神经营养因子-3的改造及其联合神经生物支架用于脊髓损伤修复的研究
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摘要
脊髓损伤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。成人脊髓损伤后,微环境的破坏致使中枢神经再生困难。近几十年来,大量实验证明中枢神经在合适的环境中可以再生,让我们看到了让脊髓损伤截瘫病人站起来的希望。然而,由于脊髓损伤病理改变的复杂性,其治疗效果一直欠佳。新近的观点多认为,联合治疗的方法,如神经营养因子和神经生物材料联合,有望达到更好的疗效。其中,神经营养因子因其可以提供营养和生长引导信号而倍受关注,而脊髓损伤后神经营养因子自身的不足使外源性给药十分必要。然而目前的研究表明,由于外源性给药方式的缺陷(如扩散及损伤区药物有效浓度维持),使得神经营养因子的广泛应用受到了限制。移植分泌营养因子的细胞可能导致免疫排斥反应；病毒载体安全性的问题还未被真正解决；微渗透泵的方法可能导致感染发生。因此,亟需找到一种理想的运载神经营养因子的方法。神经营养因子-3(neurotrohin-3,NT3)是神经营养因子家族中对促进中枢神经再生非常重要的因子,尤其是对中枢神经系统中的重要通路之一——皮质脊髓束的再生起着关键作用。通过组织工程学对NT3进行改造,使之以化学键牢固结合于神经生物材料上,使之在损伤灶区域发挥更有效的作用。本研究将构建带有胶原结合区和层粘连蛋白结合区的NT3,通过酶联免疫吸附试验(Enzyme-linked immunoadsordent assay,ELISA)检验新构建因子的结合能力,通过细胞学方法研究这些重组蛋白的生物学活性。高分子材料——聚乳酸聚乙醇酸共聚物(Poly (lactic-co-glycolic acid), PLGA)和胶原支架是两种广泛应用并且适用于脊髓损伤治疗的生物材料。PLGA是经美国食品和药物管理局认证的生物材料。本研究利用的多通道PLGA神经导管支架可以重建脊髓结构,引导神经生长,从而促进脊髓损伤修复。本课题拟利用这一生物支架与重组NT3联合治疗脊髓损伤,观察其治疗效果。胶原材料可以作为连接损伤脊髓头端和尾端的物理桥梁,也可以成为运输NT3的载体。胶原材料因其容易获取、具有良好的可塑性和生物相容性,而广泛应用于脊髓损伤的修复研究中。本研究在中科院遗传与发育研究所研制的有序胶原材料基础上,以大鼠鼠尾韧带为原材料,制做了一种适用于基础研究的纤维性胶原支架。它机械强度好,保持了天然的纤维排列,可以支持细胞生长,能够定向引导神经迁移；可以结合神经生长因子,能够构建运载药物的良好体系。本课题将利用这一纤维性有序胶原支架与带有胶原结合区的NT3联合治疗脊髓损伤。
第一章：重组NT3及其衍生物的获取和功能分析
目的：利用组织工程学技术重组NT3,通过对NT3的基因改造构建胶原靶向NT3和层粘连蛋白靶向NT3,并通过蛋白表达、复性和纯化获取这三种NT3,为进一步联合治疗脊髓损伤建立基础。
方法：从人脑组织中提取基因组cDNA,利用设计好的NT3成熟肽的上下游引物扩增NT3成熟肽的编码序列nt3,与带有组氨酸亲和标签的质粒pET28a重组成pET28a-nt3。同时,在组氨酸亲和标签和nt3之间连接胶原结合区(collagen binding domain, CBD)和层粘连蛋白结合区(laminin binding domain, LBD),构建pET28a-CBD-nt3和pET28a-LBD-nt3。NT3、胶原靶向NT3和层粘连蛋白靶向NT3在大肠杆菌中诱导表达后,经过菌落收集、超声破碎、洗涤、溶解等多个步骤在包涵体中最后表达。得到的三种因子分别进行蛋白的胶原结合实验和层粘连蛋白结合实验,并分别绘制胶原结合曲线图和层粘连蛋白结合曲线图。从孕15天的胚鼠中获取背根神经节(dorsal root ganglion,DRG),利用NT3能维持神经元存活和促进DRG轴突生长,以DRG和消化后的DRG细胞进行因子的定性和定量活性检测。最后,体外培养DRG细胞,分别检验三种因子在胶原蛋白和层粘连蛋白上的维持神经元存活和促进DRG轴突生长的能力,验证胶原靶向NT3和层粘连蛋白靶向NT3的体外功能活性。
结果：基因序列正确的nt3分别连接胶原结合区和层粘连蛋白结合区,获取重组的nt3并成功构建带有胶原结合区和层粘连蛋白结合区的nt3。蛋白经诱导表达后,在聚丙烯酰胺凝胶电泳胶上大约15.49Kd、17.69Kd和31.88Kd的位置出现三个条带；复性、纯化后亦可见纯度较高的此三种蛋白；Western Blotting结果证明这三种蛋白的确正确表达。由结合曲线图可知,在0-10μM浓度范围内,胶原靶向NT3具有比NT3更高的胶原结合能力(P<0.01)；在0-10μM浓度范围内,层粘连蛋白靶向NT3具有比NT3更高的层粘连蛋白结合能力(P<0.01)。定性和定量活性实验表明,胶原结合的NT3和层粘连蛋白结合的NT3与NT3相比,其活性没有下降。体外功能实验证明,胶原蛋白能结合更多的胶原蛋白靶向NT3,从而发挥更好的维持神经元存活(P<0.05)和促进DRG轴突生长(P<0.01)的功能；层粘连蛋白能结合更多的层粘连蛋白靶向NT3,从而发挥更好的维持神经元存活(P<0.05)和促进DRG轴突生长(P<0.05)的功能。
结论：所用的蛋白表达、复性、纯化方法方便易行。新构建的胶原靶向NT3和层粘连蛋白靶向NT3不仅活性没有下降,而且具有更高的结合能力。这种结合不单是物理方式的结合,更有化学键的结合。体外功能实验证明,胶原蛋白能够结合更多的胶原靶向NT3,层粘连蛋白也能够结合更多的层粘连蛋白靶向NT3,从而发挥更好的生物功能。
第二章：联合运用PLGA和NT3修复脊髓损伤
目的：联合运用新制作的聚乳酸聚乙醇酸共聚物(PLGA)和重组NT3治疗大鼠全横断脊髓损伤,验证这种联合方法的可行性和治疗效果,为今后的治疗策略提供参考。
方法：制做大鼠T8-T10全横断脊髓损伤模型,分组移植PLGA和NT3,术后给予大鼠悉心照料,养育16周后将大鼠灌注处死。整个实验期间,通过Basso-Beattie-Bresnahan (BBB)评分和斜坡实验正确步态百分比的计量,对大鼠运动功能进行监测和比较。灌注处死前18天每组随机选取健康大鼠各5只,在大脑感觉运动皮层(前囟前2mm,中线旁2mm以内)注射顺行追踪剂；灌注处死前10天再随机选取健康大鼠每组各5只,脊髓横断处以下10mm处注射逆行追踪剂；灌注处死前全部大鼠行皮层感觉诱发电位的电生理测试,刺激坐骨神经,在大脑感觉运动皮层记录电反应。将大鼠灌注处死后,分离脊髓组织,冰冻切片。通过神经丝染色和神经胶质原纤维酸性蛋白染色,观察、分析新生神经纤维穿越胶质疤痕和移植物的生长情况,计数神经丝染色阳性面积。利用辣根过氧化物酶显示顺行追踪剂阳性神经纤维,计数其迁移距离和阳性面积。逆行追踪大鼠的脊髓组织切片直接观察、拍照,计数逆行追踪剂阳性纤维的迁移距离和阳性面积。
结果：联合运用PLGA和NT3的治疗组大鼠,BBB评分结果有比较明显的提高,尤其在术后7周内提高更迅速,7周后评分结果进入缓慢甚至平台期。未治疗组、单纯NT3组、单纯PLGA组BBB评分结果在曲线中呈持续缓慢上升趋势。假手术组大鼠术后1周即开始表现出与正常大鼠相似的运动功能,BBB评分为21分。PLGA和NT3联合治疗组的BBB分数在7周、12周、16周显著高于未治疗组(P<0.05)、单纯NT3组(P<0.05)和单纯PLGA组(P<0.05)。术后32天、60天、150天斜坡实验正确步态百分率结果表明,PLGA和NT3联合治疗组爬斜坡的能力好于未治疗组(P<0.05)、单纯NT3组(P<0.05)和单纯PLGA组(P<0.05),且具有显著差异。BBB评分和斜坡实验说明了各组功能重建的结果,而免疫组化、顺行追踪结果、逆行追踪结果等形态学结果则证明了组织和纤维结构生长、连接情况。免疫组化结果还可见,PLGA和NT3联合治疗组的损伤灶胶质疤痕少,神经纤维可以长入移植物；而未治疗组胶质疤痕较多,单纯NT3和单纯PLGA组损伤灶处神经纤维较少；计数神经丝阳性面积,PLGA和NT3联合治疗组的结果显著大于未治疗组(P<0.05)、单纯NT3组(P<0.05)和单纯PLGA组(P<0.05)。顺行和逆行追踪结果表明,PLGA和NT3联合治疗组中,顺行和逆行追踪剂可以穿越移植物迁移,其迁移距离和阳性面积均显著大于未治疗组(P<0.05)、单纯NT3组(P<0.05)和单纯PLGA组(P<0.05)。但电生理结果表明,未治疗组、单纯NT3组、单纯PLGA组及PLGA和NT3联合治疗组的大鼠都未引出皮层感觉诱发电位,暗示再生神经的有限。
结论：PLGA和NT3联合治疗脊髓损伤,不仅能为损伤后的神经生长提供物理支持,更能提供适宜的微环境以促进轴突生长。经过PLGA和NT3的联合治疗,损伤后的大鼠运动功能部分恢复。这种新制的PLGA具有孔隙率高、连结性好的特点,有利于神经营养因子的进入,与神经营养因子发挥协同作用,能够更好地促进脊髓损伤的修复。
第三章：胶原靶向NT3结合有序胶原支架修复脊髓损伤
目的：制作新型的适合脊髓的有序胶原材料,检验此生物支架结合胶原靶向NT3体系发挥的治疗脊髓损伤的作用。有望为以后的治疗脊髓损伤提供新方法,为今后的治疗方案提供指导作用。
方法：制做大鼠T8-T10全横断脊髓损伤模型,分组移植有序胶原支架和胶原靶向NT3,术后给予大鼠悉心照料,养育16周后将大鼠灌注处死。16周内,对大鼠进行运动功能监测,通过BBB评分和斜坡实验错误步态百分比的计量,比较各组运动功能恢复的情况。灌注处死前18天每组随机选取健康大鼠各5只,大脑感觉运动皮层相对固定位置(前囟前2mm,中线旁2mm以内)注射顺行追踪剂。将大鼠灌注处死后,分离脊髓组织,冰冻切片。通过神经丝染色和神经胶质原纤维酸性蛋白染色,观察神经纤维穿越胶质疤痕和移植物的生长情况,计数神经丝染色阳性面积。计数5羟色胺神经纤维染色阳性面积。利用辣根过氧化物酶显示顺行追踪剂阳性的皮质脊髓束纤维,计数损伤灶尾端5mm以内再生皮质脊髓束纤维的阳性面积。
结果：有序胶原支架+胶原靶向NT3治疗的大鼠BBB评分结果在术后16周不断提高,提高幅度和速度均比较明显,与未治疗组(P<0.05)、单纯胶原靶向NT3组(P<0.05)、有序胶原+PBS组(P<0.05)和有序胶原+NT3组(P<0.05)相比有显著性差异；未治疗组BBB评分结果基本无提高；单纯胶原靶向NT3组、有序胶原+PBS组和有序胶原+NT3组的BBB评分结果在曲线中呈持续缓慢上升趋势。假手术组大鼠术后1周即开始表现出与正常大鼠相似的运动功能。术后32天-150天的斜坡实验正确步态百分率结果表明,有序胶原支架+胶原结合NT3治疗组大鼠爬斜坡的能力好于未治疗组(P<0.05)、单纯胶原结合NT3组(P<0.05)、有序胶原+PBS组(P<0.05)和有序胶原+NT3组(P<0.05),且具有显著性差异。BBB评分和斜坡实验表明①单纯有序胶原对治疗脊髓损伤有益②有序胶原+胶原靶向NT3组大鼠功能恢复比有序胶原+NT3组大鼠好,且具有显著性差异,说明胶原靶向NT3能够更好地结合在有序胶原上而发挥更有效的治疗作用。神经丝、顺行追踪标记的皮质脊髓束纤维和5羟色胺能神经纤维的免疫染色及其阳性面积统计结果说明有序胶原能与胶原靶向NT3形成体系,从而更有效的发挥修复脊髓损伤的作用；而且,这些再生的纤维与功能恢复具有一致性,暗示这些再生纤维对促进运动功能恢复的作用。
结论：有序胶原支架和胶原靶向NT3能构成有机的体系,从而有效运载NT3,更有效的发挥NT3治疗脊髓损伤的作用。该治疗体系能更有效地促进运动功能的恢复和神经纤维的再生。总的神经丝、5羟色胺能神经纤维和顺行追踪剂标记的皮质脊髓束的再生与功能恢复有一致的关系。
Spinal cord injury (SCI), one most serious disease in the world, results in axonal degeneration and neuronal necrosis. The microenvironment after SCI in adults is much less receptive to regrowth, thus hindering the regeneration of the damaged nerve. However, accumulating evidence these years has demonstrated that the central nerves in adults can regenerate in a supportive environment following injuries such as SCI. So we are looking forward to the patients, such as Sang Lan, standing up again. Because of the multifaceted nature of SCI, the therapeutic efficacy of current treatment strategies is not so satisfactory till now. It is believed nowadays that combinatorial measures, including neurotrophic factors and biomaterials, have been investigated in potential protocols for SCI repair. Exogenous neuretrophic factor is extensively used as they supply nutrition and guidance cues for axonal regrowth, and application of the neurotrophic factors is required as they are insufficient after SCI. But researches at present indicate that the extensive application of neurotrophic factors is limited. Transplanting genetically-modified cells may result in immunologic rejection. Adenoviral vector may have safety problems. Mini-osmotic pump could cause infection. Therefore, a more feasible and effective way for NT3 delivery is required. NT3, a member of neurotrophin family, is a most crucial factor in promoting central nerve regeneration. It is possible that when NT3 is gene-modified, it may tightly bind to the injury site or certain biomaterial to have more effective therapeutic efficiency. In this study, the NT3, the collagen-binding NT3 and the laminin-biding NT3 are recombinated. After that, the binding assay and the biological activity of DRG cells are conducted. The polymer Poly (lactic-co-glycolic acid) (PLGA) and the collagen scaffold are two suitable bioscaffolds. PLGA is approved by the U.S. Food and Drug Administration. The multi-channel PLGA nerve conduits with porous architectures in this study were recently manufactured and proved useful for SCI reconstruction. We are to use this scaffold and the recombinant NT3 to save SCI. Collagen material could serve as a physical bridge to connect the rostral and caudal of the injured spinal cord and a vehicle to deliver NT3. As collagen could be easily obtained and have good plasticity and biocompatibility, it has been extensively used in SCI repair. We are exploring a kind of fibrous collagen which is fit to the scientific researches according to the previous ordered collagen manufacture. The retained mechanical strength and fiber arrangements make it possible for the cells and axons growing and extending. Furthermore, the neurotrophic factors can bind to the collagen scaffold to construct an ideal system for NT3 delivery. In the present research, the combination of the new fibrous collagen scaffold and the collagen-binding NT3 was used for SCI repair.
Chapter One:The construction and harvest of the recombinant NT3, collagen-targeting NT3 and laminin-targeting NT3 and their function studies
Object:To recombine NT3, reconstruct collagen-targeting NT3 and laminin-targeting NT3 by fusing collagen binding domain and laminin binding domain, respectively. Obtain the three NT3 by protein expression, renaturation and purification for the future studies.
Methods:Obtain the genome cDNA from the human brain tissue, amplify the coded sequence of NT3 mature peptide (nt3) using its upstream and downstream primers, and recombinate pET28a-nt3 with nt3 and his-tagged plasmid pET28a. Construct pET28a-CBD-nt3 and pET28a-LBD-nt3 by adding collagen binding domain (CBD) and laminin binding domain (LBD) to the part between his-tag and nt3, respectively. After the expression of NT3, collagen-targeting NT3 and laminin-targeting NT3 in E.coli, the E.coli was collected, ultrasonicated, washed and dissolved. The three kinds of protein were harvested in the inclusion body, followed by renaturing and purifying. The collagen binding ability and the laminin binding ability was tested, and indicated in the curve diagram. After isolated the rat embryos from E15 rats, the dorsal root ganglion (DRG) was obtained carefully. As NT3 could maintain neuron survival and promote neurite outgrowth of DRG, the bioactivity qualitation and quantitation assays of the three NT3 were tested with DRG and DRG cells. Finally, we investigated the biological activities of the collagen-targeting NT3 and laminin-targeting NT3 on collagen and laminin in vitro, respectively. The surviving neurons and the neurite outgrowth was quantificated.
Results:The collagen binding domain and the laminin binding domain was fused with the correct gene sequence of NT3 (nt3). The recombination of nt3 was obtained, and the nt3 with collagen binding domain or laminin binding domain was constructed. The proteins were expressed at 37℃with IPTG induction. It showed that the three bonds were about 15.49KD,17.69KD和31.88KD. After renaturation and purification, the three purified proteins were in the corresponding place. Western Blotting results furtherly demonstrated the exact proteins. It was showed in the binding ability curve that the collagen binding ability of collagen-targeting NT3 was significantly higher than NT3, and the laminin binding ability of laminin-targeting NT3 was significantly higher than NT3. The bioactivity qualitation and quantitation assays indicated that the biological activities of the collagen-targeting NT3 and laminin-targeting NT3 were not decreased. It was also demonstrated in the functional study in vitro that collagen may bind more collagen-targeting NT3, and laminin may bind more lanminin-targeting NT3. In this way, these two proteins could maintain neuron surviving and promote neurite outgrowth of DRG cells more effectively.
Conclusion:The method of protein expression, renaturation and purification was feasible and convenient. The bioactivity of either reconstructed collagen-targeting NT3 or reconstructed laminin-targeting NT3 was not decreased. However, the binding abilities of the two proteins were significantly higher than that of NT3. The results of the functional study in vitro demonstrated that the collagen-targeting and laminin-targeting NT3 could have more effective biofunctions as they have significantly stronger binding ability to collagen and laminin, respectively.
Chapter Two:The combination of the PLGA and recombinant NT3 on spinal cord injury repair
Object:To repair rats' completely transected spinal cord injury with the combination of the newly manufactured PLGA and the recombinant NT3. To verify the feasibility and the effect of the combinated treatment measures, and provide information for the future treatment strategies.
Methods:Completely transect T8-T10 to make rat spinal cord injury models, and implant the PLGA and NT3 immediately into the injury site. The injured rats were given considered care, and perfused after 16 weeks post surgery. During the whole period, the locomotor functional recovery was monitored and compared through BBB rating scale and % right steps in the grid walk test.18 days before the animals sacrified, every 5 rats in each group were randomly chosen, and microinjected with anterograde tracer into the sensorimotor cortex (within 2mm according to the midline and Bregma).10 days before the animals died, every 5 rats in each group were randomly chosen, and microinjected with retrograde tracer into 10mm below the injury site. The cortical somatosensory evoked potential was tested immediately before the animals perfused, with stimulating the sciatic nerve, and recording in the sensorimotor cortex. After the rats were perfused, the spinal cord was carefully dissected and processed. Through the staining of the neurofilament and the glial fibrillary acidic protein, the neural fiber growing across the glial scar and the transplantation was analysed, and its positive area was quantified. The anterograde tracer labeled axons were visualized with horseradish peroxidase, and its migration distance and % positive area was counted. The spinal cord tissue with retrograde tracing was observed and analysed immediately, following the measurement of the migration distance and % positive area of the retrograde tracer labeling fibers.
Results:In the PLGA+NT3 group of rats, BBB score was greately improved, especially in the first 7 weeks post surgery. During the later period, BBB score was slowly going up or even plateaued. BBB rating scores continued rising gradually in the groups of non-treated, NT3 alone and PLGA alone. Animals in the sham group, scoring 21, had normal performances after 1 week post surgery. At 7th,12th and 16th week after injury, BBB scores of the PLGA+NT3 were significantly higher than those of non-treated, NT3 alone and PLGA alone groups. The % right steps in the grid walk test was measured at 32d,60d,150d, showing that the performance of the PLGA+NT3 animals in the grid walk test was significantly better compared with that of the non-treated, NT3 alone and PLGA alone animals. Functional reconstruction was indicated in the BBB rating scale and the grid walk test, meanwhile, the tissue and fiber structural connection was demonstrated in the morphology of immunohistochemisty, anterograde tracing and retrograde tracing results. The immunohistochemisty results also showed that the scarring was weaker in the PLGA+NT3 group, permiting more neural fibers growing into the transplantation; the scarring was much more intensive in the non-treated group; and there were fewer fibers in the lesion in NT3 alone and PLGA alone groups. The anterograde and retrograde tracing results demonstrated that the tracers could go across the transplantation in PLGA+NT3 group, and there was significance in the migration distance and % positive area between this group and the others. However, CSEP results indicated the limited neural regeneration, as there was no induced evoked potential.
Conclusion:To treat spinal cord injury, the combination of the PLGA and the NT3 could not only supply physical support, but also provide attractive microenvioronment for axonal regrowth. With the combination of PLGA and NT3 treatment, there was partial functional recovery in the injured rats. This manufactured PLGA was characterized as high porosity and good interconnection, thus easy for the neurotrophins entering. Therefore, the PLGA could have synergistic effect with neurotrphins, and enhance spinal cord injury repair.
Chapter Three:The collagen-targeting NT3 promote spinal cord injury repair with binding to collagen scaffolds through the collagen binding domain
Object:To manufacture new suitable ordered collagen scaffolds for spinal cord injury repair. To investigate the effect of the system of the scaffolds binding with collagen-targeting NT3 on spinal cord injury repair. Provide new strategies for spinal cord repair, or supply directions for the future treatment measures.
Methods:Completely transect T8-T10 to make rat spinal cord injury models, and implant the ordered collagen scaffolds and collagen targeting NT3 immediately into the injury site. The injured rats were given considered care, and perfused after 16 weeks post surgery. During the whole period, the locomotor functional recovery was monitored and compared through BBB rating scale and % right steps in the grid walk test.18 days before the animals sacrified, every 5 rats in each group were randomly chosen, and microinjected with anterograde tracer into the sensorimotor cortex (within 2mm according to the midline and Bregma). After the rats were perfused, the spinal cord was carefully dissected and processed. With the staining of the neurofilament and the glial fibrillary acidic protein, the neural fiber growing across the glial scar and the transplantation was analysed, and its positive area was quantified. The % positive area of 5-hydroxytryptamine (5HT) was also measured. The anterograde tracer labelling axons were visualized with horseradish peroxidase, and the positive area within 5mm caudal to the injury was counted.
Results:In the ordered collagen scaffold+collagen targeting NT3 group of animals, BBB rating scores continued rising in the 16 weeks after surgery. The functional recovery was so speedy that there was significance comparing with the transected-only group, collagen-targeting NT3 group, ordered collagen+PBS group and ordered collagen+NT3 group. The BBB scores of the transected animals hardly rised, and those of the collagen-targeting NT3 animals, ordered collagen+PBS animals and ordered collagen+NT3 animals gradually improved. The % right steps in the grid walk test was measured at 32-150d post surgery, showing that the performance of the ordered collagen+collagen targeting NT3 animals in the grid walk test was significantly better compared with that of the transected-only, collagen-targeting NT3 alone, ordered collagen+PBS and ordered collagen+NT3 animals. These two investigations indicated that①ordered collagen benefited spinal cord injury repair②functional recovery in the ordered collagen+collagen-targeting NT3 group of animals was significantly better than that in the collagen+NT3 animals. It indicated that collagen-targeting NT3 could strongly bind to the ordered collagen and have better therapeutical effect. The staining of neurofilament, BDA-labeled fibers and 5HT fibers and their positive area showed that the system of ordered collagen and collagen-targeting NT3 could repair spinal cord injury more effectively. Furthermore, these regenerating axons consisted with the functional recovery, indicated that the regenerating neural fibers could promote locomotor functional recovery.
Conclusions:The system of the ordered collagen scaffold and collagen-targeting NT3 could effectively deliver NT3 to treat spinal cord injury. This therepy could enhance functional recovery and promote neural fiber regenerating. The neurofilament,5HT fiber and BDA-labeled corticospinal tract regeneration was consistent with the functional improvement.
第一章：重组NT3及其衍生物的获取和功能分析
目的：利用组织工程学技术重组NT3,通过对NT3的基因改造构建胶原靶向NT3和层粘连蛋白靶向NT3,并通过蛋白表达、复性和纯化获取这三种NT3,为进一步联合治疗脊髓损伤建立基础。
方法：从人脑组织中提取基因组cDNA,利用设计好的NT3成熟肽的上下游引物扩增NT3成熟肽的编码序列nt3,与带有组氨酸亲和标签的质粒pET28a重组成pET28a-nt3。同时,在组氨酸亲和标签和nt3之间连接胶原结合区(collagen binding domain, CBD)和层粘连蛋白结合区(laminin binding domain, LBD),构建pET28a-CBD-nt3和pET28a-LBD-nt3。NT3、胶原靶向NT3和层粘连蛋白靶向NT3在大肠杆菌中诱导表达后,经过菌落收集、超声破碎、洗涤、溶解等多个步骤在包涵体中最后表达。得到的三种因子分别进行蛋白的胶原结合实验和层粘连蛋白结合实验,并分别绘制胶原结合曲线图和层粘连蛋白结合曲线图。从孕15天的胚鼠中获取背根神经节(dorsal root ganglion,DRG),利用NT3能维持神经元存活和促进DRG轴突生长,以DRG和消化后的DRG细胞进行因子的定性和定量活性检测。最后,体外培养DRG细胞,分别检验三种因子在胶原蛋白和层粘连蛋白上的维持神经元存活和促进DRG轴突生长的能力,验证胶原靶向NT3和层粘连蛋白靶向NT3的体外功能活性。
结果：基因序列正确的nt3分别连接胶原结合区和层粘连蛋白结合区,获取重组的nt3并成功构建带有胶原结合区和层粘连蛋白结合区的nt3。蛋白经诱导表达后,在聚丙烯酰胺凝胶电泳胶上大约15.49Kd、17.69Kd和31.88Kd的位置出现三个条带；复性、纯化后亦可见纯度较高的此三种蛋白；Western Blotting结果证明这三种蛋白的确正确表达。由结合曲线图可知,在0-10μM浓度范围内,胶原靶向NT3具有比NT3更高的胶原结合能力(P<0.01)；在0-10μM浓度范围内,层粘连蛋白靶向NT3具有比NT3更高的层粘连蛋白结合能力(P<0.01)。定性和定量活性实验表明,胶原结合的NT3和层粘连蛋白结合的NT3与NT3相比,其活性没有下降。体外功能实验证明,胶原蛋白能结合更多的胶原蛋白靶向NT3,从而发挥更好的维持神经元存活(P<0.05)和促进DRG轴突生长(P<0.01)的功能；层粘连蛋白能结合更多的层粘连蛋白靶向NT3,从而发挥更好的维持神经元存活(P<0.05)和促进DRG轴突生长(P<0.05)的功能。
结论：所用的蛋白表达、复性、纯化方法方便易行。新构建的胶原靶向NT3和层粘连蛋白靶向NT3不仅活性没有下降,而且具有更高的结合能力。这种结合不单是物理方式的结合,更有化学键的结合。体外功能实验证明,胶原蛋白能够结合更多的胶原靶向NT3,层粘连蛋白也能够结合更多的层粘连蛋白靶向NT3,从而发挥更好的生物功能。
第二章：联合运用PLGA和NT3修复脊髓损伤
目的：联合运用新制作的聚乳酸聚乙醇酸共聚物(PLGA)和重组NT3治疗大鼠全横断脊髓损伤,验证这种联合方法的可行性和治疗效果,为今后的治疗策略提供参考。
方法：制做大鼠T8-T10全横断脊髓损伤模型,分组移植PLGA和NT3,术后给予大鼠悉心照料,养育16周后将大鼠灌注处死。整个实验期间,通过Basso-Beattie-Bresnahan (BBB)评分和斜坡实验正确步态百分比的计量,对大鼠运动功能进行监测和比较。灌注处死前18天每组随机选取健康大鼠各5只,在大脑感觉运动皮层(前囟前2mm,中线旁2mm以内)注射顺行追踪剂；灌注处死前10天再随机选取健康大鼠每组各5只,脊髓横断处以下10mm处注射逆行追踪剂；灌注处死前全部大鼠行皮层感觉诱发电位的电生理测试,刺激坐骨神经,在大脑感觉运动皮层记录电反应。将大鼠灌注处死后,分离脊髓组织,冰冻切片。通过神经丝染色和神经胶质原纤维酸性蛋白染色,观察、分析新生神经纤维穿越胶质疤痕和移植物的生长情况,计数神经丝染色阳性面积。利用辣根过氧化物酶显示顺行追踪剂阳性神经纤维,计数其迁移距离和阳性面积。逆行追踪大鼠的脊髓组织切片直接观察、拍照,计数逆行追踪剂阳性纤维的迁移距离和阳性面积。
结果：联合运用PLGA和NT3的治疗组大鼠,BBB评分结果有比较明显的提高,尤其在术后7周内提高更迅速,7周后评分结果进入缓慢甚至平台期。未治疗组、单纯NT3组、单纯PLGA组BBB评分结果在曲线中呈持续缓慢上升趋势。假手术组大鼠术后1周即开始表现出与正常大鼠相似的运动功能,BBB评分为21分。PLGA和NT3联合治疗组的BBB分数在7周、12周、16周显著高于未治疗组(P<0.05)、单纯NT3组(P<0.05)和单纯PLGA组(P<0.05)。术后32天、60天、150天斜坡实验正确步态百分率结果表明,PLGA和NT3联合治疗组爬斜坡的能力好于未治疗组(P<0.05)、单纯NT3组(P<0.05)和单纯PLGA组(P<0.05),且具有显著差异。BBB评分和斜坡实验说明了各组功能重建的结果,而免疫组化、顺行追踪结果、逆行追踪结果等形态学结果则证明了组织和纤维结构生长、连接情况。免疫组化结果还可见,PLGA和NT3联合治疗组的损伤灶胶质疤痕少,神经纤维可以长入移植物；而未治疗组胶质疤痕较多,单纯NT3和单纯PLGA组损伤灶处神经纤维较少；计数神经丝阳性面积,PLGA和NT3联合治疗组的结果显著大于未治疗组(P<0.05)、单纯NT3组(P<0.05)和单纯PLGA组(P<0.05)。顺行和逆行追踪结果表明,PLGA和NT3联合治疗组中,顺行和逆行追踪剂可以穿越移植物迁移,其迁移距离和阳性面积均显著大于未治疗组(P<0.05)、单纯NT3组(P<0.05)和单纯PLGA组(P<0.05)。但电生理结果表明,未治疗组、单纯NT3组、单纯PLGA组及PLGA和NT3联合治疗组的大鼠都未引出皮层感觉诱发电位,暗示再生神经的有限。
结论：PLGA和NT3联合治疗脊髓损伤,不仅能为损伤后的神经生长提供物理支持,更能提供适宜的微环境以促进轴突生长。经过PLGA和NT3的联合治疗,损伤后的大鼠运动功能部分恢复。这种新制的PLGA具有孔隙率高、连结性好的特点,有利于神经营养因子的进入,与神经营养因子发挥协同作用,能够更好地促进脊髓损伤的修复。
第三章：胶原靶向NT3结合有序胶原支架修复脊髓损伤
目的：制作新型的适合脊髓的有序胶原材料,检验此生物支架结合胶原靶向NT3体系发挥的治疗脊髓损伤的作用。有望为以后的治疗脊髓损伤提供新方法,为今后的治疗方案提供指导作用。
方法：制做大鼠T8-T10全横断脊髓损伤模型,分组移植有序胶原支架和胶原靶向NT3,术后给予大鼠悉心照料,养育16周后将大鼠灌注处死。16周内,对大鼠进行运动功能监测,通过BBB评分和斜坡实验错误步态百分比的计量,比较各组运动功能恢复的情况。灌注处死前18天每组随机选取健康大鼠各5只,大脑感觉运动皮层相对固定位置(前囟前2mm,中线旁2mm以内)注射顺行追踪剂。将大鼠灌注处死后,分离脊髓组织,冰冻切片。通过神经丝染色和神经胶质原纤维酸性蛋白染色,观察神经纤维穿越胶质疤痕和移植物的生长情况,计数神经丝染色阳性面积。计数5羟色胺神经纤维染色阳性面积。利用辣根过氧化物酶显示顺行追踪剂阳性的皮质脊髓束纤维,计数损伤灶尾端5mm以内再生皮质脊髓束纤维的阳性面积。
结果：有序胶原支架+胶原靶向NT3治疗的大鼠BBB评分结果在术后16周不断提高,提高幅度和速度均比较明显,与未治疗组(P<0.05)、单纯胶原靶向NT3组(P<0.05)、有序胶原+PBS组(P<0.05)和有序胶原+NT3组(P<0.05)相比有显著性差异；未治疗组BBB评分结果基本无提高；单纯胶原靶向NT3组、有序胶原+PBS组和有序胶原+NT3组的BBB评分结果在曲线中呈持续缓慢上升趋势。假手术组大鼠术后1周即开始表现出与正常大鼠相似的运动功能。术后32天-150天的斜坡实验正确步态百分率结果表明,有序胶原支架+胶原结合NT3治疗组大鼠爬斜坡的能力好于未治疗组(P<0.05)、单纯胶原结合NT3组(P<0.05)、有序胶原+PBS组(P<0.05)和有序胶原+NT3组(P<0.05),且具有显著性差异。BBB评分和斜坡实验表明①单纯有序胶原对治疗脊髓损伤有益②有序胶原+胶原靶向NT3组大鼠功能恢复比有序胶原+NT3组大鼠好,且具有显著性差异,说明胶原靶向NT3能够更好地结合在有序胶原上而发挥更有效的治疗作用。神经丝、顺行追踪标记的皮质脊髓束纤维和5羟色胺能神经纤维的免疫染色及其阳性面积统计结果说明有序胶原能与胶原靶向NT3形成体系,从而更有效的发挥修复脊髓损伤的作用；而且,这些再生的纤维与功能恢复具有一致性,暗示这些再生纤维对促进运动功能恢复的作用。
结论：有序胶原支架和胶原靶向NT3能构成有机的体系,从而有效运载NT3,更有效的发挥NT3治疗脊髓损伤的作用。该治疗体系能更有效地促进运动功能的恢复和神经纤维的再生。总的神经丝、5羟色胺能神经纤维和顺行追踪剂标记的皮质脊髓束的再生与功能恢复有一致的关系。
Spinal cord injury (SCI), one most serious disease in the world, results in axonal degeneration and neuronal necrosis. The microenvironment after SCI in adults is much less receptive to regrowth, thus hindering the regeneration of the damaged nerve. However, accumulating evidence these years has demonstrated that the central nerves in adults can regenerate in a supportive environment following injuries such as SCI. So we are looking forward to the patients, such as Sang Lan, standing up again. Because of the multifaceted nature of SCI, the therapeutic efficacy of current treatment strategies is not so satisfactory till now. It is believed nowadays that combinatorial measures, including neurotrophic factors and biomaterials, have been investigated in potential protocols for SCI repair. Exogenous neuretrophic factor is extensively used as they supply nutrition and guidance cues for axonal regrowth, and application of the neurotrophic factors is required as they are insufficient after SCI. But researches at present indicate that the extensive application of neurotrophic factors is limited. Transplanting genetically-modified cells may result in immunologic rejection. Adenoviral vector may have safety problems. Mini-osmotic pump could cause infection. Therefore, a more feasible and effective way for NT3 delivery is required. NT3, a member of neurotrophin family, is a most crucial factor in promoting central nerve regeneration. It is possible that when NT3 is gene-modified, it may tightly bind to the injury site or certain biomaterial to have more effective therapeutic efficiency. In this study, the NT3, the collagen-binding NT3 and the laminin-biding NT3 are recombinated. After that, the binding assay and the biological activity of DRG cells are conducted. The polymer Poly (lactic-co-glycolic acid) (PLGA) and the collagen scaffold are two suitable bioscaffolds. PLGA is approved by the U.S. Food and Drug Administration. The multi-channel PLGA nerve conduits with porous architectures in this study were recently manufactured and proved useful for SCI reconstruction. We are to use this scaffold and the recombinant NT3 to save SCI. Collagen material could serve as a physical bridge to connect the rostral and caudal of the injured spinal cord and a vehicle to deliver NT3. As collagen could be easily obtained and have good plasticity and biocompatibility, it has been extensively used in SCI repair. We are exploring a kind of fibrous collagen which is fit to the scientific researches according to the previous ordered collagen manufacture. The retained mechanical strength and fiber arrangements make it possible for the cells and axons growing and extending. Furthermore, the neurotrophic factors can bind to the collagen scaffold to construct an ideal system for NT3 delivery. In the present research, the combination of the new fibrous collagen scaffold and the collagen-binding NT3 was used for SCI repair.
Chapter One:The construction and harvest of the recombinant NT3, collagen-targeting NT3 and laminin-targeting NT3 and their function studies
Object:To recombine NT3, reconstruct collagen-targeting NT3 and laminin-targeting NT3 by fusing collagen binding domain and laminin binding domain, respectively. Obtain the three NT3 by protein expression, renaturation and purification for the future studies.
Methods:Obtain the genome cDNA from the human brain tissue, amplify the coded sequence of NT3 mature peptide (nt3) using its upstream and downstream primers, and recombinate pET28a-nt3 with nt3 and his-tagged plasmid pET28a. Construct pET28a-CBD-nt3 and pET28a-LBD-nt3 by adding collagen binding domain (CBD) and laminin binding domain (LBD) to the part between his-tag and nt3, respectively. After the expression of NT3, collagen-targeting NT3 and laminin-targeting NT3 in E.coli, the E.coli was collected, ultrasonicated, washed and dissolved. The three kinds of protein were harvested in the inclusion body, followed by renaturing and purifying. The collagen binding ability and the laminin binding ability was tested, and indicated in the curve diagram. After isolated the rat embryos from E15 rats, the dorsal root ganglion (DRG) was obtained carefully. As NT3 could maintain neuron survival and promote neurite outgrowth of DRG, the bioactivity qualitation and quantitation assays of the three NT3 were tested with DRG and DRG cells. Finally, we investigated the biological activities of the collagen-targeting NT3 and laminin-targeting NT3 on collagen and laminin in vitro, respectively. The surviving neurons and the neurite outgrowth was quantificated.
Results:The collagen binding domain and the laminin binding domain was fused with the correct gene sequence of NT3 (nt3). The recombination of nt3 was obtained, and the nt3 with collagen binding domain or laminin binding domain was constructed. The proteins were expressed at 37℃with IPTG induction. It showed that the three bonds were about 15.49KD,17.69KD和31.88KD. After renaturation and purification, the three purified proteins were in the corresponding place. Western Blotting results furtherly demonstrated the exact proteins. It was showed in the binding ability curve that the collagen binding ability of collagen-targeting NT3 was significantly higher than NT3, and the laminin binding ability of laminin-targeting NT3 was significantly higher than NT3. The bioactivity qualitation and quantitation assays indicated that the biological activities of the collagen-targeting NT3 and laminin-targeting NT3 were not decreased. It was also demonstrated in the functional study in vitro that collagen may bind more collagen-targeting NT3, and laminin may bind more lanminin-targeting NT3. In this way, these two proteins could maintain neuron surviving and promote neurite outgrowth of DRG cells more effectively.
Conclusion:The method of protein expression, renaturation and purification was feasible and convenient. The bioactivity of either reconstructed collagen-targeting NT3 or reconstructed laminin-targeting NT3 was not decreased. However, the binding abilities of the two proteins were significantly higher than that of NT3. The results of the functional study in vitro demonstrated that the collagen-targeting and laminin-targeting NT3 could have more effective biofunctions as they have significantly stronger binding ability to collagen and laminin, respectively.
Chapter Two:The combination of the PLGA and recombinant NT3 on spinal cord injury repair
Object:To repair rats' completely transected spinal cord injury with the combination of the newly manufactured PLGA and the recombinant NT3. To verify the feasibility and the effect of the combinated treatment measures, and provide information for the future treatment strategies.
Methods:Completely transect T8-T10 to make rat spinal cord injury models, and implant the PLGA and NT3 immediately into the injury site. The injured rats were given considered care, and perfused after 16 weeks post surgery. During the whole period, the locomotor functional recovery was monitored and compared through BBB rating scale and % right steps in the grid walk test.18 days before the animals sacrified, every 5 rats in each group were randomly chosen, and microinjected with anterograde tracer into the sensorimotor cortex (within 2mm according to the midline and Bregma).10 days before the animals died, every 5 rats in each group were randomly chosen, and microinjected with retrograde tracer into 10mm below the injury site. The cortical somatosensory evoked potential was tested immediately before the animals perfused, with stimulating the sciatic nerve, and recording in the sensorimotor cortex. After the rats were perfused, the spinal cord was carefully dissected and processed. Through the staining of the neurofilament and the glial fibrillary acidic protein, the neural fiber growing across the glial scar and the transplantation was analysed, and its positive area was quantified. The anterograde tracer labeled axons were visualized with horseradish peroxidase, and its migration distance and % positive area was counted. The spinal cord tissue with retrograde tracing was observed and analysed immediately, following the measurement of the migration distance and % positive area of the retrograde tracer labeling fibers.
Results:In the PLGA+NT3 group of rats, BBB score was greately improved, especially in the first 7 weeks post surgery. During the later period, BBB score was slowly going up or even plateaued. BBB rating scores continued rising gradually in the groups of non-treated, NT3 alone and PLGA alone. Animals in the sham group, scoring 21, had normal performances after 1 week post surgery. At 7th,12th and 16th week after injury, BBB scores of the PLGA+NT3 were significantly higher than those of non-treated, NT3 alone and PLGA alone groups. The % right steps in the grid walk test was measured at 32d,60d,150d, showing that the performance of the PLGA+NT3 animals in the grid walk test was significantly better compared with that of the non-treated, NT3 alone and PLGA alone animals. Functional reconstruction was indicated in the BBB rating scale and the grid walk test, meanwhile, the tissue and fiber structural connection was demonstrated in the morphology of immunohistochemisty, anterograde tracing and retrograde tracing results. The immunohistochemisty results also showed that the scarring was weaker in the PLGA+NT3 group, permiting more neural fibers growing into the transplantation; the scarring was much more intensive in the non-treated group; and there were fewer fibers in the lesion in NT3 alone and PLGA alone groups. The anterograde and retrograde tracing results demonstrated that the tracers could go across the transplantation in PLGA+NT3 group, and there was significance in the migration distance and % positive area between this group and the others. However, CSEP results indicated the limited neural regeneration, as there was no induced evoked potential.
Conclusion:To treat spinal cord injury, the combination of the PLGA and the NT3 could not only supply physical support, but also provide attractive microenvioronment for axonal regrowth. With the combination of PLGA and NT3 treatment, there was partial functional recovery in the injured rats. This manufactured PLGA was characterized as high porosity and good interconnection, thus easy for the neurotrophins entering. Therefore, the PLGA could have synergistic effect with neurotrphins, and enhance spinal cord injury repair.
Chapter Three:The collagen-targeting NT3 promote spinal cord injury repair with binding to collagen scaffolds through the collagen binding domain
Object:To manufacture new suitable ordered collagen scaffolds for spinal cord injury repair. To investigate the effect of the system of the scaffolds binding with collagen-targeting NT3 on spinal cord injury repair. Provide new strategies for spinal cord repair, or supply directions for the future treatment measures.
Methods:Completely transect T8-T10 to make rat spinal cord injury models, and implant the ordered collagen scaffolds and collagen targeting NT3 immediately into the injury site. The injured rats were given considered care, and perfused after 16 weeks post surgery. During the whole period, the locomotor functional recovery was monitored and compared through BBB rating scale and % right steps in the grid walk test.18 days before the animals sacrified, every 5 rats in each group were randomly chosen, and microinjected with anterograde tracer into the sensorimotor cortex (within 2mm according to the midline and Bregma). After the rats were perfused, the spinal cord was carefully dissected and processed. With the staining of the neurofilament and the glial fibrillary acidic protein, the neural fiber growing across the glial scar and the transplantation was analysed, and its positive area was quantified. The % positive area of 5-hydroxytryptamine (5HT) was also measured. The anterograde tracer labelling axons were visualized with horseradish peroxidase, and the positive area within 5mm caudal to the injury was counted.
Results:In the ordered collagen scaffold+collagen targeting NT3 group of animals, BBB rating scores continued rising in the 16 weeks after surgery. The functional recovery was so speedy that there was significance comparing with the transected-only group, collagen-targeting NT3 group, ordered collagen+PBS group and ordered collagen+NT3 group. The BBB scores of the transected animals hardly rised, and those of the collagen-targeting NT3 animals, ordered collagen+PBS animals and ordered collagen+NT3 animals gradually improved. The % right steps in the grid walk test was measured at 32-150d post surgery, showing that the performance of the ordered collagen+collagen targeting NT3 animals in the grid walk test was significantly better compared with that of the transected-only, collagen-targeting NT3 alone, ordered collagen+PBS and ordered collagen+NT3 animals. These two investigations indicated that①ordered collagen benefited spinal cord injury repair②functional recovery in the ordered collagen+collagen-targeting NT3 group of animals was significantly better than that in the collagen+NT3 animals. It indicated that collagen-targeting NT3 could strongly bind to the ordered collagen and have better therapeutical effect. The staining of neurofilament, BDA-labeled fibers and 5HT fibers and their positive area showed that the system of ordered collagen and collagen-targeting NT3 could repair spinal cord injury more effectively. Furthermore, these regenerating axons consisted with the functional recovery, indicated that the regenerating neural fibers could promote locomotor functional recovery.
Conclusions:The system of the ordered collagen scaffold and collagen-targeting NT3 could effectively deliver NT3 to treat spinal cord injury. This therepy could enhance functional recovery and promote neural fiber regenerating. The neurofilament,5HT fiber and BDA-labeled corticospinal tract regeneration was consistent with the functional improvement.
引文
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