NRCF动态蛋白组学测定、生物信息学分析及其功能的初步实验研究

发布时间：2018-06-05 22:10

  本文选题：周围神经再生 + 神经再生条件液　； 参考：《郑州大学》2014年博士论文

【摘要】：周围神经损伤(peripheral nerve injuries, PNI),是一类高发生率、高致残性的常见创伤,其发生的常见原因为意外伤害、疾病或者需要分离周围神经到达毗邻手术部位的外科操作等。对周围神经损伤处理不当,可引起其部分或全部功能丧失,影响患者生活质量,甚至造成患者残疾,给社会造成巨大的经济负担。临床工作中,周围神经损伤最常用的处理方法为断端直接缝合法和自体神经移植术。但因神经功能束的错位、吻合口瘢痕等,疗效难以进一步提高,并可造成供区的继发性损伤。因此,周围神经拟生态结构再生成为了机理研究的重要方向,也是对周围神经再生研究的新思路。研究表明,周围神经再生依赖于具备自我调控、自我完善机制的微环境。目前,基于神经再生室(nerve regeneration chambers, NRC)修复为核心的,综合应用功能细胞植入及基因治疗等多种措施的生物学治疗方法,取得了突破性进展,是周围神经再生研究的方向。20世纪80年代,Lundborg G和他的合作者采用了圆筒状NRC模型成功修复了大鼠周围神经的离断缺损,从而成为研究周围神经损伤的标准动物模型。此后,研究者对Lundborg G NRC模型的管腔材料、管腔内容物、可溶性调节成分和功能细胞等进行了相关研究。NRC模型所提供的封闭微环境是研究周围神经再生的重要窗口。而神经再生条件液(nerve regeneration conditioned fluid, NRCF)是NRC模型中自神经断端分泌的功能性液体,其来源于周围神经的组成细胞,或经轴突顺向转运、逆向弥散而来的物质成分,主要组成为蛋白,其在周围神经再生不同时期的表达存在种类和数量的动态变化。研究表明,NRCF对再生神经提供支持、营养和导向等重要作用。周围神经损伤的修复是一个复杂的、精细的生理过程。周围神经功能性再生取决于特定微环境和神经元自身生长能力的共同作用。相对于激活神经元自身生长能力是其固有特性方面,对损伤所处的特定微环境研究就更为重要。神经再生室模型(NRC)所提供的封闭微环境作为研究周围神经再生的重要窗口,而神经再生条件液(NRCF)是NRC中的功能性成分,对其进行研究将是揭示周围神经再生机理的重要途径。但目前,对周围神经再生的微环境因素,如细胞外基质、神经营养因子和细胞因子等,进行了大量研究,但都是随机的、散在的和推测的。因此,对NRCF中蛋白成分谱的描绘,可为研究微环境在周围神经再生中的作用提供直接依据,对阐明再生机制具有重要指导意义。而蛋白组学技术的发展为此提供了研究的方法基础。相对和绝对定量的等量异位标签(isobaric tags for relative and absolute quantitation, iTRAQ)技术是目前先进的蛋白组学技术,可同时比较4种或8种不同样品中蛋白质的相对含量或绝对含量,w为目前研究NRCF中整体蛋白成分和时间差异表达的有效方式。鉴于微环境在周围神经损伤修复中的重要作用,对其功能性成分(NRCF)进行研究将是了解周围神经再生机理的重要途径。本课题首次采用iTRAQ技术对周围神经再生四个关键时间点(神经离断3d,7d,14d和25d)NRCF的蛋白成分做了整体的、全景的动态呈现和分析,通过比对蛋白质组数据库,对共表达蛋白进行了生物信息学分析,发现了某些关键蛋白和周围神经再生的关系。通过实验初步探讨了NRCF对雪旺细胞和成纤维细胞增殖的影响。并结合前期研究成果,讨论了NRCF中不同补体成分参与周围神经再生过程的动态变化及其意义。第一部分 NRC模型的建立、NRCF的提取及对雪旺细胞和成纤维细胞增殖的影响目的典型的神经再生室(NRC)模型为连接神经两断端的圆筒状硅胶或其他材质所形成的密闭的间隙,其所提供的封闭微环境是研究周围神经再生的重要窗口,而神经再生条件液(NRCF)是NRC模型中自神经断端分泌的功能性成分,可对再生神经提供支持、营养和导向作用。雪旺细胞(schwann cell,Sc)及成纤维细胞(fibroblast cell, Fb)是参与周围神经再生的主要功能细胞。本部分实验采用Lewis大鼠坐骨神经(sciatic nerve)建立NRC模型,提取NRCF,并采用CCK-8法及流式细胞仪检测,探讨NRCF对雪旺细胞和成纤维细胞增殖的影响。方法Lewis大鼠坐骨神经10mm离断缺损,采用特定规格硅胶管体内建立NRC模型、7d时提取NRCF。BCA法进行NRCF蛋白浓度测定。雪旺细胞和成纤维细胞细胞株的传代培养。CCK-8法和流式细胞术检测NRCF对雪旺细胞和成纤维细胞增殖的影响。结果NRCF为清澈、淡黄色、呈类血清样并稍粘稠的液体,浓度为15.01μg/μl。雪旺细胞呈类圆形或梭形胞体,部分有两个或两个以上的突起,而成纤维细胞细胞体较大,呈棱形、长条状或不规则三角形,融合后为编织状,细胞核一般呈长椭圆形。NRCF能显著促进雪旺细胞(Sc)、成纤维细胞(Fb)的增殖,并且此作用呈时间、浓度依赖性,促进该两种细胞增殖的有效浓度为10000ng/ml。结论1.采用Lewis大鼠坐骨神经建立NRC模型,模型稳定、可靠,提取NRCF质量能很好满足实验要求。Lewis Rat Sciatic Nerve-NRC模型是研究周围神经再生的有效途径。2.采用CCK-8法及流式细胞仪检测技术,初步证明神经再生条件液(NRCF)具有促进雪旺细胞及成纤维细胞增殖的生物活性。第二部分NRCF-iTRAQ蛋白组学研究和生物信息学分析目的神经再生条件液(NRCF)是NRC中神经断端分泌的功能性成分,主要为蛋白成分,对NRCF中的蛋白成分进行研究将为揭示周围神经再生机理提供一个平台。本部分实验采用iTRAQ蛋白组学技术联合质谱技术,以周围神经再生四个关键时间点(坐骨神经离断3d,7d,14d和25d)所提取的NRCF为研究对象,对NRCF每个时间点、全部时间点及其各个时间点之间的蛋白定性和定量表达情况进行分析,并通过比对蛋白质组数据库,对共表达蛋白进行了进一步的生物学信息分析。方法建立NRC模型、提取3d,7d,14d和25dNRCF材料及其NRCF样品处理。SDS-PAGE电泳。iTRAQ蛋白组学技术、质谱鉴定。GO分析、KEGG通路分析、分层聚类分析、共趋势分析、时序表达的基因筛选等生物信息学分析方法对NRCF蛋白进行定性、定量分析。结果3d,7d,14d和25dNRCF4组样品蛋白质条带分布较均一,平行度良好。3d,7d,14d和25d神经再生条件液(NRCF)中共鉴定出876个蛋白,其中,3d,7d,14d和25d神经再生条件液(NRCF)中蛋白个数分别为：536、645、376和548个,其独特蛋白质个数分别为：95、137、18和58个,共同蛋白质个数为264个。共表达蛋白定量生物信息学分析显示：KEGG Pathway所得到的TOP 10通路为：补体及凝血级联、金黄色葡萄球菌感染、系统性红斑狼疮、百日咳、粘着斑、朊病毒、阿米巴病、肌动蛋白骨架调节、糖酵解/糖异生、ECM受体作用等。共趋势分析第2组、第3组和第4组TOP 7蛋白分别为PROFILIN-1、 DESTRIN-LIKE、ACTIN-RELATED PROTEIN 2/3 COMPLEX SUBUNIT 2、 ANNEXIN A1、CORONIN-1A、PROTEIN S100-A8和PROTEIN S100-A9等；CLUSTERIN. EPIDERMAL GROWTH FACTOR RECEPTOR 、RETINOL-BINDING PROTEIN 4、COLLAGEN ALPHA-2(I) CHAIN、HIGH MOBILITY GROUP PROTEIN B2-LIKE、LIVER CARBOXYLESTERASE 1和COMPLEMENT FACTOR I等；EUKARYOTIC TRANSLATION ELONGATION FACTOR 1 ALPHA 2、HEMOGLOBIN SUBUNIT ALPHA-1/2、HEMOGLOBIN ALPHA 2 CHAIN. SUPEROXIDE DISMUTASE [CU-ZN].、GALECTIN-5、 ISOFORM 2 OF GROWTH FACTOR RECEPTOR-BOUND PROTEIN 2和CARBONIC ANHYDRASE 2等。显著差异蛋白和高连接度信息蛋白为GRB2、 ACTB、ALB、VIM、VCP、A2M、CALR、THBS1、TF和PEBP1等。结论1.采用iTRAQ蛋白组学技术联合质谱技术,对周围神经再生的四个关键时间点的NRCF蛋白进行了全景的、动态的分析,建立了一个相对完整的NRCF蛋白数据库。NRCF蛋白含量丰富,种类繁多,每个关键时间点既有特异蛋白,各个关键时间点也有共同蛋白,与周围神经再生不同阶段关系密切。2.数据分析筛选出周围神经再生的不同关键时间点的特异蛋白、共趋势分析筛选出的不同组别的高表达蛋白和时序表达的基因筛选所获得的显著表达蛋白等,为寻找影响周围神经再生的关键蛋白提供了依据。这些数据,不仅,丰富了对周围神经再生的认识,而且,为将来神经再生课题提供了众多的基因和蛋白研究靶点。3. NRCF蛋白的全景揭示和相关文献复习,对既往神经再生所取得的研究成果也进行了部分验证,并为深入研究再生理论开辟了新的途径。第三部分NRCF中补体成分及与周围神经再生关系目的NRCF蛋白谱提示,补体系统成分的大量存在,并在不同神经再生时期具有明显的动态变化。生物学分析(KEGG通路分析)表明补体通路位居前十个显著富集通路。补体系统在周围神经再生过程中所起的作用非常值得研究。补体系统(几种或一种关键成分)所介导的免疫反应、炎症反应,甚至与两者无关,但与神经再生相关的作用于PNI中亟待揭示。本部分将对NRCF-iTRAQ蛋白组学结果发现的大量补体成分及其动态变化加以描述、分析和总结,并采用Western blot技术对NRCF中部分补体成分加以验证,对其可能参与周围神经再生的作用机制加以探讨。方法建立NRC模型、提取7dNRCF及其NRCF样品处理。iTRAQ蛋白组学技术相关方法(同第二部分)。Western blot实验技术检测C1q、C3、C7和CFD在NRCF中的表达。结果NRCF-iTRAQ蛋白组学分析所得10种补体成分分别为CFI、Clq-A、Clq-B、 C2、C3、C4、C5、C7、C8β和CFD等。Western blot证实NRCF中有C1q、C3、C7和CFD的表达。结论NRCF中有10余种补体成分定性和定量信息的存在,补体C7在再生不同时间阶段量化分析有显著性变化。补体Clq、C3、C4和C5等与神经再生关系密切。
[Abstract]:Peripheral nerve injuries (PNI) is a kind of high incidence, high disability common trauma, the common cause of which is accidental injury, disease, or surgical operation that needs to separate the peripheral nerve to the adjacent surgical site. Improper treatment of peripheral nerve damage can cause some or all function loss. In clinical work, the most commonly used treatment methods for peripheral nerve injury are the direct fracture method and autologous nerve grafting in clinical work. However, because of the dislocation of the nerve function bundle, the anastomotic scar and so on, the treatment effect is difficult to be further improved and the secondary sex of the donor area can be caused. Therefore, the regeneration of the peripheral nerve quasi ecological structure has become an important direction of the mechanism research, and a new idea for the study of peripheral nerve regeneration. The study shows that the regeneration of peripheral nerve depends on the micro environment with self-regulation and self-improvement mechanism. At present, the nerve regeneration chambers (NRC) repair is the core. A variety of biological therapies, such as functional cell implantation and gene therapy, have made breakthrough advances. The direction of peripheral nerve regeneration is the direction of the.20 century in 80s. Lundborg G and his collaborators used a cylindrical NRC model to successfully repair the circumference of the rat's circumference defect, thus becoming a study of the surrounding gods. After the damage of the standard animal model, the researchers have studied the lumen materials, lumen content, soluble regulatory components and functional cells of the Lundborg G NRC model. The closed microenvironment provided by the.NRC model is an important window for the study of the regeneration of peripheral nerve (nerve regeneration conditioned f). Luid, NRCF) is a functional liquid secreted from the nerve terminal of the NRC model. It is derived from the constituent cells of the peripheral nerve, or the transshipment of the axonal process, and the reverse diffuse material, which is mainly composed of the protein. The dynamic changes of the species and quantity in the expression of the peripheral nerve regeneration at different times. The study shows that NRCF is a regenerative God. The repair of peripheral nerve injury is a complex, fine physiological process. The functional regeneration of the peripheral nerve depends on the joint action of the specific microenvironment and the ability of the neuron itself. The study of specific microenvironment is more important. The closed microenvironment provided by the neural regeneration chamber model (NRC) is an important window for the study of peripheral nerve regeneration, and the neural regeneration conditioned fluid (NRCF) is a functional component in NRC. The study of it will be an important way to reveal the regeneration mechanism of peripheral nerve. Microenvironmental factors, such as extracellular matrix, neurotrophic factor and cytokine, have been studied in a large amount, but they are random, scattered and speculative. Therefore, the description of the protein composition in NRCF can provide a direct basis for the study of the role of microenvironment in the regeneration of peripheral nerves, and is of great guiding significance for clarifying the mechanism of regeneration. The development of proteomics technology provides a basis for research. Relative and absolute quantitative heterotopic tags (isobaric tags for relative and absolute quantitation, iTRAQ) are currently advanced proteomics techniques that can simultaneously compare the relative or absolute content of protein in 4 or 8 different products, and W is the eye. In view of the important role of the protein composition and time difference expression in NRCF, in view of the important role of microenvironment in the repair of peripheral nerve injury, the study of its functional components (NRCF) will be an important way to understand the mechanism of peripheral nerve regeneration. This subject is the first time to use iTRAQ technology to regenerate four key time points for peripheral nerve regeneration. The protein components of 3D, 7d, 14d and 25d) NRCF were used as a holistic, panoramic dynamic presentation and analysis. By comparing the proteome database to the bioinformatics analysis of the co expressed proteins, the relationship between some key proteins and the regeneration of peripheral nerve was found. The experiment was conducted to explore the NRCF to Schwann cells and fibroblasts through the experiment. The effect of proliferation, combined with previous research results, discussed the dynamic changes and significance of different complement components involved in peripheral nerve regeneration in NRCF. The establishment of the first part of the NRC model, the extraction of NRCF and the effect on the proliferation of Schwann cells and fibroblasts, the typical NRC model is the connection between the two ends of the nerve. The closed space formed by cylindrical silica gel or other materials is an important window to study the regeneration of peripheral nerve, and the nerve regeneration fluid (NRCF) is a functional component secreted from the nerve terminal in the NRC model, which can provide support, nutrition and guidance to the regenerative nerve. Schwann cell (S) C) and fibroblast cell (Fb) are the main functional cells involved in peripheral nerve regeneration. This part of the experiment used Lewis rat sciatic nerve (sciatic nerve) to establish NRC model and extract NRCF. The effect of NRCF on the proliferation of Schwann cells and fibroblasts was investigated by CCK-8 method and flow cytometry. Method Lewis rats were seated. NRC model was established in specific specs of silicone tube in 10mm, and NRCF.BCA was used to determine NRCF protein concentration in 7d. The effect of NRCF on the proliferation of Schwann cells and fibroblasts was detected by.CCK-8 and flow cytometry in Schwann and fibroblast cell lines. Results NRCF was clear and yellowish. A serolike and slightly thicker liquid, with a concentration of 15.01 mu g/ Mu L. in a round or spindle cell, partially with two or more than two protrusions, and the fibroblast cell body is larger, prismatic, elongated or irregular triangle, and after fusion, a long oval.NRCF can significantly promote Schwann cells (Sc). The proliferation of fibroblasts (Fb), and this effect is time, concentration dependent, and the effective concentration of the two cell proliferation is 10000ng/ml. conclusion 1. using the Lewis rat sciatic nerve to establish the NRC model, the model is stable and reliable. The extraction of NRCF quality can meet the requirements of the experimental.Lewis Rat Sciatic Nerve-NRC model is to study the peripheral nerve. The effective pathway of regeneration,.2., using CCK-8 and flow cytometry, has preliminarily proved that the neural regeneration conditioned fluid (NRCF) has biological activity to promote the proliferation of Schwann cells and fibroblasts. Second part of NRCF-iTRAQ proteomics and bioinformatics analysis of neural regeneration conditioned fluid (NRCF) is the secretion of nerve broken ends in NRC Functional components, mainly protein components, the study of protein components in NRCF will provide a platform for revealing the mechanism of peripheral nerve regeneration. This part of the experiment uses iTRAQ proteomics technology combined with mass spectrometry technology to study the NRCF from the four key time points of peripheral nerve regeneration (the sciatic deity 3D, 7d, 14d and 25d). To analyze the qualitative and quantitative expression of protein between all time points and each time point at each time point of NRCF, and to further analyze the biological information by comparing the proteomic database to the co expressed protein. The NRC model was established to extract the 3D, 7d, 14d and 25dNRCF materials and the NRCF sample processing.SDS-PAG. E electrophoresis.ITRAQ proteomics technology, mass spectrometric identification.GO analysis, KEGG pathway analysis, stratified cluster analysis, CO trend analysis, sequence expression gene screening, and other bioinformatics analysis methods for NRCF protein were qualitatively and quantitatively analyzed. Results 3D, 7d, 14d and 25dNRCF4 group samples were more homogeneous in protein bands,.3d, 7d, 14d, and exports were in good parallelism. 876 proteins were identified in the nerve regeneration fluid (NRCF), of which the number of proteins in 3D, 7d, 14d and 25d were 536645376 and 548, respectively: 95137,18 and 58, and 264 common protein numbers. The co expressed protein quantitative bioinformatics analysis showed KEGG Pathwa The TOP 10 pathway obtained by Y is: complement and coagulation cascade, Staphylococcus aureus infection, systemic lupus erythematosus, pertussis, sticky spots, prion, amieobiasis, actin cytoskeleton regulation, glycolytic / glycosyogenesis, ECM receptor, etc., second groups of CO trend analysis, third and fourth groups of TOP 7 proteins are PROFILIN-1, DESTRIN-LIKE, ACTI, respectively. N-RELATED PROTEIN 2/3 COMPLEX SUBUNIT 2, ANNEXIN A1, CORONIN-1A, PROTEIN S100-A8 and PROTEIN S100-A9. RANSLATION ELONGATION FACTOR 1 ALPHA 2, HEMOGLOBIN SUBUNIT ALPHA-1/2, HEMOGLOBIN ALPHA 2 CHAIN. SUPEROXIDE DISMUTASE. TF and PEBP1. Conclusion 1. using the iTRAQ protein group technique combined with mass spectrometry, the NRCF protein of the four key time points of peripheral nerve regeneration was panoramic and dynamic analysis. A relatively complete NRCF protein database was established, and the.NRCF protein content was rich and varied. Each key time point had both specific protein and each key. The key time point also has common protein, which is closely related to the different stages of peripheral nerve regeneration..2. data analysis is used to screen out the specific proteins at different critical time points of peripheral nerve regeneration. Co trend analysis is used to screen the high expression proteins of different groups and the significant protein expressed in the sequence of gene screening. These data, not only, enrich the understanding of the regeneration of peripheral nerve, but also provide a panoramic view of the.3. NRCF protein of the target of gene and protein research for the future nerve regeneration and the review of relevant literature, and some of the achievements of previous neural regeneration have also been carried out. A new approach was opened up to study the theory of regeneration. In part third, the complement components and the relationship with the peripheral nerve regeneration in the third part of the NRCF protein spectrum suggest that the components of the complement system exist in large quantities and have obvious dynamic changes during the different period of nerve regeneration. Biological analysis (KEGG pathway analysis) indicates that the complement pathway is in the front. Ten significant enrichment pathways. The role of the complement system in the regeneration of peripheral nerve is very worthy of study. The immune response, inflammation, and even the relationship between the complement system (several or one of the key components) are not related to the two, but the role of the nerve regeneration in PNI needs to be revealed. This part will be used in the NRCF-iTRAQ protein group. A large number of complement components and its dynamic changes were described, analyzed and summarized, and Western blot technology was used to verify the part of the complement components in NRCF. The mechanism of its possible participation in peripheral nerve regeneration was discussed. A NRC model was established to extract 7dNRCF and its NRCF samples to deal with.ITRAQ protein components. The expression of C1q, C3, C7 and CFD in NRCF was detected by the method of.Western blot (the same second part). Results there were more than 10 complement components in NRCF-iTRAQ protein component analysis. Quantitative analysis showed that there was a significant change in complement C7 at different stages of regeneration. Complement Clq, C3, C4 and C5 were closely related to nerve regeneration.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R745
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本文编号：1983575