【作者】 周松；

【导师】 李锋；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 第一部分：利用抽吸法诱导兔椎间盘椎退变模型目的：探讨利用抽吸法构建兔椎间盘退变模型,并观察其病理及影像表现。方法：10月龄健康新西兰大耳白兔28只,对照组7只,实验造模组21只。利用21G皮肤穿刺针分别在造模组兔L4/5单节段刺入,抽吸髓核越0.008-0.012g。造模完毕,实验兔继续培养4-20周。造模前后利用,X-ray平片、MRI及Alcian blue组织染色观察退变椎间盘的变化。结果：造模前后退变椎间盘高度指数百分比(DHI%)：对照组、4周组、12周组、20周组分别为(100±0)%、(81±3.2)%、(75±2.5)%、(71±1.8)%(P<0.05)。退变椎间盘T2加权像信号明显降低,Alcian blue组织染色显示退变椎间盘组织中聚集蛋白多糖含量明显降低。结论：抽吸法构建兔椎间盘退变模型简便可靠,其退变过程的病理及影像表现与人椎间盘退变过程的病理及影像表现十分相似,该退变模型可用于人椎间盘退变的机制与临床治疗研究。第二部分：髓核细胞对骨髓基质干细胞的诱导作用目的：探讨在藻酸盐微球的介导下,髓核细胞与骨髓间充质干细胞(bone marrow stromal cells, BMSCs)非接触性共培养时,髓核细胞对骨髓间充质干细胞的诱导作用。方法：①4月龄健康新西兰大耳白兔5只,取髓核细胞与骨髓间充质干细胞原代培养,利用传代法分离纯化；②将纯化的骨髓间充质干细胞经胰蛋白酶消化、收集,并与适量的藻酸钠溶液混合,形成106个/ml的单细胞悬液,将混合好的单细胞悬液经注射器滴加至3.5%的CaCl2溶液中,形成海藻酸钙凝胶珠；③将海藻酸钙凝胶微球与髓核细胞共培养。在7天和15天时,分组溶解海藻酸钙凝胶珠,收集骨髓间充质干细胞,分别利用免疫组化技术、rt-PCR技术和、Western blot技术检测骨髓间充质干细胞中Ⅱ型胶原和聚集蛋白聚糖的表达,用以判断髓核细胞对骨髓基质干细胞在非接触条件下的诱导作用。结果：在骨髓间充质干细胞的细胞爬片免疫组化染色中可见,细胞Ⅱ型胶原和聚集蛋白聚糖染色阳性,rt-PCR和、Western blot结果显示经诱导后骨髓间充质干细胞中已有Ⅱ型胶原和聚集蛋白聚糖基因的表达,且诱导15天组的目的条带明显亮于诱导7天组的目的条带。结论：在体外非接触共同培养时,髓核细胞能够实现对骨髓基质干细胞的诱导作用,将骨髓基质干细胞分化为髓核细胞,这必将为椎间盘退行性变的移植治疗提供可靠的种子细胞来源。第三部分：骨髓间充质干细胞向类髓核细胞的分化及永生化目的：尝试利用共培养法和SV40Tag;永生化基因导入法构建一种来源于骨髓基质干细胞(MSCs)的永生化型类髓核细胞。方法：取实验白兔原代MSCs和髓核细胞(NPCs),荧光标记NPCs;将标记的NPCs与MSCs直接共培养6、9、12、15、18天；共培养完毕,利用流式细胞仪分选出荧光标记阴性细胞,即MSCs;观测共培养后MSCs细胞形态变化,检测胞内Ⅱ型胶原和聚集蛋白多糖mRNA及蛋白的表达变化；共培养15天时,将含有SV40Tag永生化基因的pCMVSV40T/PUR质粒转染MSCs,利用MTT法观测转染后MSCs增殖活性。结果：共培养后,MSCs胞内表达大量的Ⅱ型胶原和聚集蛋白多糖,细胞形态也从长椭圆形变为多角形；当共培养15天时,胞内Ⅱ型胶原和聚集蛋白多糖mRNA表达浓度达到最大值,但低于原代NPCs胞内Ⅱ型胶原和聚集蛋白多糖mRNA含量(P<0.05)。导入SV40Tag基因后,MSCs-SV增殖能力与原代NPCs相同,传代20次后其增殖能力无衰退(P<0.05)。结论：利用共培养法和SV40Tag永生化基因导入法可构建出一种来源于MSCs的永生化型类髓核细胞；该类髓核细胞可能会对椎间盘退变的细胞移植治疗研究起到一定得帮助作用。更多还原

【Abstract】 PartⅠ:The Pathology and Imaging Performance of Rabbit Disc Degeneration Model Induced by the Aspiration of Nucleus PulposusObjective:To investigate the use of liposuction to build rabbit model of disc degeneration and to observe the performance of its pathology and imaging.Methods:Healthy 10-month-old New Zealand rabbits 28,7 for control group and 21 for experimental group.21-gauge hypodermic needle was used to aspirate nucleus pulposus from L4/5 disc about 0.008～0.012g. All the experimental rabbits were continued to foster for 4-20 weeks. X-ray plain film, MRI and Alcian blue staining were used to observe the organizational changes in the intervertebral disc degeneration.Results:The percentage of disc height index (DHI%) of control group,4-week group, 12-week group and 20-week group respectively was (100±0)%, (81±3.2)%, (75±2.5)% and (71±1.8)% (P<0.05). T2-weighted image showed that the signal of the discs was significantly decreased, and the content of aggrecan was also significantly decreased.Conclusion:The puncture and aspiration is a reliable and convenient way to construct rabbit models of intervertebral disc degeneration, and these models can be used for the research of human the mechanism and clinical therapeutic research. PartⅡ:The Induction of Nucleus Pulposus Cells to Bone Marrow Stem Cells by Co-culturedObjective:To research the effect of nucleus pulposus cells to bone marrow stem cells which were encapsulated in alginate beads when they were co-cultured in vitro.Methods:①we got the primary nucleus pulposus cells and bone marrow stem cells from 5 healthy New Zealand rabbits of 4-month-old, and the nucleus pulposus cells and bone marrow stem cells were selected by their adherence to tissue culture flasks.②Cultures of MSCs were trypsinized, centrifuged, and mixed homogeneously into 1.2% alginate solution at 106 cells/mL. The alginate solution was injected into 3.5% CaCl2 solution by injector forming small droplets of cell-alginate beads.③The nucleus pulposus cells and cell-alginate beads were co-cultured in Six-well plates. At the 7th day and the 15th day, we collected the bone marrow stem cells from the cell-alginate beads. Immunohistochemical techniques, rt-PCR and Western blot technique was used to study the expression of collagen-Ⅱand aggrecan.Results:The immunohistochemical stain show that collagen-Ⅱand aggrecan has been expressed in the MSCs, and we also find that both of the collagen-Ⅱand aggrecan, our purpose strips of the 15th day’s rt-PCR result are brighter than those of the 7th day’s rt-PCR result.Conclusion:In vitro, bone marrow stem cells can be differentiated into the nucleus pulposus cell-like cells by been co-cultured with the nucleus pulposus cells. It can be served as optimal cell source for therapy of the degeneration disc. PartⅢ:Differentiation and Immortalization of Bone Marrow Strom Cells toward to Nucleus Pulposus Cell-Like CellsObjective:To attempt to use the way of co-culture and transfecting plasmid pCMVSV40T/PUR to get a type of immortalized Nucleus Pulposus Cell like cells, which came from Bone Marrow Strom Cells (MSCs).Methods:Primary Nucleus Pulposus Cells (NPCs) and primary bone Marrow Strom Cells (MSCs) were got from 20 rabbits. The NPCs were fluorescence-labeled, and were co-cultured with MSCs(co-culture ratio 1:1).After been co-cultured for 6,9,12,15 and 18 days, the MSCs (fluorescent-negative cells) were selected by Flow Cytometry from the co-culture system. Morphological changes of MSCs were observed, and real-time PCR reaction was performed to assess the mRNA expression of maker genes (Collagen II and Aggrecan) in MSCs.In the co-culture 15 days group, plasmid pCMVSV40T/PUR containing SV40Tag gene was transfected into MSCs, and the MTT assay was carried out to observe the MSCs proliferative ability.Results:Co-cultured with NPCs, MSCs began to express maker genes.15 days later, the mRNA expression of maker genes in MSCs reached the peak level at 0.90 and 0.93.But this peak level was still lower than the mRNA expression of maker genes in primary NPCs (P<0.05).Cell morphology also changed from a spindle into a polygon. After been transfected with plasmid pCMVSV40T/PUR, the MSCs almost had the same proliferative ability with the primary NPCs, and this ability would not decline in the next 20 passages (P<0.05).Conclusion:By co-cultured with NPCs and transfected with SV40Tag gene, we can get a kind of NPC-like cell. This NPC-like cell may be helpful to the cell-based therapy of intervertebral disc degeneration.更多还原