摘要
背景与目的
脊髓损伤是一种常见的中枢神经系统损伤,可造成患者的感觉、运动等功能严重障碍,给自身、家庭和国家带来沉重的经济、社会负担。虽然大量基础实验研究对动物脊髓损伤的治疗取得了令人鼓舞的结果,但是临床治疗脊髓损伤的效果始终不令人满意。因此,积极开展脊髓损伤的治疗研究有着重要的科学和社会意义。
随着研究的深入人们认识到轴突脱髓鞘改变是临床和实验脊髓损伤的重要病理变化。外力作用脊髓后通常在软脊膜下存留部分相对正常的白质区域,多种继发性损伤因素的影响进一步造成了残留神经纤维的脱髓鞘改变。研究表明脊髓损伤后轴突脱髓鞘改变与损伤局部少突胶质细胞死亡有着密切的联系。脊髓损伤不仅造成神经元的丢失,同时还导致少突胶质细胞大量的死亡进而引起神经轴突脱髓鞘改变。正常的髓鞘及髓鞘形成细胞对于神经轴突的功能发挥有着重要作用,因而脊髓损伤后少突胶质细胞死亡和轴突脱髓鞘改变进一步影响了残留神经纤维功能的正常发挥。上述研究分析表明,脊髓损后由于继发性损害和少突胶质细胞死亡而造成残留脊髓组织内轴突脱髓鞘改变,影响了存留神经纤维的功能发挥。因此,我们推测改善脊髓损伤后轴突髓鞘化可有助于损伤脊髓神经功能的恢复。
由于少突胶质细胞死亡是造成损伤脊髓轴突脱髓鞘改变的主要原因,因而补充丢失的少突胶质细胞、改善残留神经轴突髓鞘化,有利于脱髓鞘神经轴突的功能恢复、最大限度地发挥残留神经纤维的作用,从而促进损伤脊髓神经功能的恢复。少突胶质前体细胞(Oligodendrocyte Precursor Cells,OPCs)是未成熟的少突胶质细胞,能够在体内增殖、迁移、最后分化为成熟少突胶质细胞并形成髓鞘发挥功能。OPCs作为移植细胞比成熟少突胶质细胞更具有优势,也不像干细胞那样容易受到移植微环境的不利影响。基于以上分析,本实验拟通过OPCs移植治疗改善损伤脊髓轴突髓鞘化,发挥残留神经纤维的功能,进而实现促进脊髓神经功能恢复的目的。通过本课题研究进一步加深对脊髓损伤轴突脱髓鞘改变生物学意义的认识,并为今后脊髓损伤治疗研究提供一种新的思路。
方法
1.取新生48小时Sprague-Dawley大鼠大脑皮层细胞进行OPCs原代培养,通过相差显微镜和扫描电子显微镜连续观察体外条件下OPCs的生长情况。
2.实验采用振荡分离和差速贴壁等方法进一步分离OPCs,通过免疫细胞化学技术对分离后的OPCs进行分析鉴定。
3. OPCs在体外化学条件培养基中继续生长,通过相差显微镜连续观察和免疫细胞化学技术检测研究分析体外条件下OPCs的定向分化规律和分化成熟情况,并采用MTT检测法研究分析OPCs在体外条件下的分裂增殖能力。
4.采用Allen’s打击法制作大鼠脊髓挫裂损伤模型,将实验大鼠分为四组,即OPCs移植治疗组、移植对照组、单纯损伤组及假手术组。在脊髓损伤术后第7天时将体外标记的OPCs移植治疗大鼠脊髓损伤。
5.通过BBB运动功能评分法连续评估脊髓损伤后大鼠后肢运动功能的恢复情况,并采用神经电生理技术检测分析OPCs移植治疗对大鼠损伤脊髓神经传导功能恢复的影响。
6. OPCs移植治疗术后8周时实验观察比较了各组大鼠损伤脊髓组织的大体病理变化情况,研究分析了OPCs移植治疗对大鼠损伤脊髓局部组织内白质存留的影响;实验采用免疫组织化学及免疫荧光技术检测分析了移植后OPCs在体内的存活、分布及分化成熟等情况;通过免疫组织化学、髓鞘染色、RT-PCR及透射电镜技术等方法研究分析了OPCs移植治疗对大鼠损伤脊髓组织内髓鞘含量、髓鞘相关基因表达及轴突髓鞘化超微结构的影响;实验还观察研究了OPCs移植治疗对大鼠损伤脊髓内神经元、神经轴突和胶质反应的影响。
结果
1.原代培养中OPCs大量增殖生长呈典型的前体细胞形态,镜下见其胞体呈近圆形,为折光性强的小亮点,胞体具有双极细胞突起。扫描电子显微镜观察见OPCs紧密贴附在星形胶质细胞表面生长,胞体较小呈近似圆形,直径约6-10μm,胞体周围具有纤细的两极或三极细胞突起。
2.原代培养第9-10天时形成明显的细胞分层,上层为成簇生长的OPCs,下层为融合成片的星形胶质细胞。通过振荡分离和差速贴壁等方法分离获得了大量的OPCs,免疫细胞化学检测结果显示分离后特异性表达PDGFR-α的OPCs比例达95 %。
3. OPCs能够在体外化学条件培养基内继续生长分化,胞体不断增大,细胞突起逐渐增多、增长并形成大量分支。在体外培养5-7天时OPCs逐渐定向分化为成熟的少突胶质细胞,镜下见其胞体呈圆形,胞体周围具有大量网状纤细突起,呈“分枝”状或“蜘蛛网”状分布于胞体四周。免疫细胞化学技术检测分析显示分化成熟的少突胶质细胞特异性表达主要髓鞘结构蛋白MBP。
4.实验研究表明OPCs在体外培养条件下仍保持着良好的分裂增殖能力,MTT检测结果显示观察期内OPCs的细胞数量不断增加(p < 0.05),实验观察初期OPCs的数量最少,在观察后期OPCs的数量逐渐增加,于第6天时细胞数量达到最多。
5. OPCs移植治疗组大鼠后肢运动功能的恢复情况明显好于移植对照组和单纯损伤组。脊髓损伤术后第35、42、49天时OPCs移植治疗组大鼠后肢运动功能的BBB评分情况比移植对照组结果更好(p < 0.05);第56和63天时OPCs移植治疗组大鼠的后肢运动功能BBB评分结果明显好于移植对照组(p < 0.01)。
6.神经电生理检测结果表明OPCs移植治疗组大鼠损伤脊髓神经传导功能的恢复亦明显好于移植对照组和单纯损伤组。移植治疗术后第4周时OPCs移植治疗组大鼠MEPs N1波峰潜时和波幅的结果要优于移植对照组(p < 0.05);移植治疗术后第8周时OPCs移植治疗组大鼠MEPs N1波峰潜时和幅度的恢复情况均显著好于移植对照组(p < 0.01)。移植治疗术后第2和4周时OPCs移植治疗组大鼠SSEPs N1波波幅的恢复情况也较移植对照组更好(p < 0.05);移植治疗术后第8周时OPCs移植治疗组大鼠SSEPs N1波峰潜时和波幅的结果也均明显优于移植对照组(p < 0.01)。
7.移植治疗术后第8周时研究结果显示OPCs移植治疗组大鼠损伤脊髓局部组织内存留的白质较移植对照组和单纯损伤组明显更多(p < 0.05);OPCs移植治疗组大鼠损伤脊髓局部组织内可检测到BrdU阳性的移植细胞存活,移植的OPCs在大鼠损伤脊髓组织内广泛分布并与宿主组织良好整合;移植的OPCs能够在体内继续分化成熟并表达MBP;OPCs移植治疗组大鼠损伤脊髓组织内Oligo2阳性的少突胶质细胞明显多于移植对照组(p < 0.01)。
8. OPCs移植治疗组大鼠损伤脊髓组织内MBP的表达量明显多于移植对照组(p < 0.01),LFB髓鞘染色结果也表明OPCs移植治疗组大鼠脊髓组织内髓鞘的含量亦显著高于移植对照组(p < 0.01);OPCs移植治疗组大鼠损伤脊髓组织内PLP基因的表达水平较移植对照组明显增高(p < 0.01)。
9.透射电镜观察结果显示OPCs移植治疗组大鼠损伤脊髓组织内轴突髓鞘化有明显的改善。移植对照组大鼠损伤脊髓组织内存在大量脱髓鞘、肿胀退变的神经轴突,结构解离、髓鞘崩解。OPCs移植治疗组大鼠脊髓组织内脱髓鞘的退变神经轴突更少,其髓鞘结构亦更接近于正常。
10. OPCs移植治疗组大鼠损伤脊髓组织内β-TubulinШ阳性的神经元和NF200阳性的神经轴突均明显多于移植对照组(p < 0.01)。OPCs移植治疗组大鼠损伤脊髓组织局部内GFAP表达量和星形胶质细胞数量均明显低于移植对照组(p < 0.05)。
结论
1.本实验建立了OPCs体外培养体系,通过适时的原代培养、恰当的振荡分离及差速贴壁等方法处理能够有效地分离获得OPCs。
2.研究结果表明分离后OPCs能够在体外条件下继续存活生长,保持着良好的分裂增殖能力,并且能够在体外定向分化为成熟的少突胶质细胞。
3. OPCs移植治疗能够促进脊髓损伤后大鼠后肢运动功能和脊髓神经传导功能的恢复。
4.移植术后OPCs能够在损伤脊髓内长期存活、分化成熟,与宿主组织良好的整合,并补充损伤脊髓组织内的少突胶质细胞。
5. OPCs移植治疗大鼠脊髓损伤有利于损伤脊髓组织内白质存留,改善损伤脊髓轴突髓鞘化,促进损伤脊髓组织内神经元和轴突存活及减轻胶质反应。
Background and Aims
Spinal cord injury (SCI) is a trauma with high incidence in the central nervous system (CNS), which usually results in severe impairment of sensary, motor and autonomic function below the level of injury. The misfortune brings a lot of trouble and huge burden to the patients themself, their family and the whole society. Although there have been many inspiriting findings from laboratory experiments of animals, none of current treatments or therapies is able to effectively cure SCI in the clinical setting. Thus, the researches to treat SCI are of great scientific and humanity significance.
With the better knowledge of the pathological processes following SCI, the axonal demyelination is recently realized as a common pathological change in the lesioned spinal cord experimentally and clinically. There are usually some mild-impaired white matter area spared across the lesion site, however several secondary injury mechanisms further cause the axonal demyelination of these preserved nerve fibers. The related researches indicate that the demyelination of axons after SCI has a close relationship with the death of oligodendrocytes in the lesion area. Not only die a lot of neurons after SCI, but a number of oligodendrocytes are also impaired and the demyelination of axons are brought about consequently. The normal function of myelin sheaths and myelinating cells is vital to maintain function of axons, thus, the death of oligodendrocytes and axonal demyelination after SCI further hinder the neural function of the spared nerve fibers. From above mentioned findings, it is demonstrated that the loss of oligodendrocytes and the secondary injuries cause the demyelination of spared axon in the lesioned spinal cord, which further hampers the neural function of the preserved nerve fibers. So, we speculate that amelioration of axonal myelination after SCI will be favorable for the functional recovery of the injured spinal cord.
Since the axonal demyelination in the lesion area after SCI is primarily attributed to the death of oligodendrocytes, the supplement of oligodendrocytes and amelioration of the spared axons will maximize the function of the preserved and demyelinated nerve fibers and consequently enhance the functional recovery of injured spinal cord. Oligodendrocyte precursor cells (OPCs) are immature oligodendrocytes which are able to proliferate, migrate, and finally differentiate into mature oligodendrocytes and produce myelin. As immature cells, OPCs have more advantages over mature oligodendrocytes to be a transplant, and OPCs are also less influenced by the hostile microenviroment of the lesion areas as compared with stem cells. In this experiment, OPCs are intendedly transplanted to improve axonal myelination of lesioned cord and maximize the function of spared nerve fibers after SCI, and hence to enhance the functional recovery of injured spinal cord. From this investigation we will better realize the biological significance of axonal demyelination in SCI, and the study will provide a novel strategy for the treatment of SCI.
Methods
1. The cerebral cortices of 48-hour neonate Sprague-Dawle rats were harvested for the OPCs primary culture. The growth of OPCs in vitro was observed consecutively under the contrast phase microscope and scanning electron microscope.
2. After the primary culture in vitro, the OPCs were further dissociated by the shaking process and differential adhesion. Then, the separated OPCs were also identified and analyzed by the immunocytochemical technique.
3. While the OPCs further grew in the chemical defined condition medium, the differentiation and maturation of OPCs in vitro were intensively examined by contrast phase microscopy and immunocytochemistry. Then, the proliferative ability of OPCs in vitro was also investigated with the MTT assay.
4. The contusive injury of spinal cord in a rat model was produced by the means of the weight-drop impact of Allen’s method. The experimental animals were randomly divided into 4 groups, i.e. OPCs transplant group, transplant control group, plain injury group and sham operation group. The transplantation of the labelled OPCs was performed 7 days after injury to treat SCI of rats.
5. The recovery of hindlimb locomotive function of SCI rats was consecutively examined by the BBB open-field locomotion scoring. Furthermore, the recovery of the conductive ability of the injured spinal cord was also assessed electrophysiologically.
6. Eight weeks after the OPCs transplantation, the gross pathological changes of spinal cord tissue was observed and compared in all animals of 4 groups, and the spared white matter in the lesion area was further studied after OPCs transplantation. In this study, the survival, distribution, differentiation and maturation of the transplanted OPCs in vivo were investigated by immunohistochemistry and immunofluorescent technique. The effects of OPCs transplantation on the myelin at injury site, on the experssion of myelin-associated gene and on the ultrastructure of axonal myelination were further investigated by immunohistochemistry, special staining of myelin, RT-PCR and transmission electron microscopy. The influence of OPCs transplantation on the survival of neurons and the axons, and on the glial reaction in the lesion area after SCI were also studied intensively.
Results
1. The OPCs extensively grew and proliferated in the primary culture, showing the typical appearance of precursor cells. Under the observation of microscope, the OPCs were seen with round soma and bipolar processes of their bodies. The observation of scanning electron microscopy shew that OPCs grew close adhesive onto the surface of astrocytes, which had a small and round soma, 6-10μm in diameter, with two or three fine processes around the bodies.
2. Around 9-10 days in vitro, the distinct stratification of glial cells formed in the primary culture. The upper layer consisted of clustered-growing OPCs and the bottom layer was mostly constituted of confluent astrocytes. A large number of OPCs was able to be harvested through the procedures of shaking separation and differential adhesion. The PDGFR-αpositive OPCs accounted for up to 95 % of the isolated cells after the separation process.
3. The OPCs were able to grow and continue to differentiate in the chemical defined condition medium. During the differentiation, the simple morphology of OPCs progressively evolved to more complex forms with profuse outgrowth of elongated processes and extensive secondary branching. After 5-7 days in the condition medium, the OPCs gradually differentiated into mature oligodendrocytes, which were characterized by the complex profile of“ramificated”or“cobweb-like”processes reticulating in their periphery. The differentiation of OPCs was further confirmed by immunocytochemistry with the specific expression of MBP, a marker of mature oligodendrocyte.
4. This study demonstrated that the OPCs were able to still retain the proliferative ability in the culture. The number of OPCs in culture was detected by the MTT assay to constantly increase during the experiment (p < 0.05). At the beginning of the assay, the number of OPCs in culture was least, and later on the number of OPCs progressively increased and reached the peak on the 6th day in vitro.
5. The recovery of hindlimb locomotor activity of the injured rats in the OPCs transplant group was much better than that of the transplant control group and plain injury group. On the 35th, 42nd, and 49th day after SCI, the results of BBB score of the hindlimb locomotion of the OPCs transplant group were better than that of the transplant control group (p < 0.05). On the 56th and 63rd day after SCI, the locomotor function of rat hindlimb in the OPCs transplant group was significantly better than that of the transplant control group (p < 0.01).
6. The outcome of electrophysiological experiments indicated that the recovery of the conductive ability of injured spinal cord in the OPCs transplant group was also better than that of the transplant control group and plain injury group. At the 4th week after the transplantation, the results of the onset latency and amplitude of MEPs N1 wave in the OPCs transplant group were markedly better than that of the transplant control group (p < 0.05). At the 8th week after the transplantation, the recovery of both onset latency and amplitude of MEPs N1 wave in the OPCs transplant group was significantly better than that of the transplant control group (p < 0.01). At the 2nd and 4th weeks after the transplantation, the recovery of the amplitude of SSEPs N1 wave in the OPCs transplant group was better than that of the transplant control group (p < 0.05). At the 8th week after the transplantation, the outcomes of the onset latency and amplitude of SSEPs N1 wave in the OPCs transplant group were markedly better than that of the transplant control group (p < 0.01).
7. At the 8th week after the transplantation, there were more white matter spared at the injury site of the OPCs transplant group as compared with that of the transplant control group and plain injury group (p < 0.05). The BrdU positive implant cells were able to be detected in the tissue of lesioned spinal cord, and the OPCs extensively distributed within the spinal cord and well integrated into the architecture of host tissue. The implanted OPCs continued to differentiate and maturate in vivo and express MBP as well. The number of Oligo-2 positive oligodendrocytes in the lesion area of the OPCs transplant group was much more than that of the transplant control group (p < 0.01).
8. The expression of MBP at the lesioned spinal cord was enhanced in the OPCs transplant group as compared to that of the transplant control group (p < 0.01). The specific staining of myelin by LFB also shew that the amount of myelin in the injured tissue of the OPCs transplant group was much more than that of the transplant control group (p < 0.01). The expression of the PLP gene in the spinal cord of the OPCs transplant group was significantly higher than that of the transplant control group (p < 0.01).
9. The findings of transmission electron microscopy further demonstrated the improved axonal myelination in the lesioned spinal cord of the OPCs transplant group. In the transplant control group, a number of large swollen axons with broken myelin sheath were found across degeneration area. The thick and compact myelin sheaths had split and broke down, and many demyelinated axons were also seen present in the lesion area. Otherwise, the better profile of axonal myelination was seen across white matter area in the OPCs transplant group. The axonal myelination of the white matter was improved after OPCs transplantation. The broken myelin sheath and demyelinated axons were also found less across degenerating white matter area. 10. The moreβ-TubulinШpositive neurons and NF200 positive axons were preserved in the lesioned spinal cord in the OPCs transplant group than that of the transplant control group (p < 0.01). The expression of GFAP and the number of active astrocytes within the injury site of the OPCs transplant group were lower than that of the transplant control group (p < 0.05).
Conclusion
1. The OPCs culture is able to be well established in vitro. Timely primary culture, suitable shaking process and differential adhesion together do efficiently separate the OPCs in the primary culture.
2. The isolated OPCs are found to be able to survive and continue to grow in vitro. These OPCs still retain the proliferative ability and further differentiate and maturate to oligodendrocytes.
3. The transplantation of OPCs enhances the recovery of hindlimb locomotor activity and conductive ability of the lesioned spinal cord after SCI.
4. The implanted OPCs can survive for a long term and differentiate and maturate in vivo. The transplanted cells are well integrated within the host tissue and supplement the oligodendrocytes in the lesioned spinal cord.
5. The transplantation of OPCs is able to benefit the preservation of white matter, to ameliorate the axonal myelination of injured spinal cord, to improve the survival of neurons and axons and reduce the glial reaction as well.
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