【作者】 张秀英；

【导师】 刘永茂；

【作者基本信息】 吉林大学 ， 免疫学， 2007， 硕士

【摘要】 研究表明脊髓横断性损伤的组织学特点为从两断端中心灰质到离开断端的灰质发生进行性出血性坏死。脊髓横断后断端坏死,胶质代替是脊髓神经再生的主要障碍之一。因此,去除脊髓断端坏死瘢痕区,代之以神经纤维生长的支持结构,即提供脊髓内源性纤维再生的环境,成为修复脊髓的基本条件。多年的研究表明,脊髓损伤后仍存在一定的再生能力,亦发现脊髓移植对切断轴突的神经元有保护作用。脊髓损伤的移植治疗要求移植体在术后要有较丰富的血供,并逐步在移植体内外形成血管网络,才更有利于种子细胞的迁入及功能活性的保持;同时为再生轴索的生长提供了充足的营养,为其生长发育奠定了良好的物质基础。为了能够使损伤的脊髓得到修复,并使移植的器官和细胞得到存活,移植物的血管化成了目前研究的一个焦点。本文以脊髓半切损伤模型鼠为研究对象,对肌基膜管移植入脊髓后的血管化进行了研究。一、肌基膜管的制作顺椎旁肌纤维方向切取椎旁肌2 cm×1 cm×2 cm大小。按下述程序处理:在质量浓度为30 g/ L的NaCl中浸泡10 min ,放入液氮至温度平衡;在蒸馏水中浸泡融化复温,再放入液氮中至温度平衡后取出,在等渗盐水中融化复温,反复三次。轻轻挤压肌条,漂洗,制备成肌基膜管。二、模型的制作和数据的统计自制大鼠脊髓半切损伤动物模型,肌基膜管做支架,酶组织化学技术及血管面积图像分析,检测不加任何其他治疗措施肌基膜管支架在脊髓损伤模型的血管生成情况。本实验将肌基膜管移植到大鼠脊髓半切损伤模型中,术后3天、5天、7天、14天、28天取材,利用酶组织化学技术检测支架中血管的生成情况,并观察支架炎症反应情况。利用SPSS统计软件对实验数据进行单因素方差分析。三、结果对术后3天、5天、7天、14天、28天的模型进行取材,通过酶组织化学技术对血管进行染色并进行HE常规染色,经过Motic图象分析系统分析和SPSS统计软件进行统计分析。结果表明:血管生成状况随着时间的延长有着明显的增加,5天和3天相比具有明显的差异P<0.05,7天和5天相比具有非常明显的差异P<0.01,14天和7天相比也具有非常明显的差异P<0.01。28天和14天相比具有明显的差异P<0.05。综上,通过对植入大鼠脊髓的肌基膜管的血管生成的观察,证明肌基膜管可成功的血管化,为肌基膜管应用于脊髓损伤的治疗提供了理论和实验依据。更多还原

【Abstract】 It has been showed that the histological characteristic of transected spinal cord is progressive hemorrhagic necrotizing in the gray matter from the cutting end to distal part. Glial scar is the main obstacle for axonal regeneration in the transected spinal cord after the necrosis of cutting end. As a result, a supporting structure for axon regeneration is prerequisite to replace the glial scar area and to provide a regenerative environment for endogenic nerve fiber in injured spinal cord. The studies have proved that the injured spinal cord has the capability of regeneration. It has also been showed that the transplantation of the scafford into the spinal cord has protective effect on the neurons with transected axon. This require a rich blood supply to the graft after the transplantation. The network of blood vessels in the interior and outerior of graft is propitious to the migration of the seed cells and the sustainance of their activity. It also provide enough nourishment for the growth of regenerating axons, which provide a good material foundation for the axons’growth. For the repair of injured spinal cord, the revascularization of the graft is a focus of the current research works. This thesis is try to figure out the circumstances of the revascularization of muscle basal lamina transplanted into semi-transected spinal cord. In this study, the rat spinal cord is semi-transected to manufacture the rat spinal cord injury model. Muscle basal lamina is produced by frozen-thawed process. Enzyme histochemical techniques and stereological techniques are also conducted to evaluate the vascularization of the muscle basal lamina in 3,5,7,14 and 28 days after transplantation.Part 1 The manufacture of the muscle basal laminaThe muscle basal lamina has a good biocompatibility which can promote the adherent and proliferation of the cell. Also it has low immunogenicity and its degradation products are avirulent. In addition it can be made into different shape according to the requirement. The basal lamina itself represents a component of the extracellular matrix to which regenerating axons attach preferentially, and the basal lamina tubes provide spaces for the regenerating axons to grow through. Thus, basal lamina tubes, as in acellular muscles, represent favorable scaffolds for regenerating axons.1. gross observationBy gross observation, the muscle basal lamina present rosiness, which is opaque and rigid. There is little change in shape after frozen-thawed process.2.histologic structureIn longitudinal section of the muscle basal lamina with HE stains , the compoment of the muscle cell are still present. But their integrity is damaged. There are space between and parallel to muscle cells.Part 2 revascularization of muscle basal lamina transplanted into the rat semi-transected spinal cordThe revascularization is the basis of tissue regeneration. The scaffold materials for tissue engineering must have characteristic to guaranteed that the seed cells in the scaffold can get sufficient nutrition in time. So the reconstruction of the blood supply is the key point for the transition from basic research to the clinic application.1.the rat spinal cord modelThe 30 Wistar male rats(300-400g) were divided into 5 group with 6 in each group.After anesthetization the skin and muscles overlying the midthoracic cord at T10–T11 were separated and retracted, and then after laminectomy, the underlying spinal cord segment was exposed. Using both blades of the scissors, the spinal cord was semi-transected . The 5 mm block of the spinal cord tissue between the two cuts was completely removed, leaving the two stumps of spinal cord exposed. Then the scaffold was put into the gap. Then the would is closed. After the operations, the rats were sacrificed in 3,5,7,14 and 28 days.2. General status changesAll animials wake up in 2-4 hours. In the day of operation, the both hind limbs were paralyzed and can not support the body. Two weeks after operation the hind limbs can slightly moved. All rats survive and have no emiction difficulty.3. Enzyme histochemical techniques observation1) 3 days after operations, only a few blood vessels is present in the junctional area between the spinal cord and muscle basal lamina. There is no blood vessels in the scafford. The junction between the scafford and spinal cord is loose. It is easy to seperate the scafford from the spinal cord . 2) In the fifth day after the operation, the number of blood vessels were increased.In some area of the junction the blood vessels were densely distributed.But new blood vessels were arranged in a disorderly pattern.3) In the 7th,some blood vessels are present in the interior of the scafford. The scafford is connected firmly to the spinal cord.4) 14 days later, the blood vessels were densely distributed in some area of the scafford. Some area form a network of blood vessels5) 28 days later, the blood vessels growed into the deep area of the scafford. Some blood vessels go through the whole scafford. And blood vessels were mainly arranged parallel to the direction of the long axis of the scafford.In addition, it has been proved that the degradation of the scafford will affect the formation of blood vessels in the scafford. In our study, the muscle basal lamina is not degraded, and the scafford has good a good biocompatibility with the spinal cord.This study shows that the muscle basal lamina can be well vascularize after the transplantation in the rat semi-transected spinal cord. The muscle basal lamina can establish a good fusion with the spinal cord .更多还原