【作者】 徐伟伟；

【导师】 姜晓丹；

【作者基本信息】 南方医科大学 ， 神经外科学， 2012， 博士

【摘要】 脊髓损伤(spinal cord injurey, SCI)这一严重致残的重大伤病目前仍是世界性难题,其病理学基础主要是直接或间接的损伤导致轴突变性和神经元坏死。成人SCI后微环境的破坏致使中枢神经再生困难。然而,由于SCI病理改变的复杂性,其治疗效果一直欠佳,因此SCI及其修复机理是当今世界神经科学关注的热点之一。为了征服这一疾病,众多神经科学工作者致力于此研究领域,希望通过研究、能找到缓解SCI给病人及家庭和社会带来危害的有效方法。目前,诸多团队在SCI修复的治疗方法及机制研究方面做了大量工作,其中,神经干细胞(neural stem cells, NSCs)因其可以分化为神经元与胶质细胞而倍受关注,而SCI后通过静脉注射NSCs,可以在损伤灶发现移植的NSCs,并能分化为一定数量的神经元跟胶质细胞,使得NSCs被考虑可作为一种细胞替代疗法的种子细胞进行治疗应用研究,这也让我们看到了让SCI截瘫病人恢复的希望。然而迄今为止,移植NSCs的有效性尚不理想,主要存在的问题是,如果早期移植,容易因炎症反应而被体内巨噬细胞吞噬；如果移植较晚,则损伤灶容易形成瘢痕组织,影响神经元的修复。有研究表明,在SCI后第7天移植NSCs效果较好,可能与该时期可避免巨噬细胞的吞噬、疤痕组织也未开始充分形成有关。为了更好地探讨如何提高NSCs移植疗效,本研究以大鼠SCI后的NSCs移植治疗为研究理念、以microRNA为骨髓基质细胞源神经干细胞(BMSCs-D-NSCs)调控手段,进行了microRNA调节NSCs向神经元方向分化、治疗大鼠SCI的实验探讨。microRNA即小RNA,是一种存在于植物和动物基因组里的小分子RNA,约20-24nt(少数小于20nt),由一段具有发夹环结构、长度为70-80nt的单链RNA前体(Pre-miRNA)剪切后形成。它通过与其目标mRNA分子的3’端非编码区域(3’-untranslated region,3’UTR)完全或非完全互补匹配、参与基因转录后水平(蛋白表达)的调控,在生物体内各种生理和病理状态都发挥着十分重要的作用。miRNA最早由Lee等1993年在对线虫(Caenothabditis elegans)胚胎发育研究过程中发现的一种具有调控功能的非编码RNA。首先,miRNA基因的初级转录产物(Pri-miRNA)在细胞核中被RNase ш Drosha切割成为前体miRNA (pre-miRNA)。在最初的剪切后,Pre-miRNA在转运蛋白exPortin-5的作用下、由核内转到胞质中,然后由另一种RNase m Dicer进一步切割产生成熟的miRNA.这些成熟的miRNA与其他蛋白质一起组成RISC (RNA-induced sileneing complex)复合体,从而引起靶mRNA的降解或者翻译抑制。microRNAs具有重要的组织特异功能,在基因表达中发挥着总体调控作用,其中miR-124是神经系统特异表达且最富含的microRNA。在鸡胚神经节,miR-124P制性地表达于中枢神经系统(central nerve system, CNS);在小鼠神经中枢(大脑皮层,小脑和脊髓),miR-124浓集超过其它器官的100倍。但是,miR-124在CNS解剖区域上的表达存在明显差异,在大脑皮层表达最多,小脑表达率是大脑皮层的60.7%,脊髓是35.4%。同时,miR-124在细胞增殖和分化过程的表达量也会发生变化。目前,对miR-124在CNS的表达研究更多局限在果蝇、小鼠和人类相关组织,而在大鼠等动物中的研究还较少,但大鼠一般又被用来进行SCI实验研究更适宜。miR-124调控神经元的细胞周期、细胞分化、脊髓的发育等一系列重要生理活动,作为神经系统特异的microRNA,对神经系统病理损伤的调控机制尚未明了,尤其对SCI中的相关治疗(如NSCs移植治疗等)调控作用尚缺少文献报道。本课题将探讨miR-124调节NSCs在大鼠SCI中的移植治疗作用。第一部分：miR-124对BMSCs-D-NSCs的促分化作用目的：将慢病毒载体pCDH-CMV-MCS-EF1-copGFP进行EcoRI/BamHI酶切,在基因库中查询miR-124序列5’-UAAGGCA CGCGGUGAAUGCC-3’,并进行人工合成,利用PCR技术设计引物前链5’-GCCGATTC (EcoRI) CATCGCGTTCCCCAAA CCCC-3’与反义链引物5’-GCCGGATCC (BamHI) AGGGATGAAGGTG CTGGCCT,再用PCR扩增microRNA Rno-miR-124,并在上游和下游引物分别添加限制性酶切位点EcoRI和BamHI,将以上两部分酶切连接后转化到DH5-a内。挑取阳性克隆,用PCR测序鉴定。构建质粒pCDH-CMV-MCS-EF1-copGFP-rno-miR-124(其中GFP为报告基因),并转染293T细胞。收取病毒上清液感染骨髓基质细胞(bone marrow stroma cells, BMSCs),并利用RT-PCR测定miR-124转染BMSCs后及诱导成NSCs后的表达情况、进一步观察BMSCs-D-NSCs分化为神经元与胶质细胞的情况。统计学处理：所有数据均采用Mean±SD表示,Indepent samples t-test用来比较转染组与未转染组NSCs分化为神经元与胶质细胞的情况。统计学软件采用SPSS17.0,P<0.05为有显著统计学意义。结果：BMSCs的转染率可达90%以上,RT-PCR结果表明,转染后BMSCs的miR-124表达量为转染前的30-fold；经过诱导成NSCs(BMSCs-D-NSCs)后,转染组NeuN阳性细胞的比例为53.60%±3.31%,未转染组比例为34.40%±2.88%(p<0.001)；转染组GFAP阳性细胞的比例为42.20%±1.62%,未转染组为51.90%±3.03%(p<0.001)。结论：miR-124在体外可以促进NSCs向神经元分化。第二部分：miR-124调节BMSCs-D-NSCs对SCI治疗作用目的：将携带niR-124的BMSCs经鼠尾静脉注射,治疗SCI,观察miR-124-BMSCs-D-NSCs治疗效果。方法：根据改良Allen’s方法制作动物模型。取90只成年Wistar大鼠、以氯胺酮(500mg/kg)腹腔注射麻醉,俯卧固定,以T10为中心纵行切口,显露脊髓大小为3.0cmxO.4cm的打击区。以重20g、打击头直径3mm的打击器自10cm高度自由落下,打击势能为200g.cm,打击1次。次日见大鼠双后肢完全松弛视为SCI建模成功。依实验条件将SCI模型分为3组：第一组为术后未治疗组,注射生理盐水；第二组为正常移植组,注射BMSCs-D-NSCs;第三组为转染移植组,注射miR-124-BMSCs-D-NSCs。取三组第1天,1周,2周及4周SCI的标本,进行HE染色,比较正常移植组与转染移植组相对于损伤后未治疗对照组空洞的变化。大鼠脊髓功能的恢复依据BBB评分标准进行判断。统计学处理：所有数据均采用Mean±SD表示,Independent samples t-test用以比较转染组、未转染组与正常对照组之间的脊髓空洞变化。利用ANOVA中的LSD法,比较三组间BBB的差异。统计学软件采用SPSS17.0, P<0.05为有显著统计学意义。结果：移植后14天,通过免疫荧光组织化学检测法、对体内移植细胞鉴定分析,发现病变部位有较明显的miR-124-BMSCs-D-NSCs聚集(miR-124-BMSCs-D-NSCs由GFP所标记),主要在受损脊髓的周边(图11A)。此外,共聚焦图像显示,有较多表达神经元标记物神经元核蛋白(neuron nucleoprotein, NeuN)阳性的细胞(图11B)和较少表达星形胶质细胞标记物胶质纤维酸性蛋白(Glial fibrillary acidic protein, GFAP)的细胞(图11C)出现在移植后14天。NeuN染色阳性细胞存活比率占16.0%±1.3%(Mean±SD), GFAP阳性细胞的成活率为13.95%±1.2%(Mean±SD)。损伤灶的大小在SCI后第28天,利用切片进行显微测量计算。miR-124-BMSCs-D-NSCs移植组与对照组相比,减少的脊髓组织损伤范围、与单纯NSCs移植组相对于对照组减少的脊髓组织损伤范围,均有统计学差异(P<0.001),BMSCs-D-NSCs组相对于正常对照组、减小的体积为3.3%,转染组相对于正常对照组、减少的体积为6.4%。BBB评分对以下三组动物进行BBB评分：未治疗对照组(注射生理盐水),单纯NSCs移植组与miR-124-BMSCs-D-NSCs移植组。每一周每组5只大鼠,观察1-6周(n=90),比较每一周内三组之间BBB评分的关系。第1周,三组之间BBB评分无显著统计学差异(P=0.804)。第2周,单纯BMSCs-D-NSCs移植组与损伤后未治疗对照组BBB评分有显著统计学差异(9.20±0.84vs.7.8±0.84,P=0.017),损伤后未治疗对照组与miR-124-BMSCs-D-NSCs移植组之间的BBB评分有显著统计学差异(7.8±0.84vs.9.20±0.71,P=0.035),单纯BMSCs-D-NSCs移植组与miR-124-BMSCs-D-NSCs移植组之间无明显统计学差异(9.20±0.84vs.9.20±±0.71,P=0.698)。第3周,单纯BMSCs-D-NSCs移植组与损伤后未治疗对照组有显著统计学差异(10.60±1.14vs.8.80±0.45,P=0.006), miR-124-BMSCs-D-NSCs移植组与损伤后未治疗对照组亦有显著统计学差异(11.20±0.84vs.8.80±0.45, P=0.001),但是单纯BMSCs-D-NSCs组与miR-124-BMSCs-D-NSCs移植组无明显统计学差异(10.60±1.14vs.11.20±0.84,P=0.290)。第4周,单纯BMSCs-D-NSCs移植组与损伤后未治疗对照组有显著统计学差异(11.40±1.14vs.9.80±0.48,P=0.007),miR-124-BMSCs-D-NSCs移植组与单纯BMSCs-D-NSCs移植组之间有显著统计学差异(13.40±±0.55vs.11.40±1.14,P=0.002),miR-124-BMSCs-D-NSCs移植组与损伤后未治疗对照组之间也有明显统计学差异(13.40±0.55vs.9.80±0.48,P<0.001)。第5周,三组之间两两比较均有显著统计学差异(P<0.001)。第6周,三组之间的两两比较亦均有显著统计学差异(P<0.001)(表2)。结论：经miR-124转染的BMSCs诱导为NSCs后可以更有效地治疗脊髓损伤,表现在脊髓损伤组织范围、体积的减少和BBB评分的明显改善。更多还原

【Abstract】 Spinal cord injury, as the main cause of severe disability, is still a worldwide problem. Its pathology is based primarily on direct or indirect injury leading to axonal degeneration and neuronal necrosis. Adult spinal cord injury, the micro-damage to the environment results in the regeneration of central nervous difficulties. However, due to the complexity of the pathological changes of spinal cord injury, the effect of treatment has been poor. Therefore, spinal cord injury and its repair mechanism is one of the hotspots of neuroscience nowadays. To conquer this disease, many scientists committed to this area, hope to find effective way to ease the patients and family and social harm. Many laboratories have done a lot of work in the repair of spinal cord injury. According to the study of neural stem cells (neural stem cells, NSCs), stem cell therapy is expected to reach a good effect. The NSCs, it can differentiate to neurons and glial cells. After spinal cord injury, by intravenous injection of NSCs, you can find transplanted NSCs in the damaged foci, and differentiation of a certain number of neurons and glial cells. If NSCs used clinically as a cell replacement therapy, it brings big hope for the recovery paraplegic patient after spinal cord injury. The effectiveness of the transplanted NSCs, it is not ideal, the main problems is that the early graft was affected in vivo macrophage phagocytosis. If the transplantation is too late, the damage site is easy to form scar tissue and affect neuronal repair. Studies have shown that the transplantation of NSCs in the seventh day after spinal cord injury is suitable. Avoid macrophage phagocytosis, and the scar tissue does not fully form. In order to improve the effect of NSCs, how to regulate the differentiation of NSCs into more neurons is a subject worthy of study.MicroRNA as a recently discovered small RNA in post-transcriptional level of regulation of protein expression has become the focus of our attention. MicroRNAs are small RNA molecules present in the genome of plants and animals, about20-24nt (a small number of less than20nt), a hairpin loop structure consists of a section, a length of70-80nt single-stranded RNA precursors (pre-miRNA formation) after shearing. Its target mRNA molecules in the3’untranslated region (3’from a untranslated region3’UTR) is completely non-complementary match to participate in the regulation of gene transcription levels in physiological and pathological conditions and plays a very important role.miR-124as non-coding RNA was first discovered in the embryonic development of the nematodes (Caenothabditis, elegans) in1993by Lee et al. First of all, the primary transcripts of miRNA genes (the Pri-miRNAs) cutting the precursor miRNA (pre-miRNAs) by the RNase m Drosha in the nucleus. Transporter protein exPortin-5plays a role in the initial shear Pre-miRNA from the nucleus to the cytoplasm, and then further cut by another RNase in Dicer to generate mature miRNA. The mature miRNA with other proteins form RISC (RNA-induced sileneing complex) complex and they lead to the target mRNA degradation or translational repression.MicroRNAs have important tissue-specific functions and play a general regulatory role in the regulation of gene expression. miR-124is a specific expression of the nervous system, and most rich in microRNA. miR-124restrictively expresses in the CNS in the chick embryo ganglion. In mice, the miR-124concentration in the nervous system (cortex, cerebellum and spinal cord) is more than100times that of other organs. However, miR-124expression on the anatomic region of the central nervous system, there are obvious differences in the cerebral cortex. A maximum rate of60.7%in the cerebral cortex, cerebellum, and the spinal cord is35.4%. miR-124expresses in the process of cell proliferation and differentiation, its cellular localization is expressed in the development and maturation of the nervous system neurons. miR-124plays an important role in the regulation of pathological changes of the nervous system. It is reported in the literature, miR-124regulation in tumor tissue is not yet deeply studied, it is only found in the neural tube neural tube cell tumor and pleomorphic malignant glial cell tumor in the regulation of tumor growth. miR-124regulates neuronal cell cycle, cell differentiation, and development of the spinal cord of a series of important physiological activities. As a nervous system-specific microRNA, the regulatory mechanism of pathological damage of the nervous system is not yet clear, especially in the regulation of spinal cord injury it is not reported in the literature. This subject will take advantage of miR-124regulation of NSCs treatment of spinal cord injury.Part I miR-124promotes differentiation of BMSCs-D-NSCsObjective:To use viral transfection technology, miR-124was used to transfect MSCs, and which were induced into NSCs. We observed miR-124role of NSCs in vitro and establish a basis for the further treatment of spinal cord injury.Methods:miR-124sequence5’-UAAGGCACGCGGUGAAUGCC-3’was found in gene bank. The PCR primers were designed:the former primer5’-GCCGATTC (of EcoRI) CATCGCGTTCCCCAAACCCC-3’and antisense strand primer 5’-GCCGGATCC (the BamHI) AGGGATGAAGGTGCTGGCCT, amplified out of miR-124. The synthetic lentiviral the carrier pCDH-CMV-MCS-EF1-copGFP EcoRI/the BamHI digested, and then PCR amplified microRNAs Rno-miR-124, and in the upstream and downstream primers were added to the restriction point EcoRI and BamHI, finally, connect the above two partially digested and transformed into DH5-a. Picked positive clones were identified by PCR and sequencing. The plasmid was used to infect the293T cells. Supernatant was used to infected the MSCs, RT-PCR was performed to detected the expression of miR-124after transfected MSCs which were induced into NSCs. Finally we observed the differentiation of NSCs to into neurons and glial cells.Statistical analysisAll data are shown in mean±SD, the Student t-test to compare the infected group and uninfected group, the differentiation of NSCs, Statistical software using SPSS17.0, P<0.05was considered statistically significant.Results:The transfection rate of miR-124reached up to90%. RT-PCR results showed that after transfection of MSCs of miR-124expression is30-fold prior to transfection. After induced into NSCs, in the transfected group, The proportion of NeuN positive cells was53.60%±3.31%, the proportion of non-transfected group was34.40%±2.88%(p<0.001). In the transfected group, GFAP-positive cells was42.20%±1.62%, and51.90%±3.03%in the non-transfected group (p<0.001). Conclusion:miR-124can promote NSCs to differentiate into neurons in vitro.Part II the role of miR-124-NSC in spinal cord injuryObjective:To administor miR-124NSCs by tail vein injection to treat spinal cord injury and observe the therapeutic effect of miR-124-NSCs in Wistar rats.Methods:According to the modified Allen’s method to make animal models,90adult Wistar rats were taken to Ketamine Hydrochoride (50mg/kg, i.p.) by intraperitoneal injection of anesthesia, fixed prone to T10as the center longitudinal incision to expose the strike zone of the spinal cord. The size was3.0cm x0.4cm. An iron head weighted20g, and3mm in diameter falling from10cm high to strike the exposed spinal cord and the potential was200g.cm. When we observed the rat hind limbs twitched, flicked, and then fully relaxed, the SCI model was regarded as successful. SCI models were divided into three groups; the first group was untreated control group, injected with saline; the second group was normal transplantation group, injected with NSCs, and the third group was transfected group, injected with miR-124-BMSCs-D-NSCs. Take specimens of the three groups in the1day,1week,2weeks and4weeks after spinal cord injury. By HE staining, the cavity volume of transfected group and miR-124free NSCs group was compared with control group. The function recovery of rat spinal cord was assessed by BBB scores.Statistical analysisData were statistically analyzed and presented as Mean±SD. Independent samples t-test was used to assess the damaged cavity volume reduction ratios of the modeled SCI rats in Group2(NSCs graft) and Group3(miR-124-NSCs graft), comparing with Group1(sham control), and also used to evaluate the ratios of differentiation of NSCs and miR-124-NSCs in vivo and in vitro. Differences of BBB scores among the groups were assessed by ANOVA with LSD test. All statistical analyses were two-sided and performed using statistical software (SPSS version17.0; SPSS Inc, Chicago, IL). Differences were considered statistically significant at p<0.05.Results:Identification of donor cells in vivoImmunohistochemical analysis was performed to identify miR-124-NSCs in the lesions. NSCs and miR-124-NSCs that had been intravenously administered were identified14days after transplantation. We found that the miR-124-NSCs labeled with GFP were primarily in and around the damaged sites in the spinal cord (Figure4A). In addition, confocal images showed more neural markers NeuN (Figure4B) and fewer astrocytic markers GFAP (Figure4C) appeared at the14th day after transplantation. The survival ratio of NeuN-positive cells derived from miR-124-NSCs accounted for16.4%±2.1%. The survival ratio of GFAP-positive cells differentiated from miR-124-NSCs was13.5%±4.9%.The assessment of cavity volumeSpinal cords in all groups were stained with HE after SCI (Figure5)(n=30) and sample sections were obtained from the middle of the lesion at the1st,7th,14th and28th day after SCI (Figure5A-D). Necrotic cavity volume in the Group3(miR-124-NSCs graft) was significantly smaller than those in the Group3(NSCs graft) compared with Group1(sham control, saline alone) at the28th day after SCI (p <0.001). The mean reduction ration of cavity volume was3.3%and6.4%in NSCs graft group and miR-124-NSCs graft group, respectively (Figure5E)(p<0.001). The mean reduction of cavity volume was3.3%and6.4%in NSCs group and miR-124-NSCs group, respectively.BBB scoresAll animals had near complete hind limb paraplegia immediately after SCI, and gradually recovered in varying degrees of motor performance over the time course of6weeks (n=90). The first week, the BBB score between the three groups no significant statistical difference (P=0.804). The second week, NSCs transplantation group and control group, the BBB score of NSCs group statistically significant differences (P=0.017), the control group and miR-124transfection group, the BBB score was a significantdifference (P=0.035) and miR-124transfected group had no statistically significant difference (P=0.698). The third week of NSCs transplantation group and normal control group, there was a statistically significant difference (P=0.006), miR-124transfected group and the normal control group, there was a statistically significant difference (P=0.001). For NSCs group and miR-124group there was no statistically significant difference (P=0.290). The fourth week, NSCs transplantation group and normal control group, there was a statistically significant difference (P=0.007), for miR-124transfected group and NSCs transplantation group, there was a statistically significant difference (P=0.002), miR-124transfected group and the normal control group had statistically significant difference (P<0.001). In the fifth week, the pairwise comparisons among the three groups had statistically significant difference (P<0.001). In the sixth week, the pairwise comparisonsamong the three groups had a statistically significant difference (P<0.001).Conclusion:miR-124in the NSCs treatment of spinal cord injury can play a better role.更多还原