本文選題：脊髓損傷 + 細胞移植　； 參考：《浙江大學(xué)》2012年博士論文

【摘要】：背景： 脊髓損傷(Spinal cord injury, SCI)是人類所經(jīng)歷的創(chuàng)傷中最具破壞性的創(chuàng)傷之一,常常導(dǎo)致永久性殘疾,其多由車禍、墜落等高能量撞擊引發(fā)脊柱骨折、脫位所致。脊髓損傷預(yù)防保健防治基金會統(tǒng)計,目前在世界范圍內(nèi)大約有250萬人受此病影響,每年有超過13萬的新發(fā)外傷性SCI病例。美國約有45萬脊髓損傷病人,并且每年有1.1萬新增SCI病例發(fā)生。絕大多數(shù)患者均遺留部分或完全性癱瘓,損傷后患者的生活質(zhì)量嚴重下降。82%患者為是青壯年男性,給社會和家庭造成了不可估量的損失。由于SCI主要影響年輕人,且缺乏有效的治療方法,由此帶來的功能喪失往往伴隨著病人的一生。受損脊髓病理改變主要包括兩個過程,原發(fā)性損傷和繼發(fā)性損傷,主要造成不同程度的細胞死亡、組織水腫、膠質(zhì)疤痕增生和脊髓運動功能喪失,而死亡的神經(jīng)元裂解釋放的毒素,損傷部位的慢性脫髓鞘等病理改變又可導(dǎo)致?lián)p傷平面上下兩側(cè)的脊髓組織壞死,最終導(dǎo)致?lián)p傷平面以下感覺和運動功能障礙。病變壞死區(qū)形成的空洞、反應(yīng)性膠質(zhì)疤痕增生、以及軸突斷裂和脫髓鞘反應(yīng),也對神經(jīng)細胞和軸突的再生產(chǎn)生抑制作用,即使有新生的軸突,也不能通過瘢痕組織。研究SCI的病理組織學(xué)改變可促使新的治療靶點和治療方法的發(fā)現(xiàn)。在成年哺乳動物體中,雖然存在神經(jīng)干細胞,但其對SCI的修復(fù)能力非常有限,因此脊髓損傷的治療是一大挑戰(zhàn)。 大量科學(xué)研究和臨床實踐誕生了多種SCI治療方法,例如藥物治療,康復(fù)鍛煉,電刺激等等,但這些手段僅僅改善了脊髓受損后局部情況,而臨床效果相當(dāng)有限。近年研究表明,細胞移植為SCI的治療帶來新的思路,其作用有很多方面,包括取代受損的神經(jīng)元,填補空洞,改善病變的環(huán)境,激活內(nèi)源性的神經(jīng)營養(yǎng)因子分泌增加,提供髓鞘再生,結(jié)構(gòu)支持,及最終增強軸突再生。通過這些作用,移植的細胞提供一個適宜中樞神經(jīng)細胞生長的基質(zhì)平臺,可以提供足夠的營養(yǎng)因子以及增強中樞神經(jīng)元的再生能力。目前有多種細胞如嗅鞘細胞、雪旺細胞、成纖維細胞、胚胎干細胞、神經(jīng)干細胞、神經(jīng)元、神經(jīng)膠質(zhì)前體細胞和骨髓基質(zhì)干細胞等均被用于移植治療脊髓損傷的研究。雖然,細胞移植不同程度的促進了脊髓損傷的修復(fù),但目前為止適用于SCI臨床研究和開發(fā)的種子細胞依然較少,其主要問題為：細胞來源有限,細胞移植后存活率低,致瘤性等等。因此,尋找合適的種子細胞,提高移植存活率以及深入了解移植細胞的作用機制對于脊髓損傷的治療顯得非常重要。 早前的研究報告表明,從神經(jīng)嵴衍生的組織中所分離出的干細胞具有多系分化潛能,如在牙髓組織中。Hyunmi等研究報道神經(jīng)嵴來源的人類眼瞼脂肪組織的干細胞(human eyelid adipose-derived stem cells, hEASCs)具有類似神經(jīng)元的雙極的形狀,而間充質(zhì)干細胞(MSC)是紡錘形。hEASCs和MSC不僅在形態(tài)上不同,細胞的特征表現(xiàn)也不同。未分化的hEASCs能自發(fā)表達一些神經(jīng)細胞相關(guān)的基因和蛋白,其中大部分標(biāo)記物是在人類的神經(jīng)嵴細胞所能觀察到的。相比之下,軀干脂肪組織分離的脂肪干細胞不具有此類特征。因此這類從神經(jīng)嵴來源的hEASCs有其獨特的特點。據(jù)我們所知,以前的研究中尚未使用這種細胞研究治療脊髓損傷。因此,在這項研究中,我們評估了hEASCs移植在大鼠脊髓損傷模型脊髓損傷的作用。 組織損傷后,移植干細胞在病理環(huán)境中成活率較低,是干細胞治療的一大障礙。脊髓損傷急性期進行某些化學(xué)藥物治療可以有效的阻止二次損傷的蔓延,并能最大程度的保護損傷后剩余的神經(jīng)組織。其中17-beta-雌二醇(17p-Estradiol,E2)的作用尤為明顯,能促進神經(jīng)元細胞,肝臟細胞,成纖維細胞和少突膠質(zhì)細胞對抗氧化,炎癥和凋亡等不利環(huán)境。同時,最近研究表明,E2對中樞神經(jīng)疾病有一定的治療作用,能夠減少細胞死亡和減少體內(nèi)的繼發(fā)性損傷,可以減少SCI的空洞面積和細胞凋亡比率。 因此,我們提出假說,聯(lián)合給藥E2與hEASCs移植,將有利于脊髓損傷修復(fù)。研究分為體外和體內(nèi)兩個部分：體外部分為hEASCs的分離、培養(yǎng)和鑒定；體內(nèi)部分(1)大鼠脊髓損傷模型的建立和hEASCs移植；(2)觀察聯(lián)合治療的效果,(3)探討聯(lián)合治療的可能機制。 第一章體外研究部分：hEASCs的體外分離,培養(yǎng)和鑒定 目的：由成人眼瞼脂肪中分離一群特殊的脂肪干細胞,培養(yǎng)并對其特性進行鑒定。 方法與結(jié)果：體外實驗中,我們評估了hEASCs的克隆形成能力,細胞增殖能力和三系分化能力,干細胞相關(guān)基因及神經(jīng)相關(guān)特異性基因和蛋白的表達能力。結(jié)果顯示hEASCs具有干細胞的特性,如克隆形成能力,增殖能力和三系分化潛能,同時具有神經(jīng)特異性的基因和蛋白標(biāo)記的表達等。 結(jié)論：hEASCs具有干細胞的一般特性,而且表達神經(jīng)特異性的基因和蛋白標(biāo)記,并具有向神經(jīng)系細胞分化的潛能,為其是體內(nèi)促進脊髓損傷修復(fù)提供了可能。 第二章體內(nèi)研究部分：大鼠脊髓損傷模型的建立,聯(lián)合E2和hEASCs移植進行大鼠脊髓損傷模修復(fù)。 目的：研究hEASCs在大鼠脊髓損傷中的變化,以及與E：的聯(lián)合使用對大鼠脊髓損傷的修復(fù)作用。 方法與結(jié)果：大鼠胸椎T10右半橫斷損傷后,隨機分為3組,分為PBS移植組(對照組),hEASCs移植組(細胞組),hEASCs移植和E2聯(lián)合組(聯(lián)合組)。聯(lián)合組在損傷15min后進行E2注射,維持每天給藥至15d。損傷7天后,細胞組和聯(lián)合組分別將CFDA標(biāo)記的hEASCs于脊髓損傷處上下2mm處進行注射移植,對照組于同樣位置注射PBS。術(shù)后4周和6周收集樣本,體內(nèi)熒光跟蹤結(jié)果顯示移植細胞依然存活,特異性人核染色表現(xiàn)了一致的結(jié)果,相關(guān)人生長因子的表達,也證明了細胞的體內(nèi)存活,與細胞組相比,hEASCs與E2的聯(lián)合組細胞存活率更高。人核染色(hNu)或CFDA熒光與微管相關(guān)蛋白(MAP2).半乳糖苷酶(GALAC)或者膠質(zhì)纖維酸性蛋白(GFAP)進行免疫熒光共定位,結(jié)果表明,hEASCs有向神經(jīng)元和少突膠質(zhì)細胞分化的跡象,而未向星形膠質(zhì)分化。蘇木精-伊紅染色法(HE染色)和甲苯胺藍染色組織學(xué)結(jié)果顯示,細胞組和聯(lián)合治療組的空洞形成明顯減少,聯(lián)合組的髓鞘保留程度也要好于其他兩組。TUNEL染色結(jié)果表明,單純的細胞組凋亡比較嚴重,相比之下,hEASCs與E2聯(lián)合治療組細胞凋亡數(shù)量明顯減少,caspase-3表達降低,bcl-2表達升高。同時,聯(lián)合治療組大鼠的BBB功能評分和Grid walking也明顯好于其他兩組。此外,聯(lián)合組分泌的人生長因子(igf-I，，ngf1hgf)顯著高于細胞組和對照組。 結(jié)論：hEASCs與E：的聯(lián)合使用顯著的促進了脊髓損傷修復(fù),hEASCs向神經(jīng)元細胞和少突膠質(zhì)細胞的分化可能、E2對移植細胞的存活改善及其生長因子的分泌作用可能是其修復(fù)的機制。
[Abstract]:Background:
Spinal cord injury (SCI) is one of the most destructive traumas experienced by human beings. It often leads to permanent disability, which is caused by high energy crash, such as accident and falling, caused by fracture of the spine. The foundation for prevention and control of spinal cord injury prevention and control, about 2 million 500 thousand people worldwide are currently affected by this disease. There are more than 130 thousand new traumatic SCI cases each year. There are about 450 thousand patients with spinal cord injury in the United States, and 11 thousand new SCI cases occur each year. Most patients have partial or complete paralysis. After injury, the quality of life of patients with.82% is serious. Loss. Because SCI mainly affects young people and lacks effective treatment, the loss of function often accompanied by the patient's life. The pathological changes of the spinal cord mainly include two processes, primary and secondary injuries, which mainly cause different degrees of cell death, tissue edema, glial scar hyperplasia and spinal motor work. The pathological changes that can be lost, the toxin released by the dead neuron cracking and the chronic demyelination of the injured part can lead to the necrosis of the spinal cord tissue on both sides of the damaged plane, which eventually leads to the sensory and motor dysfunction below the plane of injury. The cavity of the necrotic zone, the reactive glial scar hyperplasia, and the fracture and removal of the axon Myelin reaction, which also inhibits the regeneration of nerve cells and axons, can not pass scar tissue even if there are new axons. Histopathological changes in SCI can lead to the discovery of new therapeutic targets and treatments. In adult mammals, there are neural stem cells, but their ability to repair SCI is very good. Limited, so the treatment of spinal cord injury is a major challenge.
A large number of scientific research and clinical practice have created a variety of SCI treatment methods, such as drug treatment, rehabilitation exercise, electrical stimulation and so on. But these methods only improve the local conditions of spinal cord injury, but the clinical effect is quite limited. In recent years, the study showed that cell transplantation for the treatment of SCI brought new ideas, and its role has many aspects, including taking a lot of aspects. Replacing damaged neurons, filling holes, improving the environment of the lesion, activating endogenous neurotrophic factor secretion, providing myelin regeneration, structural support, and ultimately enhancing axon regeneration. Through these effects, the transplanted cells provide a matrix platform suitable for the growth of central nervous cells, which can provide sufficient nutrition factors as well. There are many kinds of cells such as olfactory ensheathing cells, Schwann cells, fibroblasts, embryonic stem cells, neural stem cells, neurons, glial precursor cells and bone marrow stromal stem cells, all of which are used for the treatment of spinal cord injury. Although cell transplantation promotes spinal cord injury in varying degrees However, there are still few seed cells used in SCI clinical research and development so far, the main problems are: limited cell source, low survival rate after cell transplantation, tumorigenicity and so on. Therefore, finding the right seed cells, improving the survival rate of transplantation and deep understanding of the mechanism of cell transplantation for the treatment of spinal cord injury. It's very important.
Earlier studies have shown that the stem cells isolated from the neural crest derived tissues have multiple differentiation potential, such as the human eyelid adipose-derived stem cells, hEASCs, which reports the neural crest origin of the neural crest (hEASCs) in the pulp tissue, and has a bipolar shape similar to the neuron. Mesenchymal stem cells (MSC) are spindle shaped.HEASCs and MSC not only in morphological differences, but also in different cell characteristics. Undifferentiated hEASCs can spontaneously express some of the genes and proteins related to nerve cells, most of which are observed in human neural crest cells. By contrast, the fat dry separated by the trunk fat tissue. Cells do not have such characteristics. Therefore, this type of hEASCs derived from the neural crest has its unique characteristics. As we know, this cell has not been used in previous studies to treat spinal cord injury. Therefore, in this study, we evaluated the role of hEASCs transplantation in spinal cord injury in rat spinal cord injury.
After tissue injury, the survival rate of transplanted stem cells in the pathological environment is low, which is a major obstacle to the treatment of stem cells. The acute phase of spinal cord injury can effectively prevent the spread of two injuries, and the maximum protection of the remaining nerve fabric after injury. Among them, 17-beta- estradiol (17p-Estradiol, E2) In particular, it can promote neuronal cells, liver cells, fibroblasts and oligodendrocytes against oxidation, inflammation and apoptosis. Meanwhile, recent studies have shown that E2 has a certain therapeutic effect on central nervous disease, reducing cell death and reducing secondary injury in the body and reducing the cavity area of SCI. Rate of apoptosis.
Therefore, we put forward the hypothesis that combined administration of E2 and hEASCs will benefit the repair of spinal cord injury. The study is divided into two parts in vitro and in vivo: in vitro and in vitro, the separation, culture and identification of hEASCs; the establishment of the spinal cord injury model in the body (1) and the transplantation of the spinal cord in the body; (2) observe the effect of combined treatment, and (3) discuss the combined treatment. Possible mechanisms.
Chapter 1 part of in vitro research: isolation, culture and identification of hEASCs in vitro
Objective: to isolate and identify a group of special adipose derived stem cells from adult eyelid fat.
Methods and results: in vitro, we evaluated the cloning and formation ability of hEASCs, cell proliferation and three lineage differentiation, stem cell related genes and the expression ability of neural specific genes and proteins. The results showed that hEASCs has the characteristics of stem cells, such as clone formation, proliferation and three lineage differentiation potential. The expression of genes and protein markers with neural specificity.
Conclusion: hEASCs has the general characteristics of stem cells, and expresses neural specific genes and protein markers, and has the potential to differentiate into neural cells. It is possible for it to promote the repair of spinal cord injury in vivo.
The second chapter is in vivo study: the establishment of rat spinal cord injury model, combined with E2 and hEASCs transplantation to repair spinal cord injury in rats.
Objective: To study the changes of hEASCs in spinal cord injury in rats and the effect of combination with E on spinal cord injury in rats.
Methods and results: 3 groups were randomly divided into 3 groups, which were divided into 3 groups: PBS transplantation group (control group), hEASCs transplantation group (cell group), hEASCs transplantation and E2 combined group (joint group). The combined group was injected with E2 after 15min injury, and maintained the daily dose to 15d. injury, and the cell group and the combined group were CFDA tagged hEASCs respectively, respectively. The spinal cord injury was injected at the upper and lower 2mm of the spinal cord, and the control group collected samples at 4 and 6 weeks after the same injection of PBS.. The results of fluorescence tracking in the body showed that the transplanted cells were still alive and the specific human nucleus staining showed the same results. The expression of the related human growth factor also demonstrated the survival of the cells in vivo, and compared with the cell group, hEAS The cell survival rate of the combined group of Cs and E2 was higher. Human nucleus staining (hNu) or CFDA fluorescence and microtubule related protein (MAP2), galactosidase (GALAC) or glial fibrillary acidic protein (GFAP) were immunofluorescent. The results showed that hEASCs had signs of differentiation into neurons and oligodendrocytes, but not astrocytes. The histological results of red staining (HE staining) and toluidine blue staining showed that the formation of cavity in the cell group and the combined treatment group decreased obviously. The degree of myelin retention in the combined group was better than the other two groups of.TUNEL staining results, and the apoptosis in the simple cell group was more serious. In contrast, the number of cell apoptosis in the combined treatment group of hEASCs and E2 was obvious. At the same time, the BBB function score and the Grid walking in the combined treatment group were significantly better than those of the other two groups. In addition, the human growth factor (igf-I, ngf1hgf) secreted by the combined group was significantly higher than that of the cell group and the control group. The results showed that the BBB function score and the Grid walking were better than those in the other groups.
Conclusion: the combined use of hEASCs and E significantly promotes the repair of spinal cord injury. The differentiation of hEASCs into neuron cells and oligodendrocytes may be possible. The mechanism of E2 to improve the survival of the transplanted cells and the secretion of growth factors may be the mechanism of its repair.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

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3 陳亞平;楊延硯;吳同絢;張巧云;;脊髓損傷的綜合康復(fù)治療療效分析[A];第一屆全國脊髓損傷治療與康復(fù)研討會暨中國康復(fù)醫(yī)學(xué)會脊柱脊髓損傷專業(yè)委員會脊髓損傷與康復(fù)學(xué)組成立會論文匯編[C];2009年

4 王興盛;;初探手術(shù)加補腦膏治療脊髓損傷5例[A];甘肅省中醫(yī)藥學(xué)會2010年會員代表大會暨學(xué)術(shù)年會論文匯編[C];2010年

5 景小花;張云;;探討運動療法在脊髓損傷術(shù)后康復(fù)中的臨床意義[A];首屆全國中西醫(yī)結(jié)合骨科微創(chuàng)學(xué)術(shù)交流會暨專業(yè)委員會成立大會論文匯編[C];2011年

6 周治來;陳銀海;閔少雄;;康復(fù)訓(xùn)練對脊髓損傷大鼠外源性移植的骨髓源性神經(jīng)干細胞影響的實驗研究[A];中國康復(fù)醫(yī)學(xué)會運動療法分會第十一屆全國康復(fù)學(xué)術(shù)大會學(xué)術(shù)會議論文摘要匯編[C];2011年

7 王璐;王吉興;;神經(jīng)干細胞移植與氯化鋰聯(lián)合干預(yù)修復(fù)脊髓損傷的研究[A];第一屆全國脊髓損傷治療與康復(fù)研討會暨中國康復(fù)醫(yī)學(xué)會脊柱脊髓損傷專業(yè)委員會脊髓損傷與康復(fù)學(xué)組成立會論文匯編[C];2009年

8 彭敏;鄒育庭;廖公平;;探討康復(fù)對脊髓損傷患者生活自理能力的影響[A];中國康復(fù)醫(yī)學(xué)會第七次全國康復(fù)治療學(xué)術(shù)會議主題報告、學(xué)術(shù)交流論文匯編[C];2010年

9 吳軍發(fā);胡永善;吳毅;;脊髓損傷的康復(fù)治療進展[A];中國康復(fù)醫(yī)學(xué)會第四屆會員代表大會暨第三屆中國康復(fù)醫(yī)學(xué)學(xué)術(shù)大會論文匯編[C];2001年

10 盧愛蘭;張夏軍;許美飛;;高壓氧對脊髓損傷肌張力控制的臨床對照研究[A];2011年浙江省物理醫(yī)學(xué)與康復(fù)學(xué)學(xué)術(shù)年會暨康復(fù)新進展學(xué)習(xí)班論文匯編[C];2011年

相關(guān)重要報紙文章 前10條

1 林琳 寒冰;美將進行治脊髓損傷新藥臨床試驗[N];中國醫(yī)藥報;2005年

2 本報記者 吳玲娟;脊髓損傷患者的心理康復(fù)[N];保健時報;2006年

3 鄭穎t

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