【作者】 刘秀梅；

【导师】 王玉玮；

【作者基本信息】 山东大学 ， 儿科， 2008， 博士

【摘要】 研究背景Tourette综合征（Tourette syndrome,TS）是儿童期最常见的精神运动障碍之一,表现为多发的运动性抽动和发声性抽动,近年来患病率有增加趋势。抽动是指刻板的、不随意的、无目的地重复性动作,该症状在整个TS的自然病程中可以持续多年,发作频率和强度不时地发生变化。TS伴发的行为症状复杂多样,如注意缺陷多动障碍、强迫障碍、自伤行为以及情绪障碍等,不同程度地干扰儿童的认知功能和发育,影响社会适应能力。多数情况下,抽动具有自限性,并可以通过行为矫正或药物治疗得到控制。尽管TS是一种良性的神经精神障碍,仍有接近半数的患者症状迁延,治疗困难,甚至延续到成人,导致终身疾患,严重影响生活质量。对一部分患者而言,抽动可导致终生的损害,约有5%的TS患者其症状是危及生命的,可称之为恶性TS。多种药物对恶性TS的治疗效果欠佳,因此近年来人们逐渐开始尝试用神经外科手术方法治疗恶性TS。深部脑刺激（deep brain stimulation,DBS）疗法对严重的难治性抽动症具有一定疗效,但昂贵的治疗费用以及某些手术并发症限制了DBS的应用。近年来在TS的治疗方面未见有重大的突破性进展,TS的治疗问题仍是本领域研究的重点和难点,因此,我们试图找出一条不同于传统对症治疗方法的途径,即从细胞水平进行治疗,尽可能从根本上替代和修复结构和功能受损伤的神经细胞,以期获得更好的治疗效果,探寻治疗TS,尤其是恶性难治性抽动症的新方法。近年来,干细胞移植治疗中枢神经系统疾病的潜能引起人们极大的兴趣。神经干细胞（Neural stem cells,NSCs）是一种能够自我更新具有多种分化潜能的未成熟的前体细胞。动物实验表明,将神经干细胞注入动物脑内或血液内以后,细胞可以存活并迁移到受损伤的部位,整合到神经回路中,使动物的神经功能得到改善。神经干细胞移植为许多难以治疗的神经系统疾病提供了新的治疗途径,开拓了中枢神经系统疾病治疗的新视野,在生命科学研究领域中倍受重视。神经干细胞移植将成为多种中枢神经系统功能障碍及损伤后最有前途的神经组织替代性治疗策略。与传统的药物治疗相比,干细胞移植疗法具有其自身独特的优势,尤其适用于某些发病机制不清的神经系统疾病。关于TS的确切的发病机制,无论在分子水平还是细胞水平,至今仍然不是很清楚。神经影像学、神经生理学及尸检研究表明,TS是一种以基底神经节病变为基础的运动传导和调节障碍,病变部位有可能涉及皮质-纹状体-丘脑-皮质环路中的任一部分。综上所述,我们推测如果将神经干细胞移植到TS病变部位,这些细胞有可能代替中枢神经系统中功能低下或受损的细胞发挥作用,重获内环境的稳态,从而减少刻板行为发作的频率及强度。关于神经干细胞治疗TS的研究还是空白,目前国内外尚未见到有关这方面研究的论文报道。因此,本研究试图考察神经干细胞移植能否改善TS大鼠的脑功能并减少其刻板行为,并对细胞移植治疗的作用机制进行初步地探讨。目的探讨胚胎大鼠大脑纹状体神经干细胞的分离、培养、标记与鉴定方法,观察神经干细胞增殖、传代和分化的规律;建立大鼠TS模型,采用立体定向脑内注射法,将神经干细胞移植到大鼠纹状体内,观察神经干细胞原位移植后移植细胞的成活、分化以及大鼠刻板行为改善情况。方法1、神经干细胞的取材、分离、培养和鉴定本实验在无菌条件下取100只E13胚胎大鼠脑纹状体组织,解剖显微镜下用锐性刀片切割脑组织,吸管反复吹打机械分离制备单细胞悬液。调节单细胞悬液浓度,用DMEM/F12培养液调节稀释至细胞密度为5×10⁵个/ml,接种到含有EGF（20ng/ml）和bFGF（20ng/ml）的DMEM/F12无血清培养基中,置于37℃、5%CO₂培养箱中培养。根据细胞生长情况每2～3天半量换液,低速离心未贴壁生长的细胞克隆,用新鲜培养液更换半量液体,吹打分散成单细胞及小细胞团,继续培养。原代培养5～6天后,形成数目较多、个体较大约含几十个甚至上百个细胞的“神经球”,采用机械吹打与胰蛋白酶消化相结合的方法及时进行传代培养。收集细胞,离心后弃上清液,加入1ml0.25%胰蛋白酶,同时用吸管反复轻柔吹打细胞悬液,避免产生气泡,将“神经球”重新分散制成单细胞悬液,当消化液略呈乳状时加入等体积的含10%胎牛血清的DMEM/F12培养基终止消化,调节细胞接种密度分瓶传代培养。分别用抗Nestin抗体、抗MAP2抗体、抗GFAP抗体,对培养的细胞进行干细胞特性和多分化潜能的免疫组织化学鉴定。取3～4代神经干细胞用于移植,移植前24小时将BrdU（10μg/ml）加入培养基中对细胞进行标记。2、TS大鼠动物模型的建立与神经干细胞移植抽取TS患者静脉血2.5ml,离心后分离血清,采用酶联免疫吸附实验（enzymelinked immunosorbent assay,ELISA）检测患者血清中的抗神经抗体滴度。从TS患者中选取8例OD值最高的患者血清用于大鼠纹状体微量灌注,以建立TS大鼠动物模型。32只Wistar大鼠被随机分为4组,每组8只:假模型组（微量注射正常儿童血清）,TS组（微量注射TS患者血清）,TS+PBS组（纹状体内被微量注射TS患者血清后再给予PBS做治疗对照）,TS+NSCs移植组（纹状体内被微量注射TS患者血清后再进行NSC脑内移植治疗）。将大鼠麻醉后固定于立体定向仪上,无菌条件下将套管置入大鼠双侧纹状体内,套管置入位点为前囟前2.0mm、正中线左右4.0mm、颅下7.0mm。1周后通过微量渗透泵将血清以0.5μl/h的速度缓慢注入大鼠纹状体内,持续灌注72小时。血清灌注结束后,进行神经干细胞移植,在损伤位点原位注射神经干细胞悬液,每个注射位点接受2μl的细胞悬液,细胞密度为5×10⁵个/μl。在移植后1天,7天,14天和21天评估大鼠刻板动作行为,在每12小时光循环结束前的最后30分钟观察大鼠的动作行为如啮咬（用牙齿咬笼子,嚼木屑,无目的咀嚼）、将前爪举到嘴边或面部、舔（不包括理毛）、理毛行为、摇头、摇动爪子、用后腿站立及阶段性的发声,将所观察到的动作行为计数的总和作为评估刻板运动的总分。3、神经干细胞脑内移植后的存活和分化神经干细胞移植后3周,处死大鼠,进行心脏灌流固定。快速灌注PBS150～200ml,冲洗脉管系统,随后换用4℃的4%多聚甲醛溶液继续灌注。灌流结束后将大鼠断头取脑,寻找双侧脑表面的标志针孔,以针孔为中心切取标志针孔周围脑组织;将脑组织浸入4%多聚甲醛固定。制备脑组织石蜡快,采取连续切片法,以移植部位为中心连续冠状切片。采用BCIP/NBT及AEC两种不同的显色系统,分别进行BrdU抗体+Nestin抗体、BrdU抗体+MAP2抗体、BrdU抗体+GFAP抗体双染免疫组化分析,观察神经干细胞移植到大鼠纹状体后在宿主脑内的存活及分化情况。结果1、神经干细胞的取材、分离、培养和鉴定在含有EGF和bFGF的培养基中,大鼠纹状体NSCS开始增殖,细胞种植后24小时显微镜下观察可见大量分裂期的神经干细胞,细胞呈对称或不对称分裂,有的呈哑铃状,连续观察可见细胞分裂成两个完全独立的神经干细胞。随后神经干细胞进入快速增殖期,形成3～6个疏松连接的细胞团,随着时间的延长,细胞不断分裂,在48～72小时后形成含有10～20个细胞的克隆。神经球逐渐增大,细胞之间结合更加紧密,细胞光亮,折光性好,核、浆比例大。分离神经球后继续传代培养,细胞数量持续增加。神经干细胞在细胞分化液中培养,2小时后细胞贴壁,逐渐形成突起,随着培养时间延长,突起逐渐延长,显微镜下呈现神经元和胶质细胞形态,细胞染色证实细胞系巢蛋白Nestin阳性,经血清培养液诱导分化后部分呈MAP2阳性,部分呈GFAP阳性。2、TS大鼠动物模型的建立与神经干细胞移植基于抗神经抗体ELISA的OD值,从20例TS病人中选取8例OD值最高的病人的血清（0.904±0.177）用于大鼠纹状体微量灌注。同样从20例正常对照中选取8例OD值最低的血清（0.252±0.193）用于灌注。TS病人的血清中的抗神经抗体可致纹状体功能受损,纹状体内微量灌注TS血清后,成功诱导出大鼠的刻板行为。大鼠的刻板行为如啮咬、将前爪举到嘴边或面部、舔、摇头、摇动爪子、用后腿站立及阶段性的发声显著增加。灌注TS儿童血清的大鼠的刻板行为与灌注正常儿童血清的刻板行为之间差别显著,TS大鼠的啮咬、将前爪举到嘴边或面部、自咬、理毛、舔、摇头、摇动爪子、用后腿站立、阶段性的发声明显增加。在移植后1天、7天、14天和21天时监测四组大鼠的刻板行为。重复测量数据的方差分析表明组间差别（F=354.13,p＜0.0001）、时间差别（F=106.01,p＜0.0001）以及二者间的交互作用（F=21.38,p＜0.0001）差别均显著。对TS+NSCs和TS+PBS两组分析结果表明二者间其差别显著。注射神经干细胞的大鼠在14天和21天时的刻板行为明显减少,Tukey-Kramer post hoc test统计学分析表明在14天时即出现明显组间差别。移植组的刻板行为仍高于正常对照组（p＜0.0001）,提示在3周内损伤并未得到完全恢复。3、神经干细胞脑内移植后的存活和分化在移植3周后对大鼠大脑进行免疫组织化学分析。本研究采用BrdU标记移植细胞,在纹状体内检测到细胞核着色为暗紫色的BrdU阳性细胞即为移植细胞。移植位点附近的BrdU阳性细胞分布较多,表明移植细胞在宿主脑内存活。部分移植细胞表达BrdU和MAP2,部分表达BrdU和GFAP,表明神经干细胞在移植部位生长并分化为神经元或星形胶质细胞。另外,仍有少数神经干细胞未分化,呈BrdU及Nestin双阳性。结论1、本实验成功分离和培养了来源于胚胎大鼠纹状体的NSCs,并证明它们具备了NSCs的基本特征:（1）表达神经干细胞标志性蛋白Nestin;（2）在培养中存活的NSCs可由单个细胞形成神经细胞球,表明具有自我更新、自我增殖能力;（3）在诱导分化条件下具有形成神经元、神经胶质细胞的能力。NSCs的自我增殖特性和多向分化潜能不因细胞传代而改变。2、向大鼠纹状体内微量灌注含有高滴度抗神经抗体的TS患者血清,可以成功诱导出大鼠的刻板行为;纹状体内神经干细胞移植可减少TS大鼠的刻板行为。纹状体内注射神经干细胞可能对TS具有一定的治疗作用。3、神经干细胞能够在TS大鼠脑内生长存活,部分神经干细胞分化成神经元及神经胶质细胞,可促进中枢神经系统的结构和功能的修复与重建。移植区未见异常肿瘤组织生成,无明显免疫排斥反应。更多还原

【Abstract】 BackgroundTourette syndrome (TS) is a neurobehavioural disorder occurring in children and is characterized by multiple motor and vocal tics. Tics are stereotypic, involuntary, purposeless and repetitive movements which persist for years in TS and wax and wane in frequency and intensity during their natural courses. Although TS is a benign neuropsychiatric disorder, nearly half of the cohort can not be free of tics even until 18 years of age. In most cases, tics are self-limited or can be treated by behavioural or pharmacological therapy. However, for some individuals, tics can cause lifelong impairment and about 5% of TS patients have life-threatening symptoms, which were defined malignant TS. These symptoms are intractable to conservative treatment and various attempts have been made to treat these patients through neurosurgical procedures. Recently, there has been increasing interest in deep brain stimulation (DBS) as a potential treatment for patients with severe, refractory tics. Nonetheless, the use of DBS for the treatment of TS is limited because of complications related to the surgical procedure and the infrequent hardware. Therefore, new therapy should be explored for those unfortunate patients who are significantly impaired.During recent years, stem cell-based therapy has been proved promising as a potential treatment for many neurological disorders. Neural stem cells (NSCs) are considered a heterogeneous population of mitotically active, self-renewing, multipotent and immature progenitor cells. Animal experiments suggest that if NSCs are injected into the brain or even the bloodstream, the transplanted cells would survive and migrate to damaged portion of the nervous system to incorporate into working neural circuits, and the tested animals display significant functional improvement. Therefore, NSC replacement is thought to be a promising alternative therapeutic approach for treating neurological disorders. It may breakthrough some of the existing limitations of traditional pharmaceutical approaches. Most important of all, neural stem cell therapy is a good choice for the treatment of neural diseases whose exact pathogenesis are unclear.The definitive pathophysiological mechanism of tics, at molecular and cellular level, is still unknown. However, structural and functional neuroimaging, neurophysiological, and post-mortem studies have shown the dysfunction of the basal ganglia and related cortico-striato-thalamo-cortical circuits, and the dopaminergic neuronal system. Taking these scholarship into consideration, we hypothesized that intrastriatal tansplantation of NSCs could replace disfunctioned or damaged cells in the central nervous system of TS, reduce the frequency and intensity of stereotypic behaviors and further help regain homeostasis.ObjectivesTo investigate the methods of isolation, cultivation, purification, expanding, BrdU marking and identification of neural stem cells from striatum of fetal Wistar rat and observe the proliferation and differentiation of neural stem cells. Study the effect of neural stem cell transplantation on Wistar rats which were microinfused with the sera of TS patients. Observe the growth and differentiation of transplanted cells in the rat brain and the therapeutic effects of neural stem cells on the stereotypic behavior of TS rat.MethodsNSCs were isolated from rat embryonic brain (E13). Embryonic striatum tissue was microdissected under a stereo microscope and minced into small pieces, and then mechanically triturated through a 26-gauge needle and dissociated to single cell suspension. Cells were cultured in serum-free basic medium DMEM: F12 supplemented with human recombinant epidermal growth factor (EGF) (20 ng/ml), fibroblast growth factor-basic (bFGF) (20 ng/ml), B27 (2%), penicillin-streptomycin 1% . Cells were incubated with 5% CO2 at 37℃. Within 2-3 days the cells grew as free floating neurospheres and half of the growth medium was replaced. Expanded neurospheres were collected by centrifugation and dissociated into single cells once every 5-6 days with 0.25% trypsin and mechanical trituration. NSCs were co-incubated with 5-bromodeoxyuridine (BrdU, 10μg/ml) for 24h prior to transplantation. Then BrdU-labeled NSCs (passage number 3-4) were harvested and enzymatically dissociated into single cells. Cells with higher than 85% viability were centrifuged and resuspended in basic medium supplemented with B27 at a density of 5×10~5 cells/μl and then stored on ice until grafting. To determine the phenotype of cultured cells, the cells were separately immuno-stained with anti-Nestin antibody, anti-MAP2 antibody or anti-GFAP antibody.Blood samples were drawn from patients with TS and were sent for further enzyme-linked immunosorbent assays (ELISA) to a laboratory. The selection of sera for infusion was based on ELISA optical density readings against caudate homogenate, that is, we selected the highest serum OD readings for TS subjects and the lowest OD readings for the control infusion subgroup. A total of thirty-two Wistar rats were used in the study and randomly divided into four groups: sham (microinfused with normal sera), TS alone (microinfused with TS sera), TS plus PBS vehicle injection (TS+PBS) and TS plus NSCs grating (TS+NSCs) (n=32, i.e., n=8 for each group).Rats were deeply anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic apparatus with the incisor bar set at 3.5mm below the interaural line. Then using aseptic surgical technique, the skull was exposed and holes were drilled where appropriate and 28 gauge guide cannulae were implanted into bilateral striatum. Coordinates for cannulae placements were anterior-posterior 2.0mm from bregma and medial-lateral 4.0mm and dorsoventral -7.0mm from the skull. One week later, osmotic mini pump filled with PBS was connected to each cannula by a polyethylene tube loaded with 50μl of undiluted TS or control serum under sterile conditions. Sera were microinfused at a rate of 0.5μl /hour for 72 hours. The neural stem cell suspension (2μl) was injected bilaterally using a 5μl Hamilton syringe with a 26-gauge needle at the sera-infusion site after the pumps were removed. Rat movements were video and audio taped at the end of the rat’s 12-hour light cycle for 30 min. Several categories of stereotypy including bites (teeth touching the cage, wood chips, vacuous chewing or other objects except the body), taffy pulling (raises of the forepaw to the mouth and face), self-gnawing, licking not associated with grooming, grooming, head shaking, paw shaking, rearing and episodic utterances (EU) were recorded. Stereotypic movements were recorded at 1 day, 7 days, 14 days and 21 days after transplantation.Three weeks after transplantation, rats were given a lethal dose of chloride hydrate (600 mg/kg) and transcardially perfused with cold 0.9% NS followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4, 4℃). Brains were removed from the cranium and post-fixed in PFA prior to sectioning. Rat brain sections were embedded in paraffin and 4-μm coronal sections were prepared. All the immunostaining processes were finished according to the instruction of histostain?-DS kit. Primary antibodies which included mouse monoclonal to GFAP—astrocyte marker, mouse monoclonal to MAP2—neuron marker and mouse monoclonal to Nestin—neural stem cell marker were added.ResultsFollowing the isolation, a close-to-single-cell suspension was obtained, which consisted of viable small, round cells. In suspension cultures containing EGF and bFGF, rat striatal NSCs began to proliferate and adherent clonal clusters of cells (10 - 20 cells/clone) started to appear after 48 - 72 h. The clusters divided rapidly and generated large colonies, i. e. neurospheres. These floating neurospheres displayed a spherical shape, and the cells forming the neurosphere were phase bright. These spheres were successfully passaged by dissociation and reculture. This procedure resulted in a rapid increase in cell number. The neural cells dissociated from E13 rat striatum had the capacity to proliferate and form neurospheres. The neurosphere could express Nestin, gernerate MAP2 or GFAP positive neural cells.Eight children were selected from 20 patients with TS and their sera were used for rat striatal microinfusion. Similarly, eight specimens of sera were selected from 20 control samples. The selection of sera for infusion was based on ELISA optical density readings against caudate homogenate, that is, we selected the highest serum OD readings for TS subjects and the lowest OD readings for the control infusion subgroup. The mean±SD optical density readings in serum of 8 TS subjects selected for microinfusion was 0.904±0.177 and 0.252±0.193 for the 8 selected controls.Serologic studies of children with TS have detected the existence of anti-neural antibodies, which can induce striatal dysfunction. In our study, stereotypies were successfully induced in rats by intrastriatal microinfusion of TS sera under noninflammatory conditions. After infusion of TS sera, stereotypic behaviors in rats increased significantly. Marked differences were observed in stereotypies of TS rats compared to control rats after microinfusion. TS rats exhibited significant increases in bites, taffy-pulling, gnawing, licking, head shaking, paw shaking, rearing and episodic utterance. All rats in four groups were observed at 1 day, 7 days, 14 days, and 21 days after their transplantations. Stereotypic behaviors of each rat were recorded and counted through each 30 minutes of observation period. The statistical analysis suggested that TS rats had increased stereotypic behaviors compared to normal rats (Sham). Using a repeated measurements analysis of variance (ANOVA), we found that the overall model had significant group (F=354.13, p<0.0001) and day (F= 106.01, p<0.0001) effects, as well as a (group×day) interaction (F=21.38, p<0.0001), indicating varying degrees of differences among the groups and across days. A model with just TS+NSCs and TS+PBS groups indicated significant differences between groups(F=36.63, p=0.0001) and a strong day effect(F=100.23, p<0.0001). Importantly, animals with NSCs grafts showed a significant decrease in stereotypic behaviors at 14 and 21 days. Tukey-Kramer post hoc test showed significant differences between TS+NSCs and TS+PBS groups beginning at 2 weeks post-transplantation. However, rats receiving NSC grafts (TS+NSCs) still had higher stereotypic behavior counts than sham controls (p<0.0001), which indicated that the animals’ impairment did not fully recover in a short period of 3 weeks.Histological analyses of recipient rat brains transplanted with NSCs or injected with PBS (vehicle) were given 3 weeks after transplantation. In order to trace the transplanted cells, we used BrdU labeling, so that the presence of the implanted NSCs within striatum of TS rats identifiable for the nuclei of the grafted cells were BrdU-positive and stained with purple. In the TS+NSCs rats, BrdU-positive cells were prevalent in the striatum sections near the injection site, suggesting the survival of the cells. Some of the grafted cells expressed both BrdU and MAP-2 and others expressed both BrdU and GFAP. These results suggested that NSCs used as grafts grew at the injection site and differentiated into neurons or astrocytes. Our results also indicated that a considerable portion of the transplanted cells had differentiated to mature neurons in vivo, which may lead to histological and functional reconstitution in the CNS. Besides, few BrdU and Nestin double positive cells were found, indicating undiferentiation of a small number of the grafted cells.ConclusionsNeural stem cells were isolated from fetal rat striatum and were certificated as a heterogeneous population of mitotically active, self-renewing, multipotent, immature progenitor cells. These cells expressed the intermediate filament protein Nestin and grew as neurospheres in vitro. Neural stem cells had the capacity to differentiate into neurons and gilocytes..In our study, stereotypies were successfully induced in rats by intrastriatal microinfusion of TS sera under noninflammatory conditions. Our results showed that intrastriatum transplanted rat NSCs relieved stereotypic behaviors. These results suggested the potential of using NSCs intrastriatum as a clinical treatment for TS. Neural stem cells survived in the brain of TS rat and part of them differentiated into neurons and gliocytes. These newly generated neurons from the transplanted NSCs might have played a role for the neural networks in the damaged areas. No signs of immunologic rejection or non-neural tissue growth were found in the host rat brain after transplantation.更多还原