【作者】 王凌云；

【导师】 张卫平； 刘长安；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:通过建立新西兰大白兔陈旧性脊髓损伤模型,并同时抽取其自体骨髓间质干细胞进行体外培养、增殖,然后于伤后2周末移植于脊髓损伤部位并同时联合应用神经生长因子治疗,观察脊髓功能恢复情况,并于移植术后6周对实验动物的肢体肌力、感觉、括约肌功能的恢复情况进行评分,并对最后脊髓组织病理切片进行图像分析,所得数据进行统计学分析,与仅用常规治疗方法的对照组进行对比,评价自体骨髓间质干细胞移植联合神经生长因子对促进造成陈旧性脊髓损伤大白兔的脊髓功能恢复方面的治疗作用。通过该动物实验,为临床治疗陈旧性脊髓损伤积累经验。方法: 1.骨髓间质干细胞的获取和培养:所有骨髓均从成年新西兰大白兔自体髂后上嵴处抽取,为保证细胞浓度,分别从每只新西兰大白兔左右侧髂后上嵴处抽取骨髓,并在抽取骨髓过程中变换穿刺针方向,每侧各抽取1ml,共2ml,进行标记编号。所获骨髓种植于细胞培养板内。培养液为含20%胎牛血清DMEM液。培养箱温度37oC,含5%CO2,100%饱和湿度。细胞传代密度为1×106/cm2。待第三代细胞长满后,进行脊髓移植。2.实验动物分组及处理:36只新西兰大耳白兔随机分成A、B、C3组,A组12只为对照组,造成脊髓损伤模型后仅采用常规治疗;B组12只为自体骨髓间质干细胞移植组。C组12只为自体骨髓间质干细胞移植联合应用神经营养因子治疗组。分别观察各时段实验动物的行为表现,并对移植术后6周的脊髓功能进行评分测定。移植术后6周处死所有实验动物,制作脊髓标本组织切片,进行光镜观察和组织图象分析。结果: 1骨髓间质干细胞生长情况:原代细胞接种24h ,倒置显微镜下观察,培养瓶底开始出现少许贴壁细胞,培养3～4d,可见细胞开始呈集落生长,单个生长的细胞呈短梭形或多角形,随着培养时间延长,贴壁细胞明显增多,集落逐渐增大,出现形态均一、呈放射状排列的细胞集落。培养5～7天后细胞分裂增殖十分明显,伸出长短不一、粗细不均的多种形态的突起,胞核大,核轮廓不清,核仁清晰,可见较多的分裂相。悬浮细胞随着换液次数的增多而逐渐被去除,约7～10天左右,细胞融合成单层,长满整个培养瓶。2.实验动物行为学表现:术后一周实验组与对照组无明显差异,自术后两周开始,B、C组实验动物开始运动增多,双后肢肌力、痛觉及括约肌功能有一定恢复,6周时A组动物双后肢状况无明显改善,而B、C组实验动物的双后肢肌力、痛觉及括约肌功能较前有明显恢复,大部分动物有自主排尿功能,C组较B组更为明显。3.脊髓标本切片观察:见A组镜下可见脊髓损伤后轴突萎缩坏死或凋亡,细胞核固缩、碎裂及溶解,损伤的局部炎性出血反应,形成脊髓空洞、胶质瘢痕增生。而实验组B、C组见有新生神经元及胶质纤维细胞,脊髓空洞数量较A组明显少,坏死及萎缩的神经元细胞也较少,且C组好于B组。结论: 1.骨髓离心培养与贴壁培养法相结合的方法是获得骨髓间质干细胞的良好办法,骨髓间质干细胞具有良好的体外培养、增殖特性。2.在体外培养、增殖后的自体骨髓间质干细胞植入陈旧性损伤的脊髓微环境后,能够在损伤部位存活、迁移、并与脊髓组织整合,根据所处的局部微环境发生分化,先分化成神经元祖细胞和胶质祖细胞,然后再分化成神经元和神经胶质细胞,替换受损的神经元,移植的细胞具有分泌促进轴突再生的因子、促进损伤轴突的再生并与远端轴突建立联系,部分恢复脊髓的传导功能,自体骨髓间质干细胞无免疫排斥问题,具有良好的治疗效果。3.神经生长因子可以保护受损的神经元,促进受损神经元轴突的出芽再生,诱导再生轴突向神经纤维生长,对移植的骨髓间质干细胞的存活和分化具有明显促进作用,而自体骨髓间质干细胞可以在损伤的脊髓局部存活,有效抑制胶质瘢痕的形成,有利于神经纤维的再生和长入,两者具有一定的协同作用,骨髓间质干细胞移植联合应用神经生长因子治疗陈旧性脊髓损伤比单纯自体骨髓间质干细胞移植的治疗效果更为理想。4.骨髓间质干细胞移植可以部分恢复造成陈旧性脊髓损伤的试验动物的功能,联合应用神经生长因子治疗陈旧性脊髓损伤对于试验动物的脊髓功能的改善有更为理想的作用,可以有效地减轻截瘫的程度和改善脊髓功能,但再生的神经细胞尚不能完全替代损伤的神经元恢复脊髓的功能,关于陈旧性脊髓损伤这一课题还有待进一步研究。更多还原

【Abstract】 Objective: Through the establishment of New Zealand white rabbits old spinal cord injury model, and take it’s autologous bone marrow mesenchymal stem cells cultured in vitro, proliferation at the same time, transplant it into spinal cord injuried and applicate of nerve growth factors at the same time at the second weekend after injuried, then observe the experimental animals body strength, feeling sphincter function at the six weekend after transplantation experiment and give score to them, and analysis the final pathology sections of spinal cord tissue image, collect the data of them for statistical analysis, and contrast the results to the conventional treatment methods for the control group, Comparative evaluation the effect of caused the functional recovery of the spinal cord of the oldish spinal cord injury in rabbits through the treatment of the autologous bone marrow mesenchymal stem cells transplantation with the nerve growth factors.Give experience for the clinic treatment of oldish spinal cord injury through the animal tests. Methods: 1.Marrow mesenchymal stem cells’harvesting and cultivation:All are extracted from the bone marrow of adult New Zealand white rabbits’autologous iliac crest, from each side of New Zealand white rabbits’autologous iliac crest respectively in order to guarantee cell concentration, and convert the direction in the process of taking suction marrow mesenchymal stem cells from bone marrow, 1 ml each side of, 2 ml total, and labeled. All received are planted in the bone marrow cell culture plates,Mediums are DMEM containing 20% fetal bovine serum solution. Incubator’s temperature is 37oC, containing 5% CO2, 100% saturation humidity. Passaged cell density is 1×10 6 / cm2. Waiting theyare covered with the third-generation cell, transplantate them to spinal cord. 2. Experimental animals’grouping and handling: 36 New Zealand rabbits were randomly divided into A, B, C three groups medially, Group A for the control group, they are treated only with conventional treatment after they are resulted in spinal cord injury. group B is the autologous bone marrow mesenchymal stem cells transplantation group. Group C is the combined treatment group with autologous bone marrow mesenchymal stem cells transplantation and nerve growth factors. And observe the animal behaviours during experimental, and score them’s function of spinal cord at the six weekend after transplantation. All experimental animals were killed six weeks later after transplantation, making spinal cord tissue specimens, observed by light microscopy and image analysis the tissue of spinal cord.Results: 1 Mesenchymal stem cells’growth etat: It began to cultivate a little bottle adherent cells in inverted microscope observation after primary cells were inoculated in 24h, cultured for 3~4d, they were begin to growth colony, the growth of single cells were short shuttle or polygonal shape, with the extension of time for training, adherent cells markedly increased colony is gradually increasing, a uniform shape, radiation array colony. Cells division and proliferation is very clear, extending vary in length and thickness of the many forms of inequality processes, nuclear, nuclear and ill-defined contours, nucleolus clear after 5 to 7 days, we can see that more of the split. With the cell suspension for an increase in the number of gradually being removed, about 7 to 10 days, a single cell fusion, covered the entire culture bottles. 2. Experimental animal behavior: A week later the experimental group and the control group there was no significant difference, since after two weeks, group B and group C increased movement of animals, double hindlimb muscle strength, pain and sphincter function of a certain recovery, Double-A group, the situation did not improve hind legs at six weeks, but group B and group C double experimental animals hindlimb muscle strength, pain and sphincter function recovery is better than before, the most animals urinary function independently, group C is more obvious than group B. 3. Spinal cord biopsy specimens observed: axonal atrophy necrosis or apoptosis in group A after spinal cord injury in microscope, nuclear condensation, fragmentation and dissolution, damage the local inflammatory response bleeding, formation of syringomyelia, glial scar formation. The experimental group B, group C, see a newborn neurons and glial fibrillary cells, the number of syringomyelia significantly less than that in group A, necrosis and shrinkage of neurons less than hat in group A, and Group C is good in Group B.Conclusions: 1. Centrifugal Culture and bone marrow adherent culture method is the good method to obtainmesenchymal stem cells. The bone marrow mesenchymal stem cells have goog capability to cultured in vitro and proliferation. 2. When Autologous bone marrow mesenchymal stem cells cultured in vitro and proliferated in extraorgan are transplated into the oldish spinal injury micro-environment, they can survive in the damaged areas, migration, and integration with the spinal cord, which according to the local micro-environment division, first differentiated progenitor cells into neurons and glial progenitor cells, and then differentiate into neurons and glial cells to replace damaged neurons, the transplantation of cells secreted factor to promote axonal regeneration, and promote axonal regeneration and injury and distal axon establish contact and the partial resumption of the spinal cord conduction, autologous bone marrow mesenchymal stem cells without immune rejection problems, has good therapeutic effect. 3 .Nerve growth factor can protect damaged neurons damaged neurons promote axonal sprouting regeneration, axon regeneration induced by the growth of nerve fibers, the transplantation of bone marrow mesenchymal stem cell survival and differentiation has obvious role, and Autologous bone marrow mesenchymal stem cells in the spinal cord injury of local survival, effective inhibition of glial scar formation is conducive to nerve fiber regeneration and long all, the two have a certain synergy, mesenchymal stem cell transplantation in the treatment of nerve growth factor old spinal cord injury than simply autologous bone marrow mesenchymal stem cell transplantation in the treatment of more desirable. 4. Mesenchymal stem cell transplantation could be a partial resumption of old spinal cord injury in experimental animals function, the joint application of nerve growth factor in the treatment of chronic spinal cord injury in animal experiments for the improvement of the spinal cord function of a better role to be effective in alleviating paraplegia and improve the level of spinal cord function, but the regeneration of nerve cells is still not completely injury replacement neurons restore the function of the spinal cord, spinal cord injury on the subject of old remains to be further studied.更多还原