新型近红外小分子NIRCP-61用于干细胞示踪成像和促进组织修复的实验研究

发布时间：2018-01-20 14:36

本文关键词： 近红外荧光成像荧光小分子染料细胞保护干细胞示踪组织修复与再生　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景干细胞移植治疗在再生医学领域受到越来越多的关注,但是由于对干细胞在移植后的迁移、归巢、定植、分布等生物行为缺乏深刻的认识和理解以及非常有限的体内移植干细胞存活数量等原因,外源性干细胞移植治疗效果还有待进一步提高。因此,寻找一种能同时满足当前干细胞移植所面临的细胞标记示踪和细胞保护两方面需求的多功能材料,有利于促进干细胞移植治疗的发展。据检索,目前还没有这种多功能材料的相关报道。生物光学成像由于检测仪器发展成熟、灵敏度高、对比度高、分辨率高、成像直观、成像速度快和无损检测等优点被广泛应用于医学生物研究。依据检测方式的不同生物光学成像可分为荧光成像、生物发光成像、光声成像、光学断层层析成像等,其中活体动物体内光学成像主要采用生物发光成像与荧光成像。生物发光成像技术是用荧光素酶基因标记细胞或DNA,而荧光成像技术则采用荧光报告基团(GFP、RFP及Cyt等)或荧光染料进行标记。生物发光成像和部分荧光成像技术由于需要构建基因表达载体转染目标细胞,具有潜在的生物风险性,导致其在临床干细胞标记成像中的应用受到限制。而近红外(near-infrared,NIR)荧光染料标记技术由于较低的组织自发荧光和较强的组织穿透能力等特点,通过荧光染料标记可以对目标细胞进行实时、连续检测,在小动物活体成像方面具有广阔的应用前景,目前正逐步应用于细胞标记示踪研究。与量子点、碳点等无机近红外荧光材料相比,有机近红外荧光染料具有更高的荧光量子产率、结构易调、生物安全性好等优点而备受关注。但是,当前已有的包括吲哚菁绿(indocyanine green,ICG)在内的有机近红外荧光染料大都是亲脂性的细胞膜标记染料,标记效率较低,且在生物荧光成像过程中容易和体内蛋白等生物分子相互作用,存在着严重的细胞沾染以及潜在的细胞毒性作用,限制了这类染料在干细胞标记示踪中的应用。七甲川花菁染料是近红外荧光染料中的优秀代表,部分染料能够特异性地蓄积于细胞线粒体之中并具有良好的光学稳定性,多种七甲川花菁染料已经应用于生物活体中细胞标记与示踪成像研究。本课题组在之前的工作中,通过对七甲川花菁染料的化学结构进行修饰和改造,合成制备了一系列新型七甲川花菁荧光小分子化合物。通过结构学研究分析以及其他研究者的相关报道,确定这类七甲川花菁荧光小分子能够靶向蓄积于细胞线粒体之中;同时由于保留了七甲川花菁染料的近红外荧光特性,为用于细胞标记、活体示踪以及生物成像提供了基础。此外,此类七甲川花菁荧光小分子靶向蓄积于细胞线粒体之中,对线粒体功能产生一定影响,从而使细胞命运发生转变。基于当前干细胞移植治疗的迫切需求和本课题组以往的研究工作基础,发展出一种新的能够同时具有细胞标记示踪和细胞保护作用的七甲川花菁荧光小分子化合物,在干细胞治疗领域具有重要意义和潜在应用前景。研究方法1.多功能荧光小分子的合成与鉴定在以往的工作基础上,通过对七甲川花菁染料的七甲川核心周边N-烷基侧链进行结构修饰和改造,合成制备了一系列新的、保留了七甲川花菁染料细胞线粒体靶向蓄积和近红外荧光成像等原有特性的荧光小分子化合物。本研究从这些新型荧光小分子化合物中筛选出一种水溶性、荧光强度以及生物相容性较好,适用于干细胞示踪成像,同时还能够促进细胞表达抗氧化相关蛋白,增强细胞抗氧化应激损伤能力的多功能荧光小分子(NIR cell protector-61,NIRCP-61)。2.NIRCP-61用于间充质干细胞标记示踪成像利用原代分离培养的四种不同来源的间充质干细胞(mesenchymal stem/stromal cells,MSCs),包括人脐带间充质干细胞(h UCMSCs)、人真皮间充质干细胞(h DMSCs)、大鼠真皮间充质干细胞(r DMSCs)和小鼠骨髓间充质干细胞(m BMMSCs),通过近红外荧光显微镜观察和流式细胞术(flow cytometry,FCM)检测确定NIRCP-61对MSCs的最佳标记条件和标记效率;分别采用CCK-8实验、Annexin V/碘化丙啶(propidium iodide,PI)细胞凋亡检测实验、细胞迁移实验、成骨诱导分化实验、成脂诱导分化实验检测在最佳标记条件下NIRCP-61对MSCs正常生理功能的影响;利用近红外荧光显微镜检测NIRCP-61标记细胞后6天内的荧光稳定性并使用激光共聚焦检测技术确定NIRCP-61标记在细胞内的亚细胞定位;建立放创复合伤小鼠模型,利用流式细胞术、激光共聚焦检测技术和小动物活体成像技术,通过体外细胞实验和体内动物实验分别检测NIRCP-61标记MSCs对周边非标记细胞和组织的沾染情况,以及标记细胞活体示踪成像情况。3.NIRCP-61对间充质干细胞抗氧化应激损伤的实验研究通过过氧化氢(hydrogen peroxide,H2O2)处理或γ射线照射细胞,建立体外细胞氧化应激损伤模型,采用CCK-8实验、Annexin V/PI细胞凋亡检测实验和钙黄绿素/PI染色检测评价NIRCP-61减少H2O2处理造成的细胞活力降低、凋亡和死亡情况;采用集落形成实验、细胞核内γ-H2AX免疫荧光染色、中性彗星实验以及Western Blot检测评估NIRCP-61减轻电离辐射(ionizing radiation,IR)对细胞的损伤情况;采用流式细胞术和Western Blot检测NIRCP-61处理后细胞和线粒体活性氧(reactive oxygen species,ROS)生成以及细胞内抗氧化相关蛋白的表达变化情况;通过Western Blot检测NIRCP-61处理后细胞内核因子2相关因子2/抗氧化反应元件(nuclear factorerythroid 2-related factor 2/antioxidant response element,Nrf2/ARE)和磷脂酰肌醇3-激酶/蛋白激酶B(phosphoinositide 3-kinase/proteinkinase B,PI3K/AKT)信号通路相关蛋白的表达变化情况;使用人Nrf2的小干扰RNA(small interfering RNA,si RNA)以及PI3K的抑制剂LY294002反向验证Nrf2/ARE和PI3K/AKT信号通路在NIRCP-61促进细胞抗氧化应激损伤中发挥的作用。4.NIRCP-61促进间充质干细胞体内定植存活和组织修复的研究建立放创复合伤Sprague-Dawley(SD)大鼠模型和大剂量皮肤放射损伤大鼠模型,通过检测创面愈合速度、瘢痕形成质量、胶原形成情况以及观察皮肤水疱、脱屑以及坏死性溃疡形成等指标评估NIRCP-61促进间充质干细胞定植存活,减轻机体组织损伤,促进组织修复再生,提高细胞治疗潜能的情况;通过人工结扎大鼠冠状动脉左前降支建立急性心肌梗死大鼠模型,分组治疗4周后进行超声心动图检测、心脏取材固定后进行2,3,5-三苯基氯化四氮唑(2,3,5-Triphenyltetrazolium chloride,TTC)染色、免疫组织化学染色以及Masson染色检测NIRCP-61促进间充质干细胞在大鼠体内定植存活,减少左心室梗死面积,减轻组织纤维化形成,维持心脏正常功能的情况。研究结果1.通过对七甲川花菁染料进行化学结构修饰、改造,合成制备了一系列新型近红外荧光小分子化合物,并从中成功筛选出新型荧光小分子(NIRCP-61),其水溶性和生物相容性好、标记效率和荧光强度高,并且能够同时满足细胞移植治疗对细胞标记示踪成像和细胞保护两方面的需求。2.荧光小分子NIRCP-61保留了七甲川花菁染料良好的近红外荧光以及细胞线粒体靶向蓄积的特性,体外细胞标记过程简便易行,无需额外的转染试剂或操作步骤即可完成。同当前已有的其它近红外荧光探针相比,NIRCP-61标记效率高,生物相容性好,并且标记细胞对周边细胞和组织微环境的非特异性沾染明显减少,显著提升了荧光信号的特异性,在干细胞标记示踪成像中的应用前景更佳。3.荧光小分子NIRCP-61能够减轻过氧化氢处理造成的细胞活力降低,减少细胞凋亡;维持电离辐射后干细胞的成集落能力;免疫荧光染色、中性彗星实验以及Western Blot检测结果证明NIRCP-61可明显减少IR造成的细胞DNA损伤;ROS检测结果显示,NIRCP-61处理能显著降低过氧化氢或IR处理引起的细胞内ROS水平升高,减轻过量ROS对细胞造成的损伤;Western Blot检测结果表明,NIRCP-61的细胞保护作用是通过活化细胞内Nrf2和PI3K/Akt信号通路,激活细胞内源性抗氧化还原反应通路,促进细胞高表达一系列内源性抗氧化蛋白而实现。4.在三种动物损伤模型中证明荧光小分子NIRCP-61能够显著减轻机体内氧化应激微环境对移植干细胞造成的损伤,促进移植细胞在机体病损部位及周边组织内的定植和存活,减轻组织损伤和纤维化程度,加速创伤愈合,改善瘢痕形成质量,促进组织修复与再生,提高细胞移植治疗的效果。研究结论据检索,这种同时具备近红外荧光成像和细胞保护功能的荧光小分子尚属首次报道。线粒体靶向的NIRCP-61对不同种类的MSCs均具有优异的标记性能,适合用于干细胞的示踪成像;且能显著减轻氧化应激对MSCs造成的损伤,促进MSCs移植后在生物体内的定植存活,减轻组织损伤,减少纤维化形成,促进机体组织修复与再生,提高细胞治疗潜能。因此,近红外荧光小分子NIRCP-61代表了一类新颖的、可提高细胞移植治疗效果的治疗性荧光探针,在干细胞治疗的临床转化中具有潜在的应用前景。
[Abstract]:The research background of stem cell transplantation in the treatment has attracted more and more attention in the field of regenerative medicine, but due to the migration of stem cells in transplantation, homing, colonization, distribution of biological behavior and the lack of deep understanding and the understanding of stem cell transplantation in vivo very limited number of survival and other reasons, the treatment effect of exogenous stem cell transplantation remains to be further to improve. Therefore, looking for a can meet the current stem cell transplantation multifunctional material facing cell tracer and cell protection two aspects of demand, is conducive to the promotion of stem cell transplantation in the treatment of development. According to the search, there have been no reports of this kind of multifunctional materials. Because of bioluminescent imaging detection instrument development, high sensitivity, high contrast, high resolution imaging, intuitive, fast imaging and nondestructive testing is widely used in medical and biological research. According to the test Different biological optical imaging mode can be divided into fluorescence imaging, bioluminescence imaging, photoacoustic imaging, optical tomography tomography, the animal in vivo optical imaging mainly using bioluminescence imaging and fluorescence imaging. Bioluminescence imaging technology is the use of luciferase gene or DNA labeled cells, and fluorescence imaging using fluorescent (GFP, RFP and Cyt) or fluorescent dye labeled. Due to the construction of gene expression vector was transfected into target cell imaging and fluorescence imaging part of bioluminescence, with biological potential risk, resulting in the clinical application of stem cell marker imaging is limited. In the infrared (near-infrared, NIR) fluorescent dye labeled as technology the characteristics of low autofluorescence and strong tissue penetration, by fluorescent dye labeled target cells can be real-time, continuous inspection Test, has a broad application prospect in small animal imaging, is gradually being used in the study of cell labeling. Compared with quantum dots, carbon and other inorganic near infrared fluorescent materials, organic near infrared fluorescent dyes with higher fluorescence quantum yields, easy to adjust the structure, has the advantages of good bio security is attracting more and more attention however, the current including indocyanine green (indocyanine green, ICG), organic near infrared fluorescent dyes are cell membrane marker dye labeled lipophilic, low efficiency, and the interaction in biological fluorescence imaging process easily and in vivo protein and other biological molecules, there are serious contamination and cell cytotoxicity the potential and limits the type of dyes in the application of stem cell labeling in seven. Methine cyanine dye is an excellent representative of near infrared fluorescent dyes, dye specific volume In the mitochondria and has good optical stability, a variety of seven methine cyanine dyes have been applied to the study of cell biology in vivo labeling and tracing imaging. This group in the previous work, modified or altered by the chemical structure of seven methine cyanine dyes, a series of novel seven methine cyanine the fluorescence of small molecule compounds were synthesized. The related reports on analysis and other structural studies of the determination of such seven methine cyanine fluorescent molecules can accumulate in mitochondria targeting; at the same time due to the retention of the near infrared fluorescence of seven methine cyanine dyes, for cell labeling, provide based in vivo tracking and biological imaging. In addition, the seven methine cyanine fluorescent small molecules targeting accumulation in the mitochondria, have a certain impact on mitochondrial function, so that the cell fate occurred The current urgent needs and change. Do the research work based on previous group cell transplantation based on the development of a new can also have a cell labeling and cell protection seven methine cyanine fluorescent small molecule compounds, has important significance and potential application prospect in the field of stem cell therapy. Methods: 1. multifunctional fluorescent small molecule synthesis and identification based on the previous work, for structural modification through seven methine core surrounding the alkyl side chain of N- of seven methine cyanine dyes, the synthesis of a series of new compounds, fluorescent molecules retained seven a mitochondrial Sichuan cyanine dye cell targeting and accumulation of near infrared fluorescence imaging of the original characteristics. This study from these new fluorescent small molecules screened a water-soluble, fluorescence intensity and good biocompatibility, suitable for stem cells Cell imaging, but also can promote the expression of antioxidant related proteins in cells, enhance the multifunctional fluorescent small molecule cell oxidative stress damage ability (NIR cell protector-61, NIRCP-61.2.NIRCP-61) for mesenchymal stem cells labeled by imaging using four different sources of primary cultured mesenchymal stem cells (mesenchymal stem/stromal cells, MSCs), including human umbilical cord mesenchymal stem cells (H UCMSCs), human dermal mesenchymal stem cells (H DMSCs), rat dermal mesenchymal stem cells (R and DMSCs) of mouse bone marrow mesenchymal stem cells (m, BMMSCs) by near infrared fluorescence microscopy and flow cytometry. Surgery (flow cytometry, FCM NIRCP-61) testing to determine the optimal labeling conditions of MSCs and the labeling efficiency; using CCK-8 experiment, Annexin V/ propidium iodide (propidium iodide, PI) cell apoptosis assay, cell migration assay, bone induction The differentiation and adipogenic differentiation assay of NIRCP-61 in the best condition marked influence on MSCs of normal physiological function; the fluorescence stability of NIRCP-61 labeled cells 6 days after detection of near infrared fluorescence microscopy and confocal laser detection technique to determine the subcellular localization of NIRCP-61 labeled cells in mice; establishment of radiation combined wound the model, using flow cytometry, confocal laser detection technology and small animal imaging technology, NIRCP-61 marker MSCs on the surrounding non labeled cells and tissue contamination were detected by in vitro and in vivo animal experiments, and the experimental study of.3.NIRCP-61 labeled cells in vivo imaging of mesenchymal stem cells against oxidative stress injury by hydrogen peroxide (hydrogen peroxide, H2O2) or gamma ray irradiated cells, in vitro cell model establishment of oxidative stress injury, The CCK-8 experiment, the apoptosis of Annexin cells was determined by V/PI experiment and calcein /PI staining for detection and evaluation of NIRCP-61 H2O2 treatment caused a decrease in cell viability decreased, apoptosis and death; using colony formation assay, cell nucleus gamma -H2AX immunofluorescence staining, neutral comet assay and Western Blot detection and evaluation of NIRCP-61 reduced (ionizing radiation, IR of ionizing radiation) damage of cells; used after the treatment of NIRCP-61 cells and mitochondrial ROS was detected by flow cytometry and Western Blot (reactive oxygen species, ROS) generation and expression of related intracellular antioxidant; cell nuclear factor by Western Blot detection after treatment with NIRCP-61 2 related factors 2/ antioxidant response element (nuclear factorerythroid 2-related factor 2/antioxidant response element, Nrf2/ARE) and phosphatidylinositol 3- kinase / protein kinase B (Pho Sphoinositide 3-kinase/proteinkinase B, PI3K/AKT) expression of signal pathway related proteins; small interfering RNA use Nrf2 (small interfering RNA, Si RNA) and PI3K inhibitor LY294002 and PI3K/AKT reverse validation of Nrf2/ARE signal pathway in NIRCP-61 to promote the role of.4.NIRCP-61 in cell oxidative stress damage in promoting the survival of mesenchymal stem cells and tissues in vivo colonization of wound repair to establish the Sprague-Dawley (SD) rats model and large dose of radiation skin injury, wound healing through detection, the quality of scar formation, collagen formation and observation of skin blisters, desquamation and necrotic ulcer formation and evaluation index of NIRCP-61 promote mesenchymal stem cells transplanting survival, reduce the body tissue damage, promote tissue repair and regeneration, improve the therapeutic potential of the cells through the artificial ligation; In the left anterior descending coronary artery to establish acute myocardial infarction rat model group after 4 weeks of treatment, echocardiography, heart were fixed after 2,3,5- three 5-triphenyl tetrazolium chloride (2,3,5-Triphenyltetrazolium chloride, TTC) staining, immunohistochemical staining and Masson staining NIRCP-61 promote mesenchymal stem cell survival in vivo. In rats, reduce the infarct area of the left ventricle, reduce tissue fibrosis, maintain normal heart function. Results: 1. through chemical modification, modification of seven methine cyanine dyes, a series of novel near infrared fluorescent small molecule compounds were synthesized, and the successful screening of new fluorescent molecules (NIRCP-61), its water solubility and biocompatibility, fluorescence labeling efficiency and high strength, and can meet the need of cell transplantation for the treatment on cell labeling and imaging Cell protection requirements of.2. fluorescent molecules on NIRCP-61 two retained seven methine cyanine dyes with good NIR fluorescence and cell mitochondrial targeting accumulation characteristics, in vitro cell labeling process is simple and easy to complete, without additional transfection reagents or operation steps. Compared with other near infrared fluorescent probe with the existing. NIRCP-61 markers of high efficiency, good biocompatibility, and nonspecific contamination of labeled cells on the surrounding cells and tissue microenvironment significantly reduced, significantly enhance the specificity of fluorescence signal, the application prospect of stem.3. better fluorescent molecules of NIRCP-61 cells labeled by imaging can reduce the cell viability of hydrogen peroxide treatment caused a decrease in cell, reduce apoptosis; maintain after ionizing radiation stem cell colony forming ability; immunofluorescence staining, neutral comet assay and Western Blot assay results indicated that NIRCP -61 could significantly reduce DNA cell injury caused by IR; ROS assay showed that NIRCP-61 treatment can significantly reduce the elevated levels of ROS or IR treatment caused by hydrogen peroxide in cells, reduce excessive ROS causing damage to cells; Western Blot assay showed that NIRCP-61 cell protective effect by activating intracellular Nrf2 and PI3K/Akt signaling pathway activation of endogenous antioxidant, reaction pathway, and the realization of.4. in three kinds of animal models demonstrate the damage of fluorescent molecules of NIRCP-61 can significantly reduce the oxidative stress in the light microenvironment of the transplanted stem cells damage caused by the high expression of a series of endogenous antioxidant protein, promote colonization of transplanted cells in the lesion and surrounding tissues in the body and survival, reduce tissue damage and fibrosis, accelerate wound healing, improve the quality of scar formation, promote tissue repair and regeneration. High cell transplantation therapy. The research conclusion retrieval of fluorescent molecules with the near-infrared fluorescence imaging and cell protection function. This is the first report of NIRCP-61 mitochondrial targeting were tagged with excellent performance for different types of MSCs, suitable for stem cell imaging; and can significantly reduce the oxidative stress of damage caused by MSCs, survival in vivo to promote colonization of MSCs after transplantation, reduce tissue damage, reduce fibrosis, promote tissue repair and regeneration, improve the therapeutic potential of cells. Therefore, near infrared fluorescent small molecule NIRCP-61 represents a novel treatment, fluorescent probe can improve the efficacy of transplantation, with the potential application prospect in clinical translation of stem cell therapy.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R45

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