靶向HSP60的化學(xué)合成多肽及其在凋亡細(xì)胞成像中的應(yīng)用研究
發(fā)布時間：2018-09-03 12:43
【摘要】：目的：細(xì)胞凋亡在維持機(jī)體生長發(fā)育等正常生命活動中發(fā)揮著重要作用,也與許多疾病的發(fā)生、發(fā)展及治療過程息息相關(guān),比如心肌細(xì)胞、神經(jīng)元細(xì)胞的過度凋亡分別是心力衰竭等心血管系統(tǒng)疾病、阿爾茨海默癥等神經(jīng)退行性疾病的重要誘因；而組織中過少的凋亡則可能導(dǎo)致癌癥、自身免疫病等重大疾病的發(fā)生。更值得注意的是,許多藥物都是基于誘導(dǎo)或抑制細(xì)胞凋亡而發(fā)揮其治療作用。因此,實(shí)時監(jiān)測凋亡的發(fā)生,特別是建立可視化方法,對于評價藥物或治療方案的療效與毒副作用,以及深入研究和理解細(xì)胞凋亡機(jī)制、疾病的發(fā)生與發(fā)展的機(jī)理均具有重要意義�？紤]到人工設(shè)計的小分子多肽具有易化學(xué)修飾、體內(nèi)清除速率較快、以及性價比高等優(yōu)點(diǎn),本研究從一批人工設(shè)計的化學(xué)合成多肽序列中篩選了能與凋亡細(xì)胞高效結(jié)合的多肽分子,并通過多種檢測手段在多種類型的細(xì)胞凋亡模型中考察其細(xì)胞凋亡成像能力,在藥物誘導(dǎo)腫瘤凋亡和急性心肌損傷的動物模型中考察其體內(nèi)凋亡成像能力。通過蛋白質(zhì)相互作用研究方法探究并驗(yàn)證該多肽結(jié)合的靶蛋白。在細(xì)胞和動物水平初步評價多肽的安全性。方法：以腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(TRAIL)誘導(dǎo)Jurkat細(xì)胞凋亡為模型,通過激光共聚焦顯微鏡觀察多肽與傳統(tǒng)凋亡試劑共染色情況,以驗(yàn)證多肽的凋亡結(jié)合能力；通過流式細(xì)胞術(shù)分析多肽作用濃度、作用時間以及細(xì)胞凋亡程度對其結(jié)合量的影響；通過流式細(xì)胞術(shù)考察多肽與不同凋亡時期標(biāo)志物共染色情況,以分析多肽的凋亡時期特異性；通過Pull-down實(shí)驗(yàn)及MALDI-FTICR質(zhì)譜分析對多肽結(jié)合靶蛋白進(jìn)行鑒定,并通過與靶蛋白的熒光共定位分析以及westernblot方法對靶蛋白的鑒定結(jié)果進(jìn)行驗(yàn)證。進(jìn)一步,在不同凋亡誘導(dǎo)劑誘導(dǎo)的多種細(xì)胞凋亡模型中,通過流式細(xì)胞術(shù)、熒光顯微鏡、激光共聚焦顯微鏡、酶標(biāo)儀等多種檢測手段,考察多肽在細(xì)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損傷模型,尾靜脈注射FITC標(biāo)記多肽,通過小動物活體成像分析考察多肽的體內(nèi)凋亡成像能力；通過TUNEL檢測法及Caspase-3活性檢測進(jìn)行組織凋亡分析,以對多肽標(biāo)記區(qū)域進(jìn)行凋亡驗(yàn)證。通過尾靜脈注射FITC標(biāo)記多肽,考察其在健康BALB/c小鼠中的體內(nèi)分布代謝以及排出情況。通過CCK-8、Annexin V/PI雙染色考察多肽對細(xì)胞活性及凋亡的影響；通過激光共聚焦顯微鏡、酶標(biāo)儀等檢測多肽對細(xì)胞固有功能如細(xì)胞骨架、細(xì)胞粘附能力的影響,以評價多肽的體外安全性。在健康BALB/c小鼠模型中,尾靜脈注射多肽,通過組織切片的HE染色觀察多肽主要蓄積器官的形態(tài)學(xué)變化,以初步評價多肽的體內(nèi)安全性。結(jié)果：(1)篩選出的水溶性多肽序列P17在可與多種凋亡細(xì)胞株結(jié)合,包括多種人類白血病細(xì)胞(Jurkat、THP-1、K-562、 U937)、人類實(shí)體瘤細(xì)胞(Hela、MDA-MB-231、DU4475)、人類血管內(nèi)皮細(xì)胞(HUVEC)、小鼠原代心肌細(xì)胞、大鼠心肌細(xì)胞(H9c2)等。(2)P17在凋亡細(xì)胞上的信號比對照組高出10倍以上；結(jié)合迅速,孵育15分鐘后結(jié)合即可達(dá)到峰值；結(jié)合穩(wěn)定,信號強(qiáng)度可維持4小時以上：并可通過流式細(xì)胞術(shù)、普通熒光顯微鏡、激光共聚焦顯微鏡、酶標(biāo)儀等多種方法進(jìn)行觀測。(3)與不同凋亡時期標(biāo)志物共染色結(jié)果顯示,P17傾向于與晚期凋亡的細(xì)胞結(jié)合。(4)經(jīng)質(zhì)譜分析鑒定,Pull-down實(shí)驗(yàn)中P17捕獲的靶蛋白為熱休克蛋白60(HSP60)。熒光共定位及westernblot結(jié)果證實(shí),P17可與HSP60特異性結(jié)合,同時HSP60在細(xì)胞凋亡后表達(dá)量顯著增加。(5)在小鼠乳腺癌移植瘤模型中,通過尾靜脈注射FITC標(biāo)記的P17探針,可在小動物活體成像儀上觀測到小鼠腫瘤組織內(nèi)由化療藥誘導(dǎo)的凋亡區(qū)域或自發(fā)凋亡區(qū)域,并可在冰凍組織切片上清楚觀測到P17結(jié)合的凋亡區(qū)域。(6)在阿霉素誘導(dǎo)的急性心肌損傷模型中,尾靜脈注射P17后1-9小時內(nèi),阿霉素處理小鼠的全身及心臟內(nèi)可見明顯熒光信號,外周血中熒光信號強(qiáng)度平穩(wěn)下降；健康小鼠的熒光信號主要集中在膀胱區(qū)域,且心臟內(nèi)無明顯熒光信號,外周血中熒光強(qiáng)度在注射后1小時達(dá)到峰值,隨后快速降低。(7)對健康BALB/c小鼠尾靜脈注射P17,1小時后肝、腎兩個主要代謝器官的熒光信號強(qiáng)度達(dá)到峰值,隨后下降,并于注射后6小時恢復(fù)到生理鹽水對照組水平。(8)CCK-8及Annexin V/PI雙染色結(jié)果顯示,P17對細(xì)胞的增殖活性沒有影響,也不會引起細(xì)胞凋亡。同時,P17對細(xì)胞正常功能如細(xì)胞骨架、細(xì)胞粘附能力也沒有明顯影響。(9)對健康BALB/c小鼠尾靜脈注射P17后24小時,肝、腎組織細(xì)胞結(jié)構(gòu)完整,沒有發(fā)生變性腫脹、炎性細(xì)胞浸潤、充血等異常病理改變。結(jié)論：P17通過與細(xì)胞內(nèi)HSP60特異性結(jié)合而高效標(biāo)記凋亡細(xì)胞或發(fā)生凋亡的組織,并具備較快的體內(nèi)清除速率,有望發(fā)展成一種可用于外及體內(nèi)無創(chuàng)凋亡成像的新型凋亡探針,并展現(xiàn)出在評價藥物毒副作用、評價藥物療效等方面潛在的應(yīng)用價值。同時,P17揭示了HSP60與晚期凋亡之間的潛在聯(lián)系,為進(jìn)一步明確HSP60在凋亡中的具體作用提供了新思路。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R914;R96
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