成人眼眶来源脂肪干细胞联合17-beta-雌二醇对大鼠脊髓损伤的修复研究

发布时间：2018-05-27 19:36

本文选题：脊髓损伤 + 细胞移植　；参考：《浙江大学》2012年博士论文

【摘要】：背景：脊髓损伤(Spinal cord injury, SCI)是人类所经历的创伤中最具破坏性的创伤之一,常常导致永久性残疾,其多由车祸、坠落等高能量撞击引发脊柱骨折、脱位所致。脊髓损伤预防保健防治基金会统计,目前在世界范围内大约有250万人受此病影响,每年有超过13万的新发外伤性SCI病例。美国约有45万脊髓损伤病人,并且每年有1.1万新增SCI病例发生。绝大多数患者均遗留部分或完全性瘫痪,损伤后患者的生活质量严重下降。82%患者为是青壮年男性,给社会和家庭造成了不可估量的损失。由于SCI主要影响年轻人,且缺乏有效的治疗方法,由此带来的功能丧失往往伴随着病人的一生。受损脊髓病理改变主要包括两个过程,原发性损伤和继发性损伤,主要造成不同程度的细胞死亡、组织水肿、胶质疤痕增生和脊髓运动功能丧失,而死亡的神经元裂解释放的毒素,损伤部位的慢性脱髓鞘等病理改变又可导致损伤平面上下两侧的脊髓组织坏死,最终导致损伤平面以下感觉和运动功能障碍。病变坏死区形成的空洞、反应性胶质疤痕增生、以及轴突断裂和脱髓鞘反应,也对神经细胞和轴突的再生产生抑制作用,即使有新生的轴突,也不能通过瘢痕组织。研究SCI的病理组织学改变可促使新的治疗靶点和治疗方法的发现。在成年哺乳动物体中,虽然存在神经干细胞,但其对SCI的修复能力非常有限,因此脊髓损伤的治疗是一大挑战。大量科学研究和临床实践诞生了多种SCI治疗方法,例如药物治疗,康复锻炼,电刺激等等,但这些手段仅仅改善了脊髓受损后局部情况,而临床效果相当有限。近年研究表明,细胞移植为SCI的治疗带来新的思路,其作用有很多方面,包括取代受损的神经元,填补空洞,改善病变的环境,激活内源性的神经营养因子分泌增加,提供髓鞘再生,结构支持,及最终增强轴突再生。通过这些作用,移植的细胞提供一个适宜中枢神经细胞生长的基质平台,可以提供足够的营养因子以及增强中枢神经元的再生能力。目前有多种细胞如嗅鞘细胞、雪旺细胞、成纤维细胞、胚胎干细胞、神经干细胞、神经元、神经胶质前体细胞和骨髓基质干细胞等均被用于移植治疗脊髓损伤的研究。虽然,细胞移植不同程度的促进了脊髓损伤的修复,但目前为止适用于SCI临床研究和开发的种子细胞依然较少,其主要问题为：细胞来源有限,细胞移植后存活率低,致瘤性等等。因此,寻找合适的种子细胞,提高移植存活率以及深入了解移植细胞的作用机制对于脊髓损伤的治疗显得非常重要。早前的研究报告表明,从神经嵴衍生的组织中所分离出的干细胞具有多系分化潜能,如在牙髓组织中。Hyunmi等研究报道神经嵴来源的人类眼睑脂肪组织的干细胞(human eyelid adipose-derived stem cells, hEASCs)具有类似神经元的双极的形状,而间充质干细胞(MSC)是纺锤形。hEASCs和MSC不仅在形态上不同,细胞的特征表现也不同。未分化的hEASCs能自发表达一些神经细胞相关的基因和蛋白,其中大部分标记物是在人类的神经嵴细胞所能观察到的。相比之下,躯干脂肪组织分离的脂肪干细胞不具有此类特征。因此这类从神经嵴来源的hEASCs有其独特的特点。据我们所知,以前的研究中尚未使用这种细胞研究治疗脊髓损伤。因此,在这项研究中,我们评估了hEASCs移植在大鼠脊髓损伤模型脊髓损伤的作用。组织损伤后,移植干细胞在病理环境中成活率较低,是干细胞治疗的一大障碍。脊髓损伤急性期进行某些化学药物治疗可以有效的阻止二次损伤的蔓延,并能最大程度的保护损伤后剩余的神经组织。其中17-beta-雌二醇(17p-Estradiol,E2)的作用尤为明显,能促进神经元细胞,肝脏细胞,成纤维细胞和少突胶质细胞对抗氧化,炎症和凋亡等不利环境。同时,最近研究表明,E2对中枢神经疾病有一定的治疗作用,能够减少细胞死亡和减少体内的继发性损伤,可以减少SCI的空洞面积和细胞凋亡比率。因此,我们提出假说,联合给药E2与hEASCs移植,将有利于脊髓损伤修复。研究分为体外和体内两个部分：体外部分为hEASCs的分离、培养和鉴定；体内部分(1)大鼠脊髓损伤模型的建立和hEASCs移植；(2)观察联合治疗的效果,(3)探讨联合治疗的可能机制。第一章体外研究部分：hEASCs的体外分离,培养和鉴定目的：由成人眼睑脂肪中分离一群特殊的脂肪干细胞,培养并对其特性进行鉴定。方法与结果：体外实验中,我们评估了hEASCs的克隆形成能力,细胞增殖能力和三系分化能力,干细胞相关基因及神经相关特异性基因和蛋白的表达能力。结果显示hEASCs具有干细胞的特性,如克隆形成能力,增殖能力和三系分化潜能,同时具有神经特异性的基因和蛋白标记的表达等。结论：hEASCs具有干细胞的一般特性,而且表达神经特异性的基因和蛋白标记,并具有向神经系细胞分化的潜能,为其是体内促进脊髓损伤修复提供了可能。第二章体内研究部分：大鼠脊髓损伤模型的建立,联合E2和hEASCs移植进行大鼠脊髓损伤模修复。目的：研究hEASCs在大鼠脊髓损伤中的变化,以及与E：的联合使用对大鼠脊髓损伤的修复作用。方法与结果：大鼠胸椎T10右半横断损伤后,随机分为3组,分为PBS移植组(对照组),hEASCs移植组(细胞组),hEASCs移植和E2联合组(联合组)。联合组在损伤15min后进行E2注射,维持每天给药至15d。损伤7天后,细胞组和联合组分别将CFDA标记的hEASCs于脊髓损伤处上下2mm处进行注射移植,对照组于同样位置注射PBS。术后4周和6周收集样本,体内荧光跟踪结果显示移植细胞依然存活,特异性人核染色表现了一致的结果,相关人生长因子的表达,也证明了细胞的体内存活,与细胞组相比,hEASCs与E2的联合组细胞存活率更高。人核染色(hNu)或CFDA荧光与微管相关蛋白(MAP2).半乳糖苷酶(GALAC)或者胶质纤维酸性蛋白(GFAP)进行免疫荧光共定位,结果表明,hEASCs有向神经元和少突胶质细胞分化的迹象,而未向星形胶质分化。苏木精-伊红染色法(HE染色)和甲苯胺蓝染色组织学结果显示,细胞组和联合治疗组的空洞形成明显减少,联合组的髓鞘保留程度也要好于其他两组。TUNEL染色结果表明,单纯的细胞组凋亡比较严重,相比之下,hEASCs与E2联合治疗组细胞凋亡数量明显减少,caspase-3表达降低,bcl-2表达升高。同时,联合治疗组大鼠的BBB功能评分和Grid walking也明显好于其他两组。此外,联合组分泌的人生长因子(igf-I，，ngf1hgf)显著高于细胞组和对照组。结论：hEASCs与E：的联合使用显著的促进了脊髓损伤修复,hEASCs向神经元细胞和少突胶质细胞的分化可能、E2对移植细胞的存活改善及其生长因子的分泌作用可能是其修复的机制。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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