【作者】 李祥炎；

【导师】 徐皓；

【作者基本信息】 福建医科大学 ， 外科学， 2010， 硕士

【摘要】 目的：(1)分离、培养及鉴定SD大鼠BMSCs,将其诱导分化为Nestin阳性的BMSCs,并通过分子生物学技术将Ang-1基因转染入细胞内；(2)构建SD大鼠T10脊髓钳夹损伤模型,评估其可行性；(3)通过Ang-1基因修饰的Nestin阳性BMSCs局部注射对损伤脊髓进行治疗,检测Nestin阳性的BMSCs是否能够分化为神经元细胞,检测Ang-1对移植入体内的Nestin阳性的BMSCs,新生及损伤后残留的神经元细胞是否具有保护作用。方法：(1)采用全骨髓差速贴壁分离培养法将SD大鼠双后肢股骨、胫骨骨髓内的BMSCs进行分离培养。通过显微镜下观察细胞生长性状；流式细胞仪检测其细胞表面标志物CD29、CD34、CD45和CD90的表达；向成骨样细胞、成脂肪样细胞及成神经样细胞分化证明其具有多向分化潜能；同时将BMSCs诱导分化为Nestin阳性的BMSCs,并通过腺病毒载体将Ang-1基因转染入Nestin阳性的BMSCs中,Weston blot检测转染后的细胞是否表达Ang-1。(2)构建SD大鼠T10脊髓钳夹损伤模型,造模后1周内通过运动功能评价、神经电生理检测及组织病理学检测等方法检测模型的可行性；(3)造模后1周对随机分组的大鼠实施局部注射移植细胞进行治疗,在随后的1～4周内通过运动功能评价、神经电生理检测及脊髓组织的免疫组织化学检测等方法检测Ang-1基因修饰的Nestin阳性BMSCs对损伤后脊髓的相关作用。结果：(1)通过全骨髓差速贴壁分离培养的BMSCs增殖速度快,扩增能力强,传代培养3代后细胞可获较高纯度；通过流式细胞仪检测细胞表面标记物,细胞表达CD29和CD90,阳性率均达99%以上；在体外通过诱导分化后细胞可分化为成骨细胞、成脂肪细胞及成神经样细胞；通过Weston blot检测转染后的Nestin阳性BMSCs出现Ang-1条带。(2)造模后1周内,大鼠出现明显截瘫(BBB<4分),经神经电生理检测无法引出CSEP波形,脊髓组织出现大量炎性细胞浸润,脊髓空泡变性。(3)移植后1-4周,A组大鼠后肢神经功能无明显变化,B、C组大鼠均有不同程度的恢复,C组较B组明显(P<0.05)；神经电生理检测,C组亦明显优于B组(P<0.05)；损伤脊髓经免疫组织化学染色后,B、C两组均表达BrdU、Nestin、NSE,随着时间的延长,阳性细胞数逐渐减少,但移植4周后C组仍保持一定量的阳性细胞,且明显多于A、B两组(P<0.05)。C组还可观察到BrdU阳性细胞向血管腔周围聚集。结论：(1)通过全骨髓差速贴壁分离培养法能够获取较高纯度的BMSCs,细胞具有多向分化潜能,经神经诱导后,细胞能分化为Nestin阳性BMSCs,通过分子生物学技术,Ang-1基因转染入Nestin阳性BMSCs中获得成功。(2)成功构建SD大鼠T10脊髓钳夹型损伤模型。(3)经Ang-1基因修饰后的Nestin阳性BMSCs移植入体内后能进一步分化为成熟的神经元细胞,同时其分泌的Ang-1对Nestin阳性的BMSCs、新生及残留神经细胞具有保护作用,对脊髓损伤具有一定的修复作用。更多还原

【Abstract】 Objectives:(1) To isolate, culture and identify the BMSCs from the SD rats.To induce and differentiate them into Nestin-positive BMSCs. And tansfected the Ang-1 into the cells by molecular biology. (2)To establish the models of clip compressive thoracic 10 spinal cord injury in the SD rats and investigate the feasibility of the models. (3) To look forward to the possibility of the nestin-positive BMSCs differentiate into neuron in spinal cord injury, and to look forward to the protection of Ang-1 about the nestin-positive BMSCs, the neogenetic and residual neuron in spinal cord injury by tansplanted the nestin-positive BMSCs modified by Ang-1 gene.Methods:(1)We isolated and cultured the BMSCs by whole bone marrow adherence method from tibias and femurs in SD rats,We observed the morphology of BMSCs by inverted microscope. We performed flow cytometric analysis, using a large monoclonal antibody panel:CD45, CD34, CD29, CD90,and we induced the BMSCs into osteoblast cells,adipocyte and neuron-like cells.In the end we induced the BMSCs into the other cells that the surface marker were positive of Nestin.Then we tansfected the Ang-1 gene into the cells by adenovirus vector, and detected the cells expressed Ang-1 through Weston blot technology. (2)We established graded model of clip compressive thoracic 10 spinal cord injury in the SD rats. One week later, we used the methods based on the assessment of the motor function, neurophysiology monitoring,and pathology test to investigate the feasibility of the model.(3)Rats of randomized grouping were successfully local injected by the certain suspensions a week post-operation, then we assessed the correlated function of Ang-1 and Nestin positive BMSCs to the spine by the methods based on the assessment of the motor function, neurophysiology monitoring, and pathology test after one to four weeks. Results:(1) BMSCs which was isolated and cultured by the whole bone marrow culture and adherence method, inereased lastingly and had a fast multiplication speed in vitro.The purity quotient of BMSCs after the third generation. BMSCs phenotype was considered positive for CD29, CD90 after flow cytometric analysis, and negative for the other monoclonals.The positive rate of BMSCs phenotype was out of 99%. They could be differentiated into osteoblast cells, adipocyte and neuron-like cells in vitro. The result of weston blot manifest that the Nestin-positive BMSCs transfected by Ang-1 can secreted the angiopoitin protein. (2) Most of the rats became paraplegia after spinal cord injury(BBB<4), the wave shape of CSEP could not be evoked, a lot of inflammatory cells infiltrated the spinal cord which was cytoplasmic vacuolation.(3) Certain neurologial recovery occur in group B, C at 1,2,4 weeks after transplantation, but group A was not. BBB showed significant difference between group B and group C (p<0.05), The CSEP also showed significant difference between group B and group C (p<0.05). After transplanting in group B and C,the cells labled BrdU, Nestin, NSE could be observed in the injury spine,and the cells labled BrdU could be observed around blood vessel. The number of cells labled BrdU, Nestin, NSE reduced gradually in 4 weeks, but group C remain a certain amount of positive cells,and group C showed significant more than group B (p<0.05).Conclusion:(1)We could harvested the BMSCs which was isolated and cultured by the whole bone marrow culture and adherence method.It was multipotentiality and could be differentiated the BMSCs into Nestin positive cells. we could successfully tansfected the Ang-1 into the nestin-positive BMSCs by molecular biology. (2)The models of SD rats spinal cord injury was successfully established. (3)The Nestin-positive BMSCs tranfected by Ang-1 had the ability to differentiate into neuron, and they restituted and protected the Nestin-positive BMSCs and the nerve cells, cured the injury spine.更多还原