本文選題：脊髓損傷 + 神經(jīng)干細胞　； 參考：《第三軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 現(xiàn)代社會文明進步發(fā)展的同時,也帶來了人類社會第一大公害“創(chuàng)傷”的大量發(fā)生,創(chuàng)傷被稱為“發(fā)達社會病”。脊柱、脊髓損傷(spinal cord injury,SCI)是交通、勞動和運動等意外事故中常見的創(chuàng)傷類型。脊髓損傷是人類致殘率最高的疾患之一,一直是困擾醫(yī)學(xué)界的一大難題。它可直接導(dǎo)致?lián)p傷平面以下的感覺、運動功能喪失及排尿、排便功能障礙。損傷的平面越高,患者喪失的功能越多。美國有截癱患者約253000例,每年新增約11000例,每年為這些截癱患者支付的醫(yī)療費用高達60億美元,而在中國截癱患者人數(shù)約40萬,每年新增1萬人,給患者、家庭和社會帶來巨大的負擔(dān)。但到目前為止國內(nèi)外治療脊髓損傷的藥物和外科手術(shù)雖均有進展,但卻未取得滿意的臨床療效。因此,加強SCI救治與截癱康復(fù)的研究具有重要的社會現(xiàn)實需求和科學(xué)理論價值。 近年興起的利用組織工程學(xué)的方法來修復(fù)脊髓損傷給脊髓損傷患者帶來了新的希望。組織工程學(xué)是生物醫(yī)學(xué)工程學(xué)中的一個新的分支,是應(yīng)用生命科學(xué)和工程學(xué)的原理和技術(shù),構(gòu)建人工組織以修復(fù)組織器官的結(jié)構(gòu)和功能。其主要的研究內(nèi)容包括:種子細胞的篩選、支架材料的合成與塑型以及組織的構(gòu)建。目前,用于構(gòu)建脊髓組織工程的種子細胞主要包括:胚胎干細胞、神經(jīng)干細胞(neural stem cells,NSCs)、骨髓間充質(zhì)干細胞、嗅鞘細胞、雪旺細胞和成纖維細胞等。種子細胞主要通過以下方式來修復(fù)脊髓損傷:1)替代損傷或死亡的細胞;2)橋接脊髓損傷斷端,形成功能性突觸,重新建立神經(jīng)傳導(dǎo)通路;3)改善臨床癥狀,提供神經(jīng)保護和生長因子使受損的軸突易于再生;4)起到其他的間接作用:如促進血管生成,為再生和內(nèi)生細胞提供營養(yǎng)支持。 NSCs被認為是構(gòu)建脊髓組織工程最佳的種子細胞。NSCs為多能干細胞,終身具有自我更新能力和多向分化潛能,無論是在哺乳動物胚胎神經(jīng)組織還是成年個體的腦組織中都存在,是中樞神經(jīng)系統(tǒng)內(nèi)新生細胞產(chǎn)生的源泉。NSCs在一定條件下能分化為神經(jīng)元、星形膠質(zhì)細胞和少突膠質(zhì)細胞。并且,NSCs具有易于獲取、培養(yǎng)方法成熟,鑒定容易,可在體外大量增殖、分化等優(yōu)點,適合作為組織工程神經(jīng)移植物實驗的種子細胞。盡管從理論上講NSCs的功能完美無缺,但近年來研究發(fā)現(xiàn)單獨NSCs應(yīng)用于成年SCI的治療存在如下問題:移植后NSCs分化為神經(jīng)元少,無法提供大量功能性軸突連接所需的神經(jīng)元,而膠質(zhì)細胞大量增生,形成的膠質(zhì)瘢痕不利于軸突再生;由于損傷脊髓局部的血運不能得到及時恢復(fù),變性壞死組織及毒性產(chǎn)物不能很快清除,使移植物中的神經(jīng)細胞得不到充分的血管營養(yǎng)支持,導(dǎo)致細胞存活時間短,神經(jīng)干細胞增殖分化困難,達不到應(yīng)有的治療效果。因此,如何促進移植到損傷脊髓的NSCs向神經(jīng)元分化、促進損傷脊髓血管再生以保證移植細胞的血管營養(yǎng)支持,成為利用NSCs及其構(gòu)建的組織工程移植物移植修復(fù)損傷脊髓亟待解決的關(guān)鍵問題之一。 血管內(nèi)皮祖細胞(Endothelial Progenitor Cells,EPCs)是Asahara等在1997年首次從成體外周血中分離,并證實在人類出生后的外周循環(huán)血液中存在,能分化為血管內(nèi)皮細胞的前體細胞。EPCs是一群具有游走特性,并能增殖、分化為內(nèi)皮細胞的前體細胞,包括從血液血管母細胞到成熟內(nèi)皮細胞之間多個階段的細胞,亦稱為血管母細胞或血管內(nèi)皮干細胞,不僅參與胚胎時期血管發(fā)生,而且在出生后成體血管新生過程中也起重要作用。EPCs能遷移并結(jié)合到血管再生的部位,增殖、分化為成熟的內(nèi)皮細胞,參與局部新生血管的形成。EPCs的這些特性使其具有廣泛的臨床應(yīng)用價值,可作為組織工程中血管的種子細胞來源。近年來,有關(guān)EPCs在組織工程血管再生中作用的研究主要集中在缺血心肌、缺血肢體、損傷皮膚和損傷角膜等方面,還尚未用于脊髓損傷。我們擬選用NSCs和EPCs作為種子細胞,構(gòu)建血管化組織工程脊髓來修復(fù)損傷的脊髓,以同時解決脊髓損傷后的“神經(jīng)營養(yǎng)”和“血管營養(yǎng)”,這兩大問題。然而,NSCs和EPCs來源于體內(nèi)不同胚層,體內(nèi)和體外對周圍生長的環(huán)境要求不同。要同時移植這兩種細胞到宿主體內(nèi),需先在體外對這兩種種子細胞進行共培養(yǎng)以探討兩者最佳的生長條件以及共培養(yǎng)后兩者生物學(xué)特性的變化規(guī)律,從而為構(gòu)建血管化細胞組織工程移植修復(fù)脊髓損傷和中樞神經(jīng)系統(tǒng)疾病提供可行性實驗依據(jù)和理論基礎(chǔ)。本實驗擬從大鼠的外周血中分離單個核細胞、經(jīng)體外培養(yǎng)、誘導(dǎo)后獲得EPCs,在體外與NSCs進行共培養(yǎng),觀察兩者生長情況及NSCs的分化方向,然后對其作用機制進行初步探討。 主要方法和技術(shù)路線 實驗分為三個部分 1.采用孕齡14d的SD胎鼠大腦皮層組織,分離純化培養(yǎng)NSCs,血清誘導(dǎo)NSCs分化。分別采用Nestin、β-tubulin-Ⅲ和GFAP對NSCs球、分化后的神經(jīng)元和星形膠質(zhì)細胞進行鑒定。采用密度梯度離心法從重約180g的SD大鼠外周血中得到單個核細胞,通過貼壁培養(yǎng)、體外擴增獲取細胞,通過觀察細胞形態(tài)、CD34/Ⅷ因子免疫熒光反應(yīng)等方法進行鑒定。 2.在體外進行EPC與NSCs共培養(yǎng),按培養(yǎng)基不同分為NB培養(yǎng)基組、DMEM培養(yǎng)基組和NB+DMEM培養(yǎng)基組,觀察兩種種子細胞生長情況,以尋找最佳條件培養(yǎng)基。觀察EPCs對NSCs增殖和分化的調(diào)控作用,并初步探討兩者不同比例情況下,NSCs分化變化規(guī)律。 3.初步探討體外共培養(yǎng)的EPCs調(diào)控NSCs分化為神經(jīng)元的作用機制。ELISA法檢測EPCs、NSCs及兩者共培養(yǎng)的上清液VEGF、BDNF含量。觀察VEGF、bFGF聯(lián)合應(yīng)用對NSCs分化的影響,并利用VEGF的抗體進一步探討VEGF是否在EPCs與NSCs共培養(yǎng)時,促進NSCs向神經(jīng)元方向分化中起重要作用。 4.統(tǒng)計學(xué)分析:各組數(shù)據(jù)以xv±s表示,運用SPSS13.0進行統(tǒng)計分析,組間比較采用t檢驗,以P0.05為顯著水平,P0.01為非常顯著。 主要的研究結(jié)果和結(jié)論如下: 1.胎鼠大腦皮層NSCs豐富,易于獲取,培養(yǎng)的NSCs可在體外擴增,并在一定的條件下能分化為神經(jīng)元和神經(jīng)膠質(zhì)細胞。大鼠外周血來源豐富,取材方便,分離出的EPCs在特定的培養(yǎng)條件下能分化為內(nèi)皮細胞。提示本實驗方法能夠體外成功培養(yǎng)出NSCs和EPCs種子細胞。 2. EPCs與NSCs共培養(yǎng)時,最適合的條件培養(yǎng)基為NB+DMEM(1:1)。在此條件下,EPCs能明顯促進NSCs的增殖,且調(diào)控NSCs定向分化為神經(jīng)元的比例(68.40%)明顯高于單純血清誘導(dǎo)組(28.7%)。且隨著EPCs:NSCs的比例由1:10到10:1,這種分化誘導(dǎo)作用越顯著。提示本實驗所建立的EPCs與NSCs共培養(yǎng)體系是可行的,且在該體系中EPCs能明顯促使NSCs向神經(jīng)元方向分化。 3. ELISA法檢測EPCs及其與NSCs共培養(yǎng)時上清液中的VEGF分別為817.23 pg/ml、917.78 pg/ml,明顯高于NSCs單培養(yǎng)時363.67 pg/ml,表明EPCs主要分泌VEGF。 4. VEGF、bFGF誘導(dǎo)NSCs分化為神經(jīng)元的比率分別為60.3%、60.4%,兩者聯(lián)合應(yīng)用時神經(jīng)元分化比率明顯提高達80.3%。表明VEGF能明顯促進NSCs向神經(jīng)元方向分化,且與bFGF有協(xié)同作用。 5.共培養(yǎng)時EPCs對NSCs向神經(jīng)元分化的調(diào)控作用,能被VEGF抗體明顯抑制;而在加入與VEGF抗體等量的VEGF后,EPCs仍然能明顯促進NSCs向神經(jīng)元分化。表明EPCs可通過其分泌的VEGF調(diào)控NSCs向神經(jīng)元分化。
[Abstract]:At the same time, the progress and development of civilization of modern society also brought about the massive occurrence of "trauma" of the first public harm in human society. The trauma is called "developed social disease". Spinal cord injury (spinal cord injury, SCI) is the common type of trauma in traffic, labor and sports accidents. Spinal cord injury is the highest disability rate of human being. One of the problems that has been plaguing the medical community has been a direct cause of sensation below the level of injury, loss of motor function, urination, and defecation dysfunction. The higher the level of injury, the more the patients lose. There are about 253000 paraplegic patients in the United States, about 11000 new cases a year, and the higher medical costs for these paraplegic patients each year. The number of paraplegic patients in China is about 6 billion dollars, and the number of paraplegic patients in China is about 400 thousand, and 10 thousand people have added a huge burden to the patients, family and society each year. However, although there has been progress in the treatment of spinal cord injury at home and abroad so far, however, it has not achieved satisfactory clinical effect. Therefore, the study of strengthening SCI treatment and paraplegia rehabilitation is heavy. The need for social reality and the value of scientific theory.
The use of tissue engineering to repair spinal cord injury in recent years has brought new hope to the patients with spinal cord injury. Tissue engineering is a new branch of biomedical engineering. It is the application of the principles and techniques of life science and engineering to construct artificial tissues to repair the structure and function of tissues and organs. The contents include: screening of seed cells, synthesis and molding of scaffold materials and construction of tissue. At present, seed cells for the construction of spinal cord tissue engineering include embryonic stem cells, neural stem cells (neural stem cells, NSCs), bone marrow mesenchymal stem cells, olfactory ensheathing cells, Schwann cells and fibroblasts. Repair spinal cord injury in the following ways: 1) replace damaged or dead cells; 2) bridging the injured end of the spinal cord, forming functional synapses, reestablishing the nerve conduction pathway; 3) improving the clinical symptoms, providing neuroprotection and growth factors to make the damaged axons easy to regenerate; 4) other indirect effects, such as promoting angiogenesis, and re Raw and endophytic cells provide nutritional support.
NSCs is considered to be the best seed cell for the construction of spinal cord tissue engineering,.NSCs is a pluripotent stem cell. It has a lifelong self renewal capacity and multidirectional differentiation potential, both in the mammalian embryonic neural tissue and in the adult brain tissue. It is the source of the source.NSCs of the new cells in the central nervous system under certain conditions. Differentiation into neurons, astrocytes and oligodendrocytes. Moreover, NSCs has the advantages of easy acquisition, maturation, identification, proliferation and differentiation in vitro. It is suitable as a seed cell for tissue engineering nerve graft experiment. Although the function of NSCs is perfect in theory, it has been discovered in recent years. The treatment of single NSCs for adult SCI has the following problems: after transplantation, NSCs is differentiated into less neurons and can not provide a large number of neurons needed for functional axonal connections, while glial cells proliferate, and glial scar formation is not conducive to axonal regeneration; the blood transport of the injured spinal cord can not be recovered in time, degeneration and necrosis and poison. The sex products can not be removed quickly, so that the nerve cells in the grafts can not get sufficient vascular nutritional support, which leads to the short survival time and the difficulty in the proliferation and differentiation of neural stem cells. Therefore, how to promote the transplantation of NSCs to the injured spinal cord to differentiate the nerve cells and promote the regeneration of the injured spinal cord to guarantee the transplantation of the spinal cord. Cellular vascular nutritional support has become one of the key problems to be solved urgently by using NSCs and its tissue engineered graft to repair injured spinal cord.
Vascular endothelial progenitor cells (Endothelial Progenitor Cells, EPCs) are the first isolation of Asahara from the peripheral blood in 1997, and proved to exist in the peripheral circulating blood after human birth, and.EPCs, a precursor cell that can differentiate into vascular endothelial cells, is a group of precursors that have a wandering, proliferation and differentiation into endothelial cells. The cells from blood vessels to mature endothelial cells, also known as angioblastoma or vascular endothelial stem cells, not only participate in embryonic angiogenesis, but also play an important role in the process of angiogenesis after birth and.EPCs can migrate and bind to the site of vascular regeneration, proliferate, and differentiate into maturity. These characteristics of endothelial cells, which participate in the formation of.EPCs in local neovascularization, have a wide range of clinical applications and can be used as the source of seed cells in tissue engineering. In recent years, the research on the role of EPCs in vascular regeneration in tissue engineering is mainly focused on ischemic myocardium, ischemic limbs, skin injury and injury of cornea. It is not yet used for spinal cord injury. We use NSCs and EPCs as seed cells to construct vascularized tissue engineered spinal cord to repair the injured spinal cord, and to solve the two major problems of "neuronutrition" and "vascular nutrition" after spinal cord injury. However, NSCs and EPCs are derived from different germ layers in the body, in vivo and in vitro The growth environment is different. To transplant the two cells to the host at the same time, the two seed cells should be co cultured in vitro to explore the best growth conditions and the changes of the biological characteristics after co culture, so as to construct the vascularized cell tissue engineering transplantation for the repair of spinal cord injury and central God. This experiment provides the basis and theoretical basis for the feasibility of systemic disease. This experiment is to separate mononuclear cells from the peripheral blood of the rat. After culture in vitro, EPCs is obtained and co cultured with NSCs in vitro. The growth of the two and the differentiation direction of NSCs are observed. Then the mechanism of its action is preliminarily discussed.
Main methods and technical routes
The experiment is divided into three parts
1. the NSCs was isolated and cultured in the cerebral cortex of SD fetal rat of gestational age. NSCs was isolated and cultured to induce the differentiation of NSCs. Nestin, beta -tubulin- III and GFAP were used to identify the neurons and astrocytes after the differentiation of NSCs balls. The density gradient centrifugation was used to obtain mononuclear cells from the peripheral blood of SD rats weighing 180g about 180g, through the adherent culture. The cells were obtained from in vitro expansion and identified by observing the cell morphology, CD34/ VIII factor immunofluorescence reaction and so on.
2. co culture of EPC and NSCs in vitro, divided into NB medium group, DMEM medium group and NB+DMEM medium group according to the culture medium, observe the growth condition of two seed cells in order to find the best condition medium, observe the regulation of EPCs on NSCs proliferation and differentiation, and discuss the differentiation and change regularity of NSCs under the condition of the two different proportions.
3. preliminary study on the role of EPCs in vitro co cultured in vitro to regulate NSCs differentiation into neurons..ELISA method was used to detect EPCs, NSCs and the content of VEGF and BDNF in the co culture supernatant. The effect of VEGF, bFGF combined application on NSCs differentiation was observed. It plays an important role in differentiation.
4. statistical analysis: the data of each group were expressed as XV + s, and SPSS13.0 was used for statistical analysis. The t test was used in the comparison between the groups, and P0.05 was the significant level. P0.01 was very significant.
The main results and conclusions are as follows:
The cerebral cortex of 1. fetal rats is rich in NSCs and easy to obtain. The cultured NSCs can be amplified in vitro, and can be differentiated into neurons and glial cells under certain conditions. The peripheral blood of rats is rich in peripheral blood, and the isolated EPCs can be differentiated into endothelial cells under specific culture conditions. This method can be successfully cultured in vitro. NSCs and EPCs seed cells were produced.
2. EPCs and NSCs co culture, the most suitable conditioned medium is NB+DMEM (1:1). Under this condition, EPCs can significantly promote the proliferation of NSCs, and the proportion of NSCs directed differentiation into neurons (68.40%) is significantly higher than that of the pure serum induction group (28.7%). And with the proportion of EPCs:NSCs from 1:10 to 10:1, the more significant differentiation induction. The co culture system of EPCs and NSCs established in the experiment is feasible. In this system, EPCs can obviously induce NSCs to differentiate into neurons.
The VEGF of the supernatant in the co culture of EPCs and NSCs by 3. ELISA method was 817.23 pg/ml and 917.78 pg/ml respectively, which was significantly higher than that of 363.67 pg/ml during the single culture of NSCs, indicating that EPCs secreted VEGF..
4. VEGF, the ratio of NSCs differentiation into neurons by bFGF was 60.3%, 60.4% respectively. The differentiation ratio of neurons was significantly increased by 80.3%., indicating that VEGF could obviously promote the differentiation of NSCs into the neuron and have synergistic effect with bFGF.
5. in co culture, the regulation of EPCs on the differentiation of NSCs to neuron can be significantly inhibited by VEGF antibody, and EPCs can obviously promote the differentiation of NSCs into neurons after adding VEGF to VEGF antibody. It indicates that EPCs can differentiate into neurons by its secreted VEGF regulation NSCs.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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