【作者】 任长乐；

【导师】 吕德成；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 脊髓损伤(spinal cord injury,SCI)是骨科领域致残率、死亡率较高的创伤之一,是运动意外、交通、事故中常见的损伤。SCI包括原发性损伤和继发性损伤。原发性损伤为不可逆性改变,继发性损伤的病理改变包括轴突断裂、髓鞘崩解以及神经细胞的坏死和凋亡,治疗要素为解除脊髓压迫、重建脊柱的稳定性、避免继发性损伤。一个多世纪以来,很多学者对脊髓损伤的病因、病理、预防、治疗进行了研究,并就其损伤后的生长环境、自身的修复能力、促进再生修复的方法论述了不同的看法和观点。总结如下:(1)移植组织或细胞如神经干细胞、骨髓间充质干细胞、胚胎组织等桥接损伤部位;(2)应用神经营养因子及药物减轻继发性损伤;(3)消除抑制脊髓修复的各种因素,如降解胶质斑痕,消除抑制轴突再生的各种因素因子;(4)使脱髓鞘的轴突重新髓鞘化;(5)研究细胞的转导机制,刺激或调节细胞内生长锥的靶器官途径。而如何桥接脊髓断端并促进神经传导信号通路的重建是脊髓损伤再生修复的重要研究方向。活性巨噬细胞(Activated Macrophages AMPs)来源丰富,取材简便,易分离培养,来源于自身无免疫排斥。将活性巨噬细胞作为移植材料以促进轴突的再生是最近几年对炎症机制中巨噬细胞重新认识的结果,外源活性巨噬细胞聚集可改善局部微环境,减轻脊髓继发性损伤。我们将活性巨噬细胞移植到损伤大鼠脊髓的部位,观察损伤脊髓的功能恢复情况。实验研究证明,活性巨噬细胞对脊髓感觉及运动神经的损伤有一定的恢复作用。活性巨噬细胞在宿主脊髓中存活并促进脊髓功能恢复,其可能的机制有:(1)通过细胞移植有效的减少脊髓损伤后断端胶质瘢痕的形成。(2)清除损伤局部坏死的细胞及组织,清除轴突再生抑制物质。(3)通过炎症机制保护、恢复部分损伤的组织。(4)刺激和分泌细胞再生所需的营养物质,并调控轴突再生所需的细胞底物和蛋白酶。骨髓间充质干细胞(mesenchymal stem cells,MSCs)是成年动物或人骨髓中的非造血组织干细胞,具有多能干细胞的特点,能分化成多种类型的细胞,实验研究证实,MSCs具有诱导分化为神经细胞的潜能并且能向神经干细胞一样在中枢神经组织里迁移和整合,能分泌神经营养因子、细胞因子和其他生物活性因子,为治疗脊髓损伤展示了一种全新和理想的方法,将MSCs移植入大鼠脊髓损伤部位,显示MSCs可以存活,并能很好地整合到宿主组织中,并在没有基因修饰时也能促进轴突的生长。MSCs较其他移植细胞具有来源广泛、取材容易、在体外长期培养过程中始终保持其多向分化潜能、体内移植反应弱、克服了有关伦理学及免疫排斥反应问题等优点,因此探索MSCs移植治疗SCI具有重要的临床意义。研究已发现由氧自由基介导的脂质过氧化反应对脊髓继发性损伤起主要作用。脂质过氧化损伤的程度对移植细胞的命运影响也很大。祖国医学将SCI所致的截瘫,归属于体惰和痿证的范畴,治疗上以活血化瘀、疏通督脉、通经活络、益气补血、补肾填精为主。肾主骨生精髓,精髓伤则肾伤,精血互生,肾所生精髓需靠后天之本、气血化生之源的脾胃源源不断地充盈。滋补脾阴方药(NPR nourishing piyin remedy)具有益气补血、补肾填精、充盈脾胃等功效,可以改善脊髓细胞线粒体ATP酶活性,缓解脂质过氧化反应;并能通过调节磷脂代谢,增强线粒体膜抗氧化能力以保护脑神经细胞。本实验探讨了滋补脾阴方药含药血清对MSCs细胞脂质过氧化损伤的保护作用及机制,旨在探索联合应用含药血清和MSCs细胞移植对脊髓损伤的修复作用。实验一活性巨噬细胞移植促进脊髓损伤后功能恢复的实验研究目的:探讨活性巨噬细胞移植对脊髓损伤修复的促进作用。方法:用改良Allen法制成大鼠脊髓损伤动物模型,随机分成对照组(A组),脊髓损伤后脊髓内微量注射生理盐水溶液20μl。活性巨噬细胞移植组(B组),脊髓损伤后脊髓内注射活性巨噬细胞悬液20μl。移植后7、14、28d采用斜板实验、脊髓运动功能BBB评分法(Basso,Beattie and Bresnahan locomotor rating scale,BBB scale)观察大鼠运动功能恢复情况,脊髓诱发电位的检测观察神经功能恢复,HE染色观察脊髓损伤处空洞面积的改变情况,免疫组化法观察移植的活性巨噬细胞的存活情况及损伤部位神经纤维的再生情况。结果:术后28d,两组斜板倾斜角度差异有统计学意义(A组44.96°±5.70°,B组52.72°±6.51°,p<0.05)。术后28d,两组BBB评分差异有统计学意义(A组6.8±1.2,B组11.8±2.2,p<0.05)。术后28d,两组MEP潜伏期差异有统计学意义〔A组( 4.69±0.47 ) ms , B组(3.62±1.29)ms, p<0.05〕。两组SEP潜伏期差异有统计学意义〔A组(4.19±1.97)ms,B组(2.01±0.55)ms,p<0.05〕。两组神经轴突计数差异有统计学意义〔A组(32.8±6.1)条/mm2,B组(40.8±9.0)条/mm2,p<0.05〕。实验组可见损伤区巨噬细胞存在,脊髓损伤处的空洞面积明显减小,明显的神经纤维再生。结论:活性巨噬细胞可在脊髓损伤处存活并减轻脊髓损伤,能够减小脊髓损伤处的空洞面积,促进受损轴突的再生和运动功能的恢复。实验二骨髓间充质干细胞移植对大鼠脊髓损伤后功能恢复的影响目的:观察骨髓间充质干细胞(MSCs)移植对脊髓损伤修复的促进作用。方法:用改良Allen法制成大鼠脊髓损伤动物模型。随机分成对照组(A组),脊髓损伤9d后脊髓内微量注射生理盐水溶液5μl;骨髓间充质干细胞移植组(B组),脊髓损伤9d后脊髓内注射骨髓间充质干细胞悬液5μl。移植后7、14、28d采用斜板实验、脊髓运动功能BBB评分法(Basso,Beattie and Bresnahan)观察大鼠运动功能恢复情况,脊髓诱发电位的检测观察神经功能恢复,HE染色观察脊髓损伤处空洞面积的改变情况,免疫组化法观察移植的骨髓间充质干细胞的存活及分化情况,损伤部位神经纤维的再生情况。结果:移植后28d,两组斜板倾斜角度差异有统计学意义〔A组(44.96±5.70)°,B组(53.19±6.51)°,p<0.05〕;两组BBB评分差异有统计学意义〔A组(6.8±1.2),B组(10.1±3.5),p<0.05〕。同时,两组MEP潜伏期差异有统计学意义〔A组( 4.69±0.47) ms, B组(3.97±0.83)ms,p<0.05〕,两组SEP潜伏期差异有统计学意义〔A组(4.19±1.97)ms,B组(2.60±0.92)ms,p<0.05〕。两组神经轴突计数差异有统计学意义〔A组(32.8±6.1)条/mm2,B组(39.0±4.6)条/mm2,p<0.05〕。实验组可见明显星形胶质细胞和神经纤维再生,脊髓损伤处的空洞面积明显减小。结论:骨髓间充质干细胞可在脊髓损伤处分化为神经元和神经胶质细胞,能够减小脊髓损伤处的空洞面积,促进受损轴突的再生和运动功能的恢复。实验三滋补脾阴方药联合骨髓间充质干细胞移植对脊髓损伤影响的实验研究目的:探讨脊髓损伤后即刻移植骨髓间充质干细胞对功能恢复的影响,及滋补脾阴方药含药血清对移植细胞在体内存活、迁移和分化的影响。方法:成年雌性SD大鼠90只,随机分成A组(对照组)、B组(骨髓间充质干细胞移植组)、C组(骨髓间充质干细胞与空白血清共移植组)、D组(骨髓间充质干细胞与中药血清共移植组),利用自制电控打击器制作动物模型。移植后7、14、28d采用斜板实验、脊髓运动功能BBB评分法(Basso,Beattie and Bresnahan)观察大鼠运动功能恢复情况,脊髓诱发电位的检测观察神经功能恢复,HE染色观察脊髓损伤处空洞面积的改变情况,免疫组化法观察移植的骨髓间充质干细胞的存活及分化情况,损伤部位神经纤维的再生情况。结果:移植后28d,D组斜板倾斜角度与A、B、C组差异有统计学意义〔A组(44.96±5.69)°,B组(48.11±5.47)°,C组(49.59±5.76)°,D组(55.75±4.14)°,p<0.05〕; BBB评分各组的差异均有统计学意义〔A组(6.9±1.3),B组(8.5±1.1),C组(10.7±0.9),D组(12.3±10.6),p<0.05〕。同时,D组MEP潜伏期与A、B、C组差异有统计学意义〔A组(4.57±0.33)ms,B组(4.49±0.47)ms,C组(4.39±0.65)ms,D组(3.77±0.76)ms,p<0.05〕,D组SEP潜伏期与A、B、C组差异有统计学意义〔A组(4.19±0.93)ms,B组(3.71±1.15)ms,C组(3.63±0.77)ms,D组(2.52±0.57)ms,p<0.05〕。D组神经轴突计数与A、B、C组差异有统计学意义〔A组(32.8±6.21)条/mm2,B组(34.5±5.5)条/mm2,C组(36.2±5.4)条/mm2,D组(40.9±3.1)条/mm2,p<0.05〕。D组28d可见脊髓损伤处的空腔大部分被充填,神经纤维和胶质细胞再生。结论:骨髓间充质干细胞移植后,可以在体内存活、迁移并分化成熟,促进大鼠脊髓损伤后神经纤维的修复与再生。滋补脾阴方药含药血清可以改善损伤脊髓内环境,有利于移植细胞的存活、迁移及分化。二者联合应用具有协同效应。综上所述,本实验通过对滋补脾阴方药保护骨髓间充质干细胞移植治疗大鼠脊髓损伤的研究,获得了以下成果:1.活性巨噬细胞来源丰富,取材简便,易分离培养,大量巨噬细胞聚集可改善局部微环境,减轻脊髓继发性损伤。2.骨髓间充质干细胞在脊髓损伤后移植,可以在体内存活、迁移并分化为成熟的神经元、星形胶质细胞及少突胶质细胞,从而促进大鼠脊髓损伤后神经纤维的修复与再生;是治疗脊髓损伤较有前景的细胞移植物。3.滋补脾阴方药含药血清可以改善损伤脊髓的内环境,减轻移植细胞的过氧化损伤反应,有利于脊髓损伤后即刻移植的骨髓间充质干细胞在体内的存活、迁移和分化。滋补脾阴方药含药血清与骨髓间充质干细胞移植联合应用治疗脊髓损伤,具有协同效应。4.如何进一步增强滋补脾阴方药含药血清的抗氧化损伤作用,还有待今后的实验研究与完善。骨髓间充质干细胞与组织工程支架的联合应用,与基因转染技术的联合应用方面还有广阔的研究空间,待广大学者研究探讨。更多还原

【Abstract】 Spinal cord injury (SCI) is a common injury during traffic, working and sports accident with high rate of disability and mortality. SCI contains primary injury and secondary injury. Primary injury is inconvertible. Pathological changes of secondary injury contain axonotmesis, myelin disintegration, neurons necrosis and apoptosis. The main clinical treatments includes reconstruct spinal stability, solve spinal compression symptom and control secondary injury. In recent years, domestic and foreign scholars research on the treatment of spinal cord injury in a wide range, elaborate a series of views on its own renewable capacity, the growth of the environment as well as the factors which promote regeneration. To sum up, including: Applicant drugs and neurotrophic factors to control secondary injury; (2) transplant neural stem cells, olfactory ensheathing cells, spinal stromal cells, embryonic tissue or gene cells of regulation functions, and bridge connection damage site; (3) study cell signal transduction mecha- nisms, stimulate or regulate signaling pathways of cell growth cone; (4) eliminate inhibition in spinal cord regeneration in a variety of factors, such as the degradation of glial mark, the elimination of inhibition of axon regeneration in a variety of factors; (5) demyelizate the axons re- myelination. And how to bridge connection spinal cord stump and to promote the reconstruction of nerve conduction pathway is an important research direction. Activated Macrophages Transplantation (AMT) is in rich sources, based on simple materials, easy isolation and culture. Local micro- environment can be improved to reduce secondary spinal cord injury by gathered a large number of AMT. We transferred AMT to rats’site of spinal cord injury to observe the function of recovery. In recent years, people’s awareness of macrophages is macrophages as a transplantation material to promote regeneration of axons. Experimental studies have proved that the AMT have a certain role in the recovery of the spinal cord sensory and motor nerve damage. AMT is survived in the host spinal cord and promoted functional recovery of spinal cord, and its possible mechanisms: (1) protect and restore some of the organizations injury; (2) removal partial damage of the cell and tissue necrosis of the wreckage, in particular, inhibit axon regeneration material– sphingomyelin derived renewable inhibitory factor; (3) stimulate and secret nutrient requirements to cells regeneration, and regulate cells substrate and protease requirements to axon regeneration; (4) reduce stump after spinal cord injury in the formation of glial scar effectively.Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells in the adult animal or human bone marrow with the characteristics of multi-potent stem cells can differentiate into multiple types of cells. Experimental research has shown that, MSCs have differentiated into neural cells induced by the potential, and the same as neural stem cells to migration and integration in the central nervous system (brain and spinal cord), can secrete neurotrophic factors, cytokines and other biologically active factor, and demonstrate a new and ideal method for the treatment of spinal cord injury. MSCs transplanted into the rat spinal cord injury site, showing MSCs can survive, and well integrated into the host tissue, and also can promote the growth of axons in the absence of gene-modified. Compared to other transplanted cells, MSCs have a wide range of sources, based on simple materials, have always been to maintain its multi- directional differentiation potential in long-term culture in vitro process, have weak response in vivo transplantation, overcome ethics and the problem of immune rejection, etc., so explore the MSCs transplantation in the treatment of SCI have important clinical significance. Some studies had shown that lipid peroxidation mediated by oxygen free radical played a major role on the secondary spinal cord injury. The extent of lipid peroxidation also affects the fate of engrafted cells in great part. According to the theory of traditional Chinese medicine, the para- plegia caused by SCI belongs to the field of body laziness and paralysis. Its treatment is mainly depended on activating blood circulation, removing blood stasis, deoppilating du channel, dredging meridian, tonifying pneuma, invigorating kidney and replenishing essence. The kidney in charge of bones produces essence depending on the incessant supplying of spleen and stomach, which is the foundation of acquiring nutriments and the source of metaplasia between pneuma and blood. Renal injury can make essence lost. Blood and essence may transform each other. Nourishing piyin remedy (NPR) has the function of tonifying pneuma, activating blood, invigorating kidney, replenishing essence, engorging spleen and stomach and so on. It can protect neurocyte by enhancing antioxygenic activity of the mitoc- hondria, accommodating phospholipid metabolism, inhibiting responsive gliosis and inflammatory reaction. The present study investigated the protective role and mechanism of serum nourishing piyin remedy on MSC slipid peroxidation injury.Part 1 Experimental Study of Function Recovery of Rat Spinal Cord Injuries Using Activated Macrophages TransplantationObjective To investigate the promoting effects of activated macro- phages transplantation on function recovery of rat spinal cord injuries (SCI).Methods The spinal cord of adult rats was injured with modified Allen method, and the rats were randomly divided into 2 groups. In one group (A group), slight saline solution (20μl) was injected into the spinal cord injury site, while in the other group (B group) activated macrophages (20μl) were transplanted instead. The inclined plane test and Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) were used to observe the recovery of rats’behavioral function on 7, 14 and 28 days after the transplantation, while spinal evoked potential tests were used to observe the recovery of spinal cord nervousness function, hematoxylin- eosin (HE) staining to reflect the change of cavity volume, and immunohistochemical method to observe the survival of activated macro- phages transplantation and regeneration of damaged nerve fibers.Results On the 28th day after transplantation, inclined plane critical point was significantly different between group A and B, which were 44.96°±5.70°and 52.72°±6.51°respectively (p<0.05). On the 28th day after transplantation, BBB scale had significant difference between group A and group B, which were 6.8±1.2 and 10.2±4.1 (p<0.05). On the 28th day after transplantation, MEP and SEP latencies had significant differences. The MEP latencies of group A and group B were 4.69±0.47 (ms) and 3.62±1.29, respectively (p<0.05) and the SEP latencies of group A and group B were 4.19±1.97 and 2.51±0.76 respectively (p<0.05). On the 28th day after transplantation, nerves axons count (strip/mm2) was significantly different between group A and group B, which were 32.8±6.1 and 40.8±9.0 respe- ctively (p<0.05). In B group, macrophages survived in the injury area, cavity volume of the injured site was significantly reduced and there was significant regeneration of nerve fiber.Conclusions Activated macrophages can survive in the injury site to relieve spinal cord injuries, reduce cavity volume and promote axonal regeneration and improve functional recovery.Part 2 The effects of mesenchymal stem cells transplantation on the function recovery of rat with spinal cord injuriesObjective To investigate the promoting effects of mesenchymal stem cells (MSCs) transplantation on function recovery of rat spinal cord injuries (SCI).Methods The spinal cord of adult rats was injured with modified Allen method, and the rats were randomly divided into 2 groups:the control group(group A) on the 9th day after SCI, were injected into the spinal cord injury site with slight saline solution (5μl), while the experimental group (group B) were injected into the spinal cord injury site with mesenchymal stem cells (MSCs) (5μl). The inclined plane test and Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) were used to observe the recovery of rats’behavioral function on 7th, 14th and 28th days after the transplantation, spinal evoked potential tests were used to observe the recovery of spinal cord nervousness function, hematoxylin-eosin (HE) staining to reflect the change of cavity volume, and immunohistochemical method to observe the survival of mesenchymal stem cells ( MSCs ) transplantation and regeneration of damaged nerve fibers.Results On the 28th day after transplantation,inclined plane critical point was significantly different between group A and B, which were 44.96°±5.70°and 53.19°±6.51°respectively (p<0.05). On the 28th day after transplantation, BBB scale had significant difference between group A and group B, which were 6.8±1.2 and 10.1±3.5 (p<0.05). On the 28th day after transplantation, MEP and SEP latencies had significant differences. The MEP latencies of group A and group B were 4.69±0.47 (ms) and 3.97±0.83, respectively (p<0.05) and the SEP latencies of group A and group B were 4.19±1.97 and 2.60±0.92 respectively (p<0.05). On the 28th day after transplantation, nerves axons count (strip/mm2) was significantly different between group A and group B, which were 32.8±6.1 and 39.0±4.6. respectively (p<0.05). In B group, there was significant regeneration astrocytes and nerve fiber,cavity volume of the injured site was significantly reduced.Conclusions Mesenchymal stem cells(MSCs) can differentiate into neurons and astrocytes, reduce cavity volume and promote axonal regeneration and improve functional recovery.Part 3 Experimental Study of Nourishing Piyin Remedy (NPR) to Protect Mesenchymal Stem Cells (MSCs) Transplantation in the Treatment of Rats with Spinal Cord InjuryObjective To investigate the promoting effects of mesenchymal stem cells (MSCs) transplantationon function recovery of rat spinal cord injuries (SCI), and the protective effectiveness of serum nourishing piyin remedy on the survival, migration and differentiation of transplanted cells in vivo.Method Ninety adult female SD rats were randomly divided into fore groups as Group A (control group), Group B (MSCs transplantationon group), Group C (MSCs engrafted group combined with blank serum) and Group D (MSCs nourishing piyin remedy serum). Injury models were established by self-designed electronic impact device. The inclined plane test and Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) were used to observe the recovery of rats’behavioral function on 7, 14 and 28 days after the transplantation, while spinal evoked potential tests were used to observe the recovery of spinal cord nervousness function, hematoxylin-eosin (HE) staining to reflect the change of cavity volume, and immunohistochemical method to observe the survival and differentiation of MSCs and regeneration of damaged nerve fibers. Results On the 28th day after transplantation, inclined plane critical point was significantly different among group A, B, C and D, which were 44.96°±5.69°, 48.11°±5.47°, 49.59°±5.76°and 55.75°±4.14°respectively (p<0.05); BBB scale had significant difference among groups, which were 6.9±1.3, 8.5±1.1 10.7±0.9 and 12.3±10.6 respectively (p<0.05). At the same time, MEP latencies had significant differences among group A, B, C and D, which were 4.57±0.33 (ms), 4.49±0.47 (ms), 4.39±0.65 (ms) and 3.77±0.76 (ms) respectively (p<0.05), SEP latencies had significant differences among group A, B, C and D, which were 4.19±0.93 (ms), 3.71±1.15 (ms), 3.63±0.77 (ms) and 2.52±0.57 (ms) respectively (p<0.05). Nerves axons count (strip/mm2) was significantly different among group A, B, C and D, which were 32.8±6.21, 34.5±5.5, 36.2±5.4 and 40.9±3.1 respectively (p<0.05). On the 28th day after transplantation, most cavity volume of the injured site was filled; tastrocytes and nerve fiber here were regenerated in group D.Conclusion The MSCs can survive, migrate and differentiate into mature phenotypes after transplantation, improve repair and regeneration of rat nerve fibers after SCI. The serum NPR can benefit the survival, migration and differentiation of transplanted MSCs by ameliorating the microenvironment of injured spinal cord. Transplantation with MSCs could facilitate recovery and reorganization of nerve fiber in moderate SCI models, and reveal synergistic effect when used combined with serum NPR.To sum up, through nourishing piyin remedy (NPR) to protect mesenchymal stem cells (MSCs) transplantation in the treatment of rats with spinal cord injury researched by this experiment, obtain the following results:1. Activated Macrophages Transplantation (AMT) is in rich sources, based on simple materials, easy isolation and culture. Local micro-environment can be improved to reduce secondary spinal cord injury by gathered a large number of AMT.2. The MSCs could retain survival, migrate and differentiate into mature phenotypes-neuron, astrocyte and oligodentrocyte, after trans- plantation into the moderate contused spinal cord, thus, facilitate the recovery and regeneration of nerve fiber.3. The serum nourishing piyin remedy could improve the SCI microenvironment, decrease the lipid peroxidation injury, benefit the survival, migration and differentiation of transplanted MSCs, and demo- nstrate synergistic effect when used in combination.4. How to enhance the antioxygenic activity of nourishing piyin remedy, still need to be further investigated. There are many prospective aspects for the transplantation of MSCs combined with tissue engineering scaffold and gene transfection technique.更多还原