【作者】 柴勇；

【导师】 杨成；

【作者基本信息】 滨州医学院 ， 人体解剖与组织胚胎学， 2009， 硕士

【摘要】 目的：探讨胚胎脊髓源性神经干细胞(Neural stem cells, NSCs)移植联合电针治疗对大鼠脊髓全横断损伤的修复作用。方法：实验1、取11.5 d的SD胎鼠分离神经管后段,分散细胞进行原代和传代培养,倒置相差显微镜下观察细胞生长状态。对其进行神经干细胞标志抗原Nestin免疫细胞化学染色鉴定；用含10%胎牛血清的DMEM/F12诱导,并进行神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、髓磷脂碱性蛋白(MBP)的免疫细胞化学染色,激光共聚焦显微镜下观察其分化情况。用BrdU标记第二代培养7-8 d的NSCs悬液,进行BrdU免疫细胞化学染色,观察标记情况,收集已标记的细胞备用。实验2、将54只成年雌性SD大鼠随机分为三组：脊髓损伤(SCI)组；NSCs移植组；NSCs移植联合电针治疗(E+NSCs)组。制作脊髓全横断损伤模型,在大鼠脊髓横断处填放明胶海绵,将BrdU标记的NSCs各10μl分别吸附到NSCs移植组和E+NSCs组损伤处的明胶海绵上,SCI组用生理盐水代替NSCs。E+NSCs组在细胞移植后每天的固定时间给予电针治疗。各组在7 d、14 d、28 d后分别进行BBB运动功能评分观察大鼠后肢运动功能恢复情况；取损伤脊髓分别进行大体观察、光镜观察和电镜观察,分析脊髓形态学改变,通过免疫荧光染色检测脊髓损伤处的神经元、星形胶质细胞及少突胶质细胞的变化情况。结果：1、11.5 d胎鼠的神经管后段培养的细胞,有良好的增殖能力,易获得大量细胞；其表达神经干细胞特异抗原Nestin,且诱导分化后表达NSE、GFAP. MBP阳性；BrdU免疫荧光观察显示,新形成的细胞克隆球中的细胞呈BrdU免疫阳性。2、脊髓损伤后大鼠后肢BBB运动功能评分显示,随时间推移,NSCs联合电针治疗组与其他各组之间均存在显著差异(P<0.01),动物肢体功能恢复显著。3、大体观察显示,损伤区的脊髓由于瘢痕化颜色变深,植入脊髓断端的明胶海绵有不同程度的吸收,损伤区近头端脊髓轻度萎缩,近尾端明显萎缩。NSCs移植组与E+NSCs组明胶海绵吸收程度较好,基本与脊髓组织融合,且萎缩程度较轻。而SCI组部分大鼠损伤处脊髓变性坏死,明胶海绵吸收较差。4、光镜观察显示,术后28 d,SCI组脊髓损伤区形态不完整,神经组织残缺；有大量泡沫细胞和明显空洞形成以及大量炎症细胞浸润。NSCs组部分神经元细胞形态较规则,少数神经元可见细胞核及核仁,但部分神经元内尼氏小体较少,分布不均匀。E+NSCs组神经元细胞数量较多,形态基本正常,核仁清晰可见,尼氏小体较多,且分布均匀,神经纤维排列较规则。5、透射电镜观察显示,SCI组髓鞘松散、破裂,少部分完全崩解,轴突固缩,部分轴突空泡变,线粒体含量较少且有肿胀；NSCs移植组髓鞘松散程度减轻,散在分布,仅有少量髓鞘破裂,轴突消失,其间有薄髓的再生髓鞘；E+NSCs组再生纤维增多,髓鞘松散程度明显减轻,部分髓鞘结构规整,排列有序,线粒体肿胀减轻,微管空泡样变较轻,可发现大量突触,薄髓的再生髓鞘明显增多。6、免疫荧光染色检测脊髓损伤处的神经元、星形胶质细胞及少突胶质细胞数量随时间推移逐渐增加,E+NSCs组与其他各组之间均存在差异(P＜0.01)。结论：1、从11.5 d胎鼠的神经管后段分离、培养,可获得足量的NSCs,血清诱导能够分化为神经元和胶质细胞；2、NSCs移植联合电针治疗能促进脊髓损伤大鼠后肢运动功能的恢复；3、NSCs移植联合电针治疗能促进损伤处脱髓的轴突再髓鞘化和神经纤维再生；4、NSCs移植联合电针治疗能促进细胞的分化,增加损伤脊髓神经元、胶质细胞的数量,改善损伤处的微环境,促进结构的修复。更多还原

【Abstract】 Objective:To explore the effect of neural stem cells transplantation in combination with electro-acupuncture treatment on repair of traumatic spinal cord injury rats.Methods:Experiment 1, The posterior segment of neural tube was dissected from SD embryonic 11.5d rats, cultured primarily in vitro and observed the growth state of cells under inverted phase contrast microscope. They were identified with immunocytochemistry for nestin which is the marker antigen of neural stem cell. Meanwhile, the differentiation was induced by 10% fetal bovine serum and cells were examined by immunocytochemical stain including neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) under confocal laser scanning microscopy. Neural stem cells (made from the culturing cells of the second generation 7-8d) were marked by BrdU immunocytochemical stain, and marked cells were collect to reserve.Experiment 2,54 adult female SD rats were randomly divided into three groups:spinal cord injury group (SCI), NSCs transplantation group, electro-acupuncture treatment with NSCs transplantation group (E+NSCs). SCI rat model was constructed by transecting spinal cord thoroughly. NSCs labeled with BrdU were adsorbed to the gelatin sponge and then transplanted into injured part of both NSCs and E+NSCs groups, yet normal saline into SCI group instead correspondingly. After transplantation, rats in E+NSCs group were given electro-acupuncture treatment at a fixed time every day. The motor functions of recovery of rat hind limb in each group were detected in 7d, 14d,28d, respectively, by methods of BBB score. The injury spinal cords were admitted to analyze morphological changes through general observation, light microscope and electron microscope respectively, and changes of neurons, astrocytes and oligodendrocytes keratinocyte were detected through immunofluorescence.Results:1. The cells derived from the posterior segment of neural tube of E11.5d rat were successfully isolated and cultured with good proliferation. The cultured cells were identified as NSCs by expressing specific antigen Nestin of NSCs and can be induced to differentiate to neurons, astrocytes and oligodendrocytes. The cells in neurosphere newly formed were proved BrdU-positive cells by immunofluorescence.2. Difference between E+NSCs group and other groups respective were obviously observed by methods of BBB score of hind limb motor function over time (P<0.01). The recovery of animal limb function was significant.3. It was observed that the color of the spinal cord in the injured site became darker as a result of scarring, the gelatin sponge migrated into the injured site was absorbed in various degree, the atrophy of the injury spinal cord close to the head atrophies was mildly while the part far from the head atrophies markedly. The gelatin sponge was absorbed in a better degree in both NSCs group and E+NSCs group and integrated nearly with the spinal cord tissue, and the spinal cord atrophied mildly. While the injury spinal cord of some rats in SCI group was metamorphic and necrotic with poor absorption of gelatin sponge.4. Under light microscope, HE staining showed that the spinal cord injury zone in SCI group was incomplete after 28d and the nervous tissue was fragmentary. Large numbers of foam cells and marked cavities emerged and the injury area was intiltrated with many chronic inflammatory cells. In NSCs group, the morphological changes of some neurons was relatively regular, nucleus and nucleolus in the minority of neurons could be seen, but Nissl bodies within some neurons were fairly less and uneven. In E+NSCs group, the number of neurons increased obviously, the shape of neurons was close to normal, nucleolus were clearly visible, more Nissl bodies with even distribution, and the nerve fibers were regular.5. Transmission Electron Microscope (TEM) showed:In SCI group, the myelin was loose, distorted and broken, with axonal shrinkage and some axonal vacuolate, mitochondrial swelling and there was less content. In NSCs group, the extent of myelin loose reduced and scattered, only a few myelin breakdown, and the axon disappeared and there were thin myelin regeneration of myelinated. In E+NSCs group, regenerated fiber increased, the extent of myelin loose reduced distinctly, the structure of myelin was clear and neat and the arrangement orderly, the swelling of mitochondria became lightened, vacuolar degeneration of microtubules got less, a lot of synapses were found, thin myelin sheath regeneration increased significantly.6. The number of neurons, astrocytes and oligodendrocytes keratinocyte in injured site gradually increased over time by immunofluorescence stain. Difference between E+NSCs group and other groups respective was obvious (P<0.01).Conclusion:1. NSCs derived from the posterior segment of neural tube of E11.5d rat were successfully isolated, cultured to sufficient quantities and induced to differentiate to neurons and glial cells by serum.2. NSCs transplantation with electro-acupuncture treatment increased the recovery of hind limb motor function of spinal cord injury in rats3. The treatment of NSCs transplanted with electro-acupuncture promoted demyelination axons in injured site re-myelination and nerve fibers regeneration.4. NSCs transplantation with electro-acupuncture treatment promoted cells differentiation, increased the number of neurons and glial cells in injured spinal cord, improved the micro-environment of the damaged site and promoted structural repair.更多还原