本文選題：周圍神經(jīng)再生 + 神經(jīng)再生條件液��； 參考：《鄭州大學》2014年博士論文

【摘要】：周圍神經(jīng)損傷(peripheral nerve injuries, PNI),是一類高發(fā)生率、高致殘性的常見創(chuàng)傷,其發(fā)生的常見原因為意外傷害、疾病或者需要分離周圍神經(jīng)到達毗鄰手術(shù)部位的外科操作等。對周圍神經(jīng)損傷處理不當,可引起其部分或全部功能喪失,影響患者生活質(zhì)量,甚至造成患者殘疾,給社會造成巨大的經(jīng)濟負擔。臨床工作中,周圍神經(jīng)損傷最常用的處理方法為斷端直接縫合法和自體神經(jīng)移植術(shù)。但因神經(jīng)功能束的錯位、吻合口瘢痕等,療效難以進一步提高,并可造成供區(qū)的繼發(fā)性損傷。因此,周圍神經(jīng)擬生態(tài)結(jié)構(gòu)再生成為了機理研究的重要方向,也是對周圍神經(jīng)再生研究的新思路。研究表明,周圍神經(jīng)再生依賴于具備自我調(diào)控、自我完善機制的微環(huán)境。目前,基于神經(jīng)再生室(nerve regeneration chambers, NRC)修復(fù)為核心的,綜合應(yīng)用功能細胞植入及基因治療等多種措施的生物學治療方法,取得了突破性進展,是周圍神經(jīng)再生研究的方向。20世紀80年代,Lundborg G和他的合作者采用了圓筒狀NRC模型成功修復(fù)了大鼠周圍神經(jīng)的離斷缺損,從而成為研究周圍神經(jīng)損傷的標準動物模型。此后,研究者對Lundborg G NRC模型的管腔材料、管腔內(nèi)容物、可溶性調(diào)節(jié)成分和功能細胞等進行了相關(guān)研究。NRC模型所提供的封閉微環(huán)境是研究周圍神經(jīng)再生的重要窗口。而神經(jīng)再生條件液(nerve regeneration conditioned fluid, NRCF)是NRC模型中自神經(jīng)斷端分泌的功能性液體,其來源于周圍神經(jīng)的組成細胞,或經(jīng)軸突順向轉(zhuǎn)運、逆向彌散而來的物質(zhì)成分,主要組成為蛋白,其在周圍神經(jīng)再生不同時期的表達存在種類和數(shù)量的動態(tài)變化。研究表明,NRCF對再生神經(jīng)提供支持、營養(yǎng)和導(dǎo)向等重要作用。周圍神經(jīng)損傷的修復(fù)是一個復(fù)雜的、精細的生理過程。周圍神經(jīng)功能性再生取決于特定微環(huán)境和神經(jīng)元自身生長能力的共同作用。相對于激活神經(jīng)元自身生長能力是其固有特性方面,對損傷所處的特定微環(huán)境研究就更為重要。神經(jīng)再生室模型(NRC)所提供的封閉微環(huán)境作為研究周圍神經(jīng)再生的重要窗口,而神經(jīng)再生條件液(NRCF)是NRC中的功能性成分,對其進行研究將是揭示周圍神經(jīng)再生機理的重要途徑。但目前,對周圍神經(jīng)再生的微環(huán)境因素,如細胞外基質(zhì)、神經(jīng)營養(yǎng)因子和細胞因子等,進行了大量研究,但都是隨機的、散在的和推測的。因此,對NRCF中蛋白成分譜的描繪,可為研究微環(huán)境在周圍神經(jīng)再生中的作用提供直接依據(jù),對闡明再生機制具有重要指導(dǎo)意義。而蛋白組學技術(shù)的發(fā)展為此提供了研究的方法基礎(chǔ)。相對和絕對定量的等量異位標簽(isobaric tags for relative and absolute quantitation, iTRAQ)技術(shù)是目前先進的蛋白組學技術(shù),可同時比較4種或8種不同樣品中蛋白質(zhì)的相對含量或絕對含量,w為目前研究NRCF中整體蛋白成分和時間差異表達的有效方式。鑒于微環(huán)境在周圍神經(jīng)損傷修復(fù)中的重要作用,對其功能性成分(NRCF)進行研究將是了解周圍神經(jīng)再生機理的重要途徑。本課題首次采用iTRAQ技術(shù)對周圍神經(jīng)再生四個關(guān)鍵時間點(神經(jīng)離斷3d,7d,14d和25d)NRCF的蛋白成分做了整體的、全景的動態(tài)呈現(xiàn)和分析,通過比對蛋白質(zhì)組數(shù)據(jù)庫,對共表達蛋白進行了生物信息學分析,發(fā)現(xiàn)了某些關(guān)鍵蛋白和周圍神經(jīng)再生的關(guān)系。通過實驗初步探討了NRCF對雪旺細胞和成纖維細胞增殖的影響。并結(jié)合前期研究成果,討論了NRCF中不同補體成分參與周圍神經(jīng)再生過程的動態(tài)變化及其意義。第一部分 NRC模型的建立、NRCF的提取及對雪旺細胞和成纖維細胞增殖的影響目的典型的神經(jīng)再生室(NRC)模型為連接神經(jīng)兩斷端的圓筒狀硅膠或其他材質(zhì)所形成的密閉的間隙,其所提供的封閉微環(huán)境是研究周圍神經(jīng)再生的重要窗口,而神經(jīng)再生條件液(NRCF)是NRC模型中自神經(jīng)斷端分泌的功能性成分,可對再生神經(jīng)提供支持、營養(yǎng)和導(dǎo)向作用。雪旺細胞(schwann cell,Sc)及成纖維細胞(fibroblast cell, Fb)是參與周圍神經(jīng)再生的主要功能細胞。本部分實驗采用Lewis大鼠坐骨神經(jīng)(sciatic nerve)建立NRC模型,提取NRCF,并采用CCK-8法及流式細胞儀檢測,探討NRCF對雪旺細胞和成纖維細胞增殖的影響。方法Lewis大鼠坐骨神經(jīng)10mm離斷缺損,采用特定規(guī)格硅膠管體內(nèi)建立NRC模型、7d時提取NRCF。BCA法進行NRCF蛋白濃度測定。雪旺細胞和成纖維細胞細胞株的傳代培養(yǎng)。CCK-8法和流式細胞術(shù)檢測NRCF對雪旺細胞和成纖維細胞增殖的影響。結(jié)果NRCF為清澈、淡黃色、呈類血清樣并稍粘稠的液體,濃度為15.01μg/μl。雪旺細胞呈類圓形或梭形胞體,部分有兩個或兩個以上的突起,而成纖維細胞細胞體較大,呈棱形、長條狀或不規(guī)則三角形,融合后為編織狀,細胞核一般呈長橢圓形。NRCF能顯著促進雪旺細胞(Sc)、成纖維細胞(Fb)的增殖,并且此作用呈時間、濃度依賴性,促進該兩種細胞增殖的有效濃度為10000ng/ml。結(jié)論1.采用Lewis大鼠坐骨神經(jīng)建立NRC模型,模型穩(wěn)定、可靠,提取NRCF質(zhì)量能很好滿足實驗要求。Lewis Rat Sciatic Nerve-NRC模型是研究周圍神經(jīng)再生的有效途徑。2.采用CCK-8法及流式細胞儀檢測技術(shù),初步證明神經(jīng)再生條件液(NRCF)具有促進雪旺細胞及成纖維細胞增殖的生物活性。第二部分NRCF-iTRAQ蛋白組學研究和生物信息學分析目的神經(jīng)再生條件液(NRCF)是NRC中神經(jīng)斷端分泌的功能性成分,主要為蛋白成分,對NRCF中的蛋白成分進行研究將為揭示周圍神經(jīng)再生機理提供一個平臺。本部分實驗采用iTRAQ蛋白組學技術(shù)聯(lián)合質(zhì)譜技術(shù),以周圍神經(jīng)再生四個關(guān)鍵時間點(坐骨神經(jīng)離斷3d,7d,14d和25d)所提取的NRCF為研究對象,對NRCF每個時間點、全部時間點及其各個時間點之間的蛋白定性和定量表達情況進行分析,并通過比對蛋白質(zhì)組數(shù)據(jù)庫,對共表達蛋白進行了進一步的生物學信息分析。方法建立NRC模型、提取3d,7d,14d和25dNRCF材料及其NRCF樣品處理。SDS-PAGE電泳。iTRAQ蛋白組學技術(shù)、質(zhì)譜鑒定。GO分析、KEGG通路分析、分層聚類分析、共趨勢分析、時序表達的基因篩選等生物信息學分析方法對NRCF蛋白進行定性、定量分析。結(jié)果3d,7d,14d和25dNRCF4組樣品蛋白質(zhì)條帶分布較均一,平行度良好。3d,7d,14d和25d神經(jīng)再生條件液(NRCF)中共鑒定出876個蛋白,其中,3d,7d,14d和25d神經(jīng)再生條件液(NRCF)中蛋白個數(shù)分別為：536、645、376和548個,其獨特蛋白質(zhì)個數(shù)分別為：95、137、18和58個,共同蛋白質(zhì)個數(shù)為264個。共表達蛋白定量生物信息學分析顯示：KEGG Pathway所得到的TOP 10通路為：補體及凝血級聯(lián)、金黃色葡萄球菌感染、系統(tǒng)性紅斑狼瘡、百日咳、粘著斑、朊病毒、阿米巴病、肌動蛋白骨架調(diào)節(jié)、糖酵解/糖異生、ECM受體作用等。共趨勢分析第2組、第3組和第4組TOP 7蛋白分別為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。顯著差異蛋白和高連接度信息蛋白為GRB2、 ACTB、ALB、VIM、VCP、A2M、CALR、THBS1、TF和PEBP1等。結(jié)論1.采用iTRAQ蛋白組學技術(shù)聯(lián)合質(zhì)譜技術(shù),對周圍神經(jīng)再生的四個關(guān)鍵時間點的NRCF蛋白進行了全景的、動態(tài)的分析,建立了一個相對完整的NRCF蛋白數(shù)據(jù)庫。NRCF蛋白含量豐富,種類繁多,每個關(guān)鍵時間點既有特異蛋白,各個關(guān)鍵時間點也有共同蛋白,與周圍神經(jīng)再生不同階段關(guān)系密切。2.數(shù)據(jù)分析篩選出周圍神經(jīng)再生的不同關(guān)鍵時間點的特異蛋白、共趨勢分析篩選出的不同組別的高表達蛋白和時序表達的基因篩選所獲得的顯著表達蛋白等,為尋找影響周圍神經(jīng)再生的關(guān)鍵蛋白提供了依據(jù)。這些數(shù)據(jù),不僅,豐富了對周圍神經(jīng)再生的認識,而且,為將來神經(jīng)再生課題提供了眾多的基因和蛋白研究靶點。3. NRCF蛋白的全景揭示和相關(guān)文獻復(fù)習,對既往神經(jīng)再生所取得的研究成果也進行了部分驗證,并為深入研究再生理論開辟了新的途徑。第三部分NRCF中補體成分及與周圍神經(jīng)再生關(guān)系目的NRCF蛋白譜提示,補體系統(tǒng)成分的大量存在,并在不同神經(jīng)再生時期具有明顯的動態(tài)變化。生物學分析(KEGG通路分析)表明補體通路位居前十個顯著富集通路。補體系統(tǒng)在周圍神經(jīng)再生過程中所起的作用非常值得研究。補體系統(tǒng)(幾種或一種關(guān)鍵成分)所介導(dǎo)的免疫反應(yīng)、炎癥反應(yīng),甚至與兩者無關(guān),但與神經(jīng)再生相關(guān)的作用于PNI中亟待揭示。本部分將對NRCF-iTRAQ蛋白組學結(jié)果發(fā)現(xiàn)的大量補體成分及其動態(tài)變化加以描述、分析和總結(jié),并采用Western blot技術(shù)對NRCF中部分補體成分加以驗證,對其可能參與周圍神經(jīng)再生的作用機制加以探討。方法建立NRC模型、提取7dNRCF及其NRCF樣品處理。iTRAQ蛋白組學技術(shù)相關(guān)方法(同第二部分)。Western blot實驗技術(shù)檢測C1q、C3、C7和CFD在NRCF中的表達。結(jié)果NRCF-iTRAQ蛋白組學分析所得10種補體成分分別為CFI、Clq-A、Clq-B、 C2、C3、C4、C5、C7、C8β和CFD等。Western blot證實NRCF中有C1q、C3、C7和CFD的表達。結(jié)論NRCF中有10余種補體成分定性和定量信息的存在,補體C7在再生不同時間階段量化分析有顯著性變化。補體Clq、C3、C4和C5等與神經(jīng)再生關(guān)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.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R745
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本文編號：1983575