【作者】 周婧；

【导师】 凌树才； 欧阳宏伟；

【作者基本信息】 浙江大学 ， 人体解剖与组织胚胎学， 2012， 博士

【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。更多还原

【Abstract】 IntroductionSpinal cord injury (SCI) is one of the most devastating forms of trauma experienced by humans, often resulting in permanent disability. As reported by the Foundation for Spinal Cord Injury Prevention, Care, and Cure, there are approximately450,000people living with SCI, and an additional11,000new SCI cases occur every year in the US. The repair of SCI is still a major therapeutic challenge at present, because endogenous repair following SCI in adult mammals is restricted. Damage caused by inflammatory cells, glia scar formation and decreased intrinsic growth drive of adult neurons, as well as the inhibitory effect of myelin in the central nervous system, may all play a part.Exogenous intervention strategies are necessary to enhance recovery. As many studies performed, cell transplantation therapy has emerged as a powerful and promising repair strategy for enhancing restitution of the lost function. The goals of cell transplantation therapy were vary widely include replacing damaged neurons, filling the cystic cavity, enhancing axonal regeneration by creating a regenerative environment, and supporting or inducing remyelination. Different types of cells have been evaluated as therapeutic strategies for post-SCI cell transplantation including embryonic stem cells, neural or glial precursor cells, genetically modified fibroblasts, mesenchymal stem cells, olfactory ensheathing cells, and Schwann cells. Although recent dramatic progress in cellular transplantation has heightened the optimism about future cures for such injuries, development of powerful strategies to treat SCI is still a major clinical challenge.Previous studies report that stem cells isolated from neural crest-derived tissue have multidifferentiation ability, such as in dental tissue. Haekwon Kim et al isolated neural crest-like stem cells from human eyelid adipose tissue. These human eyelid adipose-derived stem cells (hEASCs) are of the bipolar neuronal shape, whereas other Mesenchymal stem cells (MSCs) are spindle shaped. hEASCs and MSC are not only different in morphology, but cells characteristics are also different. Undifferentiated hEASCs spontaneously express many neural cell-related mRNAs and proteins, most of which are observed in human neural crest cells. In contrast, MSCs from the trunk adipose tissues do not show most of these neural cell-like characters unless be induced. Therefore hEASCs have unique characteristics that favor their use in transplantation strategies for SCI repair. To the best of our knowledge, no previous study used this cell for treatment SCI. Thus, in this study we conducted to assess the effect of hEASCs transplantation in a rat SCI model for SCI repair. However, previous studies also show that the low survival rate of graft stem cells alone after transplantation into lesion tissue is a major obstacle for successful stem cell therapy.Accordingly, several experimental studies have shown17-β-estradiol (E2) has neuroprotective properties and produces therapeutic effects in various models of central nervous diseases. E2has a protective effect against oxidant, inflammatory and apoptotic, able to attenuate cell death in vitro and reduce secondary damage in vivo investigations. Recent research suggests that E2decreases lesion volume and attenuates apoptotic cell death following SCI.Previous many studies have reported that estrogen alone plays the role of SCI repair. However, the protective potential for combination E2and stem cell has not yet been investigated in SCI. Therefore, we put forward a hypothesis that combination pre-administration E2with hEASCs transplantation after SCI in a rat model will promote functional recovery of paralyzed rats, beneficial for SCI repair. A broader understanding of the histopathology and functional outcomes of thoracic SCI could hasten the identification of appropriate therapeutic targets for this injury and support the translation of potential therapeutics to the sizable thoracic SCI population.Stage1Isolation and characterization of hEASCsAim:hEASCs were isolated from human eyelid adipose after eyelid reshaping surgery, cultured and characterized.Methods and results:Cell culture and fluorescence-activated cell sorting (FACS) analysis.The multi-differentiation potential, gene expression profile and proliferation assay, neural differentiation capacity and neural specific genes and protein markers of hEASCs were investigated in vitro. The results showed that hEASCs exhibited some parallel characteristics typical of MSCs (CD105, CD29, CD166and CD44), and null expression of hemopoietic stem cell marker(CD34)and bone marrow stromal cell maker (CD18). hEASCs have the capbiltiy of osteogenic, adipogenic and chondrogenic differentiation, and spontaneous expressed many neural cell-related mRNAs and proteins which would be enhanced after induced.Conclusion:This study thus demonstrated that the use of hEASCs as a source for human transplant populations not only possess the inherently broad capacity of expansion and differentiation, but also offers advantages over other cell types, and do not violate ethical. Stage2Transplantation hEASCs into rat spinal cord model in biodegradable scaffold with E2for spinal cord repairAim:The aim of this study was to investigate their therapeutic potentials for SCI repair, and whether the combination of HEACs and E2is a potential therapy method for SCI.Methods and results:We first set up the animal model of SCI at10th thoracic vertebras (T10) by hemisection at the right side. A lateral slit in the dura was generated, and then right hemisection was created at T10. The SCI rats were randomly divided into three groups,17animals for each group. The first group (sham control) underwent sterile phosphate buffer saline (PBS) injection. The second group was injected with hEASCs after SCI7days. The third group was treated with the combination of E2subcutaneous injection and hEASCs transplantation. In the third group E2was also administered at a dose of100μg/kg15min after SCI, daily for the next14days. After6weeks, hEASCs in vivo continue to express motor neuron marker microtubule associated protein (MAP2), and oligodendrocyte marker galactosidase (GALAC), do not express astrocyte markers glial fibrillary acidic portein (GFAP). These results suggested the implanted hEASCs maybe differentiate to neurons and oligodendrocytes, however not contribute to astrocyte, which is beneficial to the SCI repair. Furthermore, the hEASCs groups obviously reduced cavity formation compared SCI vehicle groups. The xenograft cell expressing growth factors (igf-1, ngf, hgf) in vivo might have the effect of improve local environment, reduce hollow, and promote axonal regeneration in transplantation treatment of SCI. These results suggested this cells hEASCs can be used as a promising seed cell for the treatment of SCI. Compared the pure cells group, the E2-hEASCs group have obvious improvement. We detected the human (3-actin expression after transplanted cell7days. The combination significantly promotes the survival of hEASCs at acute phase of SCI when plenty of cell death happens. Such survival should conduce to the consequence of increased remyelination, and subsequently significantly improvement in hind limb motor function as determined by Basso, Beattie, and Bresnahan locomotor open field behavioral rating test (BBB) and Grid-walking test. From Toluidine Blue staining, E2increased peripheral mylinated axons in grafts. Furthermore, the E2-hEASCs group effectively reduced the apoptotic cell death and caspase-3activity, compared the only cells group after SCI.Conclusion:This study demonstrated that unique properties of hEASCs combined with E2might cooperatively work, significantly increase hEASCs survival after transplantation, promote axonal regeneration and improves histological outcomes that correlate with improved recovery. This finding highlights that the combination of E2and hEASCs transplantation may be a highly efficient therapeutic approach for SCI.更多还原