体外构建PLGA—神经干细胞—雪旺细胞组织工程学复合物桥接修复大鼠喉返神经损伤的研究

发布时间：2018-05-17 06:29

本文选题：神经干细胞 + 雪旺细胞共培养　；参考：《上海交通大学》2015年博士论文

【摘要】：第一部分大鼠雪旺细胞与神经干细胞共培养的体外实验研究目的:观察大鼠雪旺细胞(Schwann cells,SCs)与神经干细胞(Neural Stem Cells,NSCs)体外共培养时对神经干细胞存活、分化方向以及BDNF、GDNF分泌量的影响。材料和方法:取p2-p4代雪旺细胞与p2代神经干细胞在诱导培养基中共培养。共分为三组:雪旺细胞组、神经干细胞组、雪旺细胞与神经干细胞共培养组。倒置相差显微镜下,每天观察三组细胞形态并计数;细胞免疫荧光化学染色:SCs用S100标记,NSCs用Nestin巢蛋白标记,分化的神经元细胞用Map2、Neu N标记,星形胶质细胞用GFAP标记;MTT(四甲基偶氮唑蓝)比色法检测各细胞组存活率;酶联免疫吸附法(ELISA)检测各组细胞悬液1,3,5,7天BDNF、GDNF分泌情况。结果:雪旺细胞与神经干细胞共培养组细胞状态更好、细胞突起细长、交织成网状,神经干细胞大多分化为神经元细胞;共培养组细胞存活率及BDNF、GDNF也高于其他两组(p0.05)。结论:雪旺细胞与神经干细胞共培养可以促进神经干细胞向神经元分化,两者共培养可以通过分泌更多神经营养因子改善神经再生微环境。第二部分组织工程化人工神经的体外构建目的:通过比较雪旺细胞和神经干细胞共同培养与单独一种细胞(雪旺细胞或神经干细胞)种植于三种材料上,细胞形态学变化及细胞增殖能力的变化,以探索进行构建组织工程化人工神经体外实验的可行性。材料和方法:雪旺细胞、神经干细胞以及雪旺细胞-神经干细胞(1:1)分别与PLGA、c PLGA、lc PLGA三种材料共同于分化培养基下培养,构建层粘连蛋白(Laminin)及壳聚糖(Chitosan)涂层的内置纳米纤维细丝的新型PLGA神经导管。MTT法检测雪旺细胞和神经干细胞共培养以及单独一种细胞的生物相容性和细胞毒性。结果:细胞与生物材料共培养七天后,扫描电子显微镜下观察发现,所有细胞组在lc PLGA材料上相较其他两种材料上具有更好的形态学表现,细胞沿管腔内纵行排列的纳米纤维细丝定向生长。而倒置相差显微镜下观察到,不论在哪一种材料上生长,雪旺细胞与神经干细胞共培养组细胞密度更大,细胞的突起更为细长且交织成网状。这项研究表明lc PLGA具有良好的生物相容性和较小的细胞毒性,而管腔内的纳米纤维细丝具有引导细胞定向生长的作用。结论:雪旺细胞和神经干细胞共同培养较单一细胞种植于神经导管生物材料上更为有利,这些发现为人工神经导管移植修复周围神经损伤提供了一个生物学基础。第三部分以PLGA为支架的神经干细胞-雪旺细胞组织工程人工神经桥接技术修复大鼠喉返神经损伤的研究目的:采用形态学、分子生物学和电生理学等方法,检测PLGA-神经干细胞-雪旺细胞组织工程人工3D神经桥接修复大鼠喉返神经缺损的功能效果。材料和方法:将72只150 g健康SD大鼠随机均分为6组。制作大鼠10 mm的喉返神经缺损模型;A组为神经干细胞+雪旺细胞+PLGA导管组,B组为神经干细胞+PLGA导管组,C组为雪旺细胞+PLGA导管组,D组为PLGA空导管组,E组为自体神经移植组。F组为假手术组。术后8周及12周,切取再生神经中间片段切片,用于免疫荧光染色。并在透射电镜下观察导管周围的神经细胞的形态学改变。术后8周及12周检测再生神经干动作电位,并计算其潜伏期和振幅。结果:免疫荧光实验结果显示,在共培养组中,有大量神经微丝免疫反应性的纤维和S100免疫反应性纤维,并围绕在神经纤维周围,提示髓鞘化的神经纤维已成功再生。透射电镜下可见不同阶段细胞类型和有髓鞘结构的显著差异,手术8周和12周后,CO组显示有大量再生神经纤维,并且其髓鞘较厚,发育成熟,纤维束之间结缔组织较少,再生轴突发育好且排列有序。术后8周的动作电位显示,潜伏期:共培养组(CO)小于神经干细胞(NSC),雪旺细胞(SC)、空导管(null),和SC组有显著差异(p0.0001),但和自体神经移植组(atograft)无差异;波幅:各组之间无明显差异性。术后12周的动作电位显示,潜伏期:共培养组(CO)小于神经干细胞(NSC),雪旺细胞(SC)、空导管(null)以及自体神经移植组(atograft);.波幅:自体神经移植组优于共培养、雪旺细胞、神经干细胞以及空导管组,其它几组之间无明显差异性。结论:PLGA-神经干细胞-雪旺细胞组织工程化人工3D神经可增强大鼠喉返神经的再生
[Abstract]:Part 1: in the first part of the co culture of rat Schwann cells and neural stem cells in vitro, the effects of Schwann cells (SCs) and neural stem cells (Neural Stem Cells, NSCs) on the survival of neural stem cells, the direction of differentiation, and the effect of BDNF and GDNF secretion were observed. Materials and methods: take the P2-P4 generation of Schwann cells. P2 generation neural stem cells were cultured in the inducible medium. They were divided into three groups: Schwann cell group, neural stem cell group, Schwann cell and neural stem cell co culture group. Under the inverted phase contrast microscope, three groups of cell morphology were observed and counted. Cell immunofluorescence staining: SCs was marked with S100, and NSCs was marked with Nestin nestin. Neuron cells were labeled with Map2, Neu N, astrocytes were labeled with GFAP, MTT (four methylazazolium blue) was used to detect the survival rate of each cell group; enzyme linked immunosorbent assay (ELISA) was used to detect the 1,3,5,7 day BDNF and GDNF secretion in each group of cells. Results: the cell state of the co culture group of Schwann cells and neural stem cells was better, and the cell protruding was fine. Long, interwoven into reticulate, neural stem cells mostly differentiate into neuron cells; the survival rate of cell and BDNF and GDNF in co culture group are also higher than that of other two groups (P0.05). Conclusion: co culture of Schwann cells and neural stem cells can promote neural stem cells to differentiate into neurons, and the co culture can improve the nerve by secreting more neurotrophic factors. Second parts of tissue engineered artificial nerve in vitro construction objective: To explore the construction of tissue engineering human by comparing Schwann cells and neural stem cells together with a single cell (Schwann cells or neural stem cells) planted on three materials, cell morphological changes and cell proliferation energy changes. Materials and methods: the materials and methods: Schwann cells, neural stem cells and Schwann cells - neural stem cells (1:1), together with PLGA, C PLGA, LC PLGA, respectively, were cultured under the differentiation medium, and constructed a new PLGA neuro conductance of nanofiber filaments of laminin (Laminin) and chitosan (Chitosan) coating. .MTT method was used to test the co culture of Schwann cells and neural stem cells and the biocompatibility and cytotoxicity of a single cell. Results: seven days after co culture of cells and biomaterials, it was observed under scanning electron microscope that all the cell groups had better morphological features on the LC PLGA material than the other two materials. Nanofiber filaments aligned vertically in the cavity were directed to grow. Under the inverted phase contrast microscope, the cell density was larger and the cell protruding was more elongated and reticulate, no matter which material was grown on one of the material, and the LC PLGA had good biocompatibility and smaller size. Conclusion: the co culture of Schwann cells and neural stem cells is more favorable than the single cell cultivation on the nerve conduit biomaterials. These findings provide a biological basis for the repair of peripheral nerve injury by artificial nerve conduit transplantation. The three part of the study on the repair of recurrent laryngeal nerve injury in rats with PLGA supported neural stem cells - Schwann cell tissue engineering artificial nerve bridging technique: morphological, molecular and electrophysiologic methods were used to detect the repair of recurrent laryngeal nerve defect in rats by PLGA- neural stem cells - Schwann cell tissue engineering human 3D nerve bridging Functional effects. Materials and methods: 72 150 g healthy SD rats were randomly divided into 6 groups. The rat model of recurrent laryngeal nerve defect was made by 10 mm. Group A was neural stem cells + Schwann cell +PLGA catheter group, B group was neural stem cell +PLGA catheter group, C group was a +PLGA catheter group of Schwann cells, D group was PLGA air catheter group, E group was autotransplantation group of autologous nerve group. In the sham operation group, 8 and 12 weeks after the operation, the intermediate segments of the regenerated nerve were cut and used for immunofluorescence staining. The morphological changes of the nerve cells around the catheter were observed under the transmission electron microscope. The regenerative nerve action potential was detected at 8 and 12 weeks after the operation, and the incubation period and amplitude were calculated. Results: the results of immunofluorescence test showed that in common. In the culture group, there are a large number of neurofilament immunoreactive fibers and S100 immunoreactive fibers and around the nerve fibers, suggesting that myelinated nerve fibers have been successfully regenerated. Under transmission electron microscopy, there are significant differences in cell types and myelin structures in different stages. 8 weeks and 12 weeks after hand surgery, group CO shows a large number of regenerative nerves. Fiber, and its myelin sheath is thicker, mature and less connective tissue between the fiber bundles, and the regeneration axons are well developed and ordered. The action potential 8 weeks after the operation shows that the incubation period is less than the neural stem cells (NSC), SC, null, and SC group (P0.0001), but with the autologous nerve graft group (atograf). T) no difference. There was no significant difference between each group. The action potential of 12 weeks after operation showed that the incubation period: the co culture group (CO) was less than the neural stem cells (NSC), the Schwann cells (SC), the empty catheter (null) and the autologous nerve graft group (atograft); the amplitude: the self body nerve graft group was superior to co culture, Schwann cells, neural stem cells, and empty catheterization groups. Conclusion: PLGA- neural stem cell Schwann cell tissue engineered artificial 3D nerve can enhance the regeneration of the recurrent laryngeal nerve in rats.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.3

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