脱细胞神经支架复合PMSCs修复大鼠横断性脊髓缺损的实验研究
发布时间：2018-08-11 17:27
【摘要】：脊髓损伤是临床常见的高致残率的疾病,由于损伤神经元的再生能力极其微弱,加之微环境中抑制性因素的存在,导致脊髓损伤后的神经再生及功能恢复非常有限。目前脊髓损伤的治疗理念是重塑损伤脊髓的空间结构,改善神经元再生环境进而诱导轴突再生,组织工程是实现这一理念的有效途径。本研究应用脱细胞神经支架复合胎盘间充质干细胞(PMSCs),构建组织工程化神经支架复合体,治疗大鼠脊髓横断性缺损,为临床应用组织工程治疗脊髓损伤提供理论基础。首先,选择改良脱细胞方法,制备理想的脱细胞神经支架。行HE染色、甲苯胺蓝染色、Laminin免疫荧光染色,观察脱细胞神经支架组织形态特征；扫描电镜、透射电镜观察脱细胞神经支架超微结构,DNA含量测定检测脱细胞神经支架细胞残留,细胞增殖率和肌袋埋入反应检测支架的细胞毒性和生物相容性。随后,构建组织工程化复合体,应用散点注射法将PMSCs注入脱细胞神经支架,构建细胞支架复合体,37℃,5%C02培养箱中培养。最后,制作大鼠横断性脊髓缺损模型,移植神经支架复合体,术后4W,免疫荧光检钡PMSCs在体生长、迁移及分化情况,BBB评分、脚印分析及电生理检测损伤大鼠的功能恢复,透射电镜观察轴突及髓鞘再生。HE染色、甲苯胺蓝染色、透射电镜结果显示,改良法制备的脱细胞神经支架,细胞脱除彻底,ECM蛋白含量丰富；扫描电镜结果显示,支架三维空间结构完整,管道通畅；细胞毒性检测结果显示,支架无胞毒性；生物组织相容性检测结果显示,无炎症细胞浸润,细胞相容性检测结果显示,细胞长入支架并大量存活,生长状态良好。神经支架复合体移植术后4W,取材行冰冻切片,荧光显微镜观察显示,PMSCs生长状态良好,向损伤区两端双向迁移,兔疫荧光结果显示植入的PMSCs分化为神经元细胞。透射电镜观察显示,与对照组相比,支架复合体组大鼠新生髓鞘数目明显增多,厚度明显增厚,直径明显增大；BBB评分显示,支架复合体组大鼠BBB评分明显提高,术后8w达到13分；脚印分析测定表明支架复合体组大鼠前后足协调性明显改善,术后8w, ILC、AR分别为1.8,12；电生理检测结果显示,支架复合体组运动诱发电位振幅为0.54±0.05mV,潜伏期为9.98±0.33ms, PMSCs组可记录到轻微电信号,生理盐水组无电信号记录。综上所述,改良法制备的脱细胞神经支架,是构建神经支架复合体的理想材料；改良脱细胞支架复合PMSCs构建神经支架复合体,治疗大鼠横断性脊髓损伤,有助于促进大鼠功能恢复；本研究为临床上应用组织工程化桥接神经元治疗脊髓损伤提供理论基础和技术支撑。
[Abstract]:Spinal cord injury (sci) is a common disease with high disability rate. Because of the very weak regeneration ability of injured neurons and the existence of inhibitory factors in microenvironment, the regeneration and functional recovery of injured neurons are very limited. At present, the idea of spinal cord injury treatment is to reconstruct the spatial structure of injured spinal cord, improve the regeneration environment of neurons and induce axon regeneration. Tissue engineering is an effective way to realize this idea. In this study, acellular nerve scaffolds combined with placental mesenchymal stem cells (PMSCs),) were used to construct tissue-engineered nerve scaffolds for the treatment of spinal cord transverse defects in rats, which provided a theoretical basis for clinical application of tissue engineering in the treatment of spinal cord injury. Firstly, an improved acellular method was chosen to prepare an ideal acellular nerve scaffold. He staining, toluidine blue staining and laminin immunofluorescence staining were used to observe the morphological characteristics of acellular nerve scaffolds, and the ultrastructure of acellular nerve stents was observed by scanning electron microscope and transmission electron microscope. The cytotoxicity and biocompatibility of the scaffold were examined by cell proliferation rate and muscle bag embedding reaction. Then, the tissue engineering complex was constructed, PMSCs was injected into acellular nerve scaffold by scattered point injection, and the cell scaffold complex was cultured in 52 incubator at 37 鈩，
本文编号：2177714
[Abstract]:Spinal cord injury (sci) is a common disease with high disability rate. Because of the very weak regeneration ability of injured neurons and the existence of inhibitory factors in microenvironment, the regeneration and functional recovery of injured neurons are very limited. At present, the idea of spinal cord injury treatment is to reconstruct the spatial structure of injured spinal cord, improve the regeneration environment of neurons and induce axon regeneration. Tissue engineering is an effective way to realize this idea. In this study, acellular nerve scaffolds combined with placental mesenchymal stem cells (PMSCs),) were used to construct tissue-engineered nerve scaffolds for the treatment of spinal cord transverse defects in rats, which provided a theoretical basis for clinical application of tissue engineering in the treatment of spinal cord injury. Firstly, an improved acellular method was chosen to prepare an ideal acellular nerve scaffold. He staining, toluidine blue staining and laminin immunofluorescence staining were used to observe the morphological characteristics of acellular nerve scaffolds, and the ultrastructure of acellular nerve stents was observed by scanning electron microscope and transmission electron microscope. The cytotoxicity and biocompatibility of the scaffold were examined by cell proliferation rate and muscle bag embedding reaction. Then, the tissue engineering complex was constructed, PMSCs was injected into acellular nerve scaffold by scattered point injection, and the cell scaffold complex was cultured in 52 incubator at 37 鈩，
本文编号：2177714
本文链接：https://www.wllwen.com/yixuelunwen/waikelunwen/2177714.html
最近更新
教材专著
