity of liver and kidney reached the peak value at 17 and 1 hours after injection of P17, then decreased, and returned to normal saline control level at 6 hours after injection. (8) CCK-8 and Annexin V/PI double staining showed that P17 had no effect on cell proliferation activity and did not induce cell apoptosis. Functions such as cytoskeleton and cell adhesion ability were not significantly affected. (9) 24 hours after tail vein injection of P17 into healthy BALB/c mice, the cell structure of liver and kidney was intact, no abnormal pathological changes such as degeneration and swelling, inflammatory cell infiltration, congestion occurred. Conclusion: P17 marked apoptotic cells by specific binding with HSP60. In addition, P17 reveals the potential relationship between HSP60 and late-stage apoptosis. Further clarifying the specific role of HSP60 in apoptosis provides a new way of thinking.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R914;R96
[Abstract]:OBJECTIVE: Apoptosis plays an important role in maintaining normal life activities such as growth and development, and is also closely related to the occurrence, development and treatment of many diseases, such as cardiomyocytes, neuronal excessive apoptosis is the heart failure and other cardiovascular diseases, Alzheimer's disease and other neurodegenerative diseases. More importantly, many drugs are based on inducing or inhibiting apoptosis. Therefore, real-time monitoring of the occurrence of apoptosis, especially the establishment of visual methods, can be used to evaluate drugs or treatment options. It is of great significance to study the mechanism of apoptosis and to understand the mechanism of the occurrence and development of diseases. Considering the advantages of easy chemical modification, rapid in vivo clearance, and high cost-effectiveness of the synthetic peptides, a series of synthetic peptides were designed. Polypeptide molecules that can bind to apoptotic cells efficiently were screened, and the imaging ability of apoptotic cells was investigated in various apoptotic models by various detection methods. The imaging ability of apoptotic cells in vivo was investigated in animal models of drug-induced tumor apoptosis and acute myocardial injury. To explore and validate the target protein binding to the peptide. To evaluate the safety of the peptide at cellular and animal levels. Methods: The apoptosis of Jurkat cells induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was used as a model. The co-staining of the peptide with traditional apoptotic reagents was observed by laser confocal microscopy to verify the apoptotic node of the peptide. The binding capacity of peptides was analyzed by flow cytometry; the binding capacity of peptides was determined by concentration, time and degree of apoptosis; the co-staining of peptides with different apoptotic markers was investigated by flow cytometry to analyze the specificity of apoptotic phase of peptides; the effect of peptides was analyzed by Pull-down test and MALDI-FTICR mass spectrometry. The target proteins were identified by fluorescence co-localization analysis and Western blot. Furthermore, in the apoptosis models induced by different apoptosis inducers, the hands were examined by flow cytometry, fluorescence microscopy, laser confocal microscopy, enzyme labeling and other methods. To establish a mouse model of breast cancer transplantation and acute myocardial injury, FITC-labeled peptides were injected into tail vein, and the imaging ability of apoptosis in vivo was examined by imaging analysis of small animals; the tissue apoptosis was analyzed by TUNEL and Caspase-3 activity assay, in order to get more than one. The distribution, metabolism and excretion of FITC-labeled peptides in healthy BALB/c mice were investigated by tail vein injection. The effects of peptides on cell activity and apoptosis were investigated by CCK-8 and Annexin V/PI double staining. The intrinsic work of peptides on cells was detected by laser confocal microscopy and enzyme labeling apparatus. In a healthy BALB/c mouse model, polypeptides were injected into the tail vein to observe the morphological changes of the main accumulation organs of polypeptides by HE staining in tissue sections, so as to preliminarily evaluate the in vivo safety of polypeptides. It can bind to a variety of apoptotic cell lines, including a variety of human leukemia cells (Jurkat, THP-1, K-562, U937), human solid tumor cells (Hela, MDA-MB-231, DU4475), human vascular endothelial cells (HUVEC), mouse primary cardiomyocytes, rat cardiomyocytes (H9c2), etc. (2) P17 signal on apoptotic cells was more than 10 times higher than the control group. It can be observed by flow cytometry, common fluorescence microscopy, laser confocal microscopy, enzyme labeling and other methods. (3) Co-staining with different apoptotic markers showed that P17 tended to be associated with late apoptosis. The results of fluorescence co-localization and Western blot confirmed that P17 could specifically bind to HSP60, and the expression of HSP60 increased significantly after apoptosis. (5) FITC labeling was injected into tail vein in mouse breast cancer xenograft model. The P17 probe can be used to observe the apoptotic region or spontaneous apoptotic region induced by chemotherapeutics in tumor tissue of mice in vivo, and the P17-binding apoptotic region can be clearly observed in frozen tissue sections. (6) In the model of adriamycin-induced acute myocardial injury, 1-9 hours after injection of P17, adriamycin was injected into tail vein. The fluorescence signal intensity in the whole body and heart of the treated mice decreased steadily, and the fluorescence signal intensity in the peripheral blood of the healthy mice was mainly concentrated in the bladder region, and there was no obvious fluorescence signal in the heart. The fluorescence intensity in the peripheral blood reached the peak one hour after injection, and then decreased rapidly. The fluorescence signal intensity of liver and kidney reached the peak value at 17 and 1 hours after injection of P17, then decreased, and returned to normal saline control level at 6 hours after injection. (8) CCK-8 and Annexin V/PI double staining showed that P17 had no effect on cell proliferation activity and did not induce cell apoptosis. Functions such as cytoskeleton and cell adhesion ability were not significantly affected. (9) 24 hours after tail vein injection of P17 into healthy BALB/c mice, the cell structure of liver and kidney was intact, no abnormal pathological changes such as degeneration and swelling, inflammatory cell infiltration, congestion occurred. Conclusion: P17 marked apoptotic cells by specific binding with HSP60. In addition, P17 reveals the potential relationship between HSP60 and late-stage apoptosis. Further clarifying the specific role of HSP60 in apoptosis provides a new way of thinking.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R914;R96
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