脱细胞脊髓支架的制作及其体内安全性评价的实验研究

发布时间：2018-09-12 10:18

【摘要】：目的本研究旨在应用脱细胞的方法将细胞完全去除,成功制作出大鼠脱细胞脊髓支架,并且最大限度的保留了脊髓细胞外基质的空间三维立体结构。将制备好的脱细胞脊髓支架磨碎后用生理盐水溶解制成脱细胞脊髓支架颗粒悬浊液,通过大鼠尾静脉注射,来评估其在大鼠体内的安全性,从而为脱细胞脊髓支架应用于治疗脊髓损伤提供依据。方法1.取Wistar大鼠4只,麻醉后手术取出脊髓,采用脱细胞方法将脊髓中神经细胞脱掉后,制作出脱细胞支架。将制备的支架进行HE染色及髓鞘染色光镜下观察脱细胞脊髓组织结构,验证大鼠脊髓支架是否成功脱掉细胞成分。2.取制备的脱细胞脊髓支架,磨碎后放入500ml生理盐水中,制成脱细胞脊髓支架悬浊液。取Wistar大鼠48只,平均分成2组,一组为实验组,另一组为对照组,每组24只,再分为6个时间节点,每个时间节点4只,分别对应1天、2天、3天、7天、10天、14天,实验组每只大鼠经尾静脉注射脱细胞脊髓悬浊液2ml,对照组每只大鼠经尾静脉注射生理盐水2ml,分别于尾静脉注射后1天、2天、3天、7天、10天、14天取血后处死,检测血清中谷丙转氨酶(ALT)和肌酐(CR)含量,进行统计学分析,处死的大鼠分别取肺脏、肝脏、脾脏、心脏及肾脏制作成组织切片进行HE染色后观察其组织学变化。结果1.采用化学萃取法制备的Wistar大鼠脱细胞脊髓支架,呈白色,短柱状,大小有所减小,触之质地柔韧。HE染色、髓鞘染色显示成功制备的支架由直径不同的孔隙组成,细胞已彻底脱掉,已不含细胞成分,髓鞘已被脱除,各个孔洞的壁由细胞外基质组成。2.实验组大鼠经尾静脉注射脱细胞脊髓悬浊液后,肺部出现明显的炎症反应和增生表现。实验组肝细胞较对照组肿胀,相邻细胞之间边界显示不清,胞核变大,胞浆相对松散,表现为空泡变性。此现象在经尾静脉注射后第1天较重,随时间延长不断减轻,到第10天肝细胞恢复到正常形态。通过测定血液中谷丙转氨酶(ALT)的含量,发现前两天谷丙转氨酶含量处在较高水平,之后逐渐降低到正常值。肾脏病理检查未见明显改变,血清学检查发现肌酐(CR)值在实验组与对照组之间无差异。实验组大鼠脾脏组织切片表现为淋巴小结较对照组增大,边缘区界限模糊,这种现象随着时间的延长而逐渐减轻。实验组心脏组织切片无显著异常。结论1.本实验采用化学萃取的方法脱掉脊髓中的细胞成分,成功制作出大鼠脱细胞脊髓支架,将细胞成分彻底脱除,并且最大限度的保留了脊髓细胞外基质的空间三维立体结构。2.将脱细胞脊髓支架悬浊液经大鼠尾静脉注射后,对大鼠体内部分器官存在一过性的毒性反应,对肝脏组织形态和肝功能以及肺的组织形态具有一过性损害,在脾脏一过性引发由B细胞参与的体液免疫,对心脏细胞的组织形态无损害,对肾脏的组织形态及肾功能无损伤。相对传统的生物材料支架,脱细胞脊髓支架具有较好的生物相容性、机械强度及形态。为将来进行脱细胞脊髓支架移植治疗脊髓损伤提供了依据。
[Abstract]:Objective To prepare acellular spinal cord scaffolds by acellular method and to preserve the three-dimensional structure of extracellular matrix of spinal cord. To evaluate the safety of acellular spinal stent in rats by tail vein injection, so as to provide basis for the application of acellular spinal stent in the treatment of spinal cord injury. Methods 1. Four Wistar rats were taken out of spinal cord after anesthesia. The acellular scaffold was made after the nerve cells in spinal cord were removed by acellular method. E staining and myelin sheath staining were used to observe the structure of acellular spinal cord and to verify whether the acellular spinal cord scaffolds were successfully removed. 2. The acellular spinal cord scaffolds were prepared and ground into 500 ml normal saline to form acellular spinal cord scaffold suspension. The control group, 24 rats in each group, were divided into 6 time nodes, each time node 4, corresponding to 1 day, 2 days, 3 days, 7 days, 10 days, 14 days, the experimental group each rat through the tail vein injection of acellular spinal cord suspension 2 ml, the control group each rat through the tail vein injection of normal saline 2 ml, respectively in the tail vein injection 1 day, 2 days, 3 days, 7 days, 10 days, 14 days to take Serum ALT and CR levels were measured and analyzed statistically. Lung, liver, spleen, heart and kidney were taken from the sacrificed rats and stained with HE. The histological changes were observed. Results 1. The acellular spinal cord scaffold of Wistar rats prepared by chemical extraction was white and short. HE staining and myelin sheath staining showed that the scaffolds were composed of pores with different diameters, the cells had been completely removed, and the myelin sheath had been removed. The walls of the holes were composed of extracellular matrix. 2. After injection of acellular spinal cord suspension into the tail vein, the rats in the experimental group were pulmonary. The hepatocytes in the experimental group were swollen, the boundary between adjacent cells was unclear, the nucleus was enlarged and the cytoplasm was relatively loose, showing vacuolar degeneration. Glutamic-pyruvic aminotransferase (ALT) levels in the blood were determined, and the ALT levels were higher two days ago, and then gradually decreased to normal values. There was no significant abnormality in the cardiac tissue sections of the experimental group. Conclusion 1. The acellular spinal cord scaffold was successfully made by chemical extraction, and the acellular spinal cord scaffold was completely removed and preserved to the maximum extent. The three-dimensional structure of spinal extracellular matrix was preserved. 2. After the acellular spinal cord scaffold suspension was injected into the tail vein of rats, transient toxic reactions were found in some organs of rats, which caused transient damage to liver morphology, liver function and lung morphology, and B cells participated in the transient damage of spleen. Compared with traditional biomaterial scaffolds, acellular spinal cord scaffolds have better biocompatibility, mechanical strength and morphology, which provide a basis for the future treatment of spinal cord injury with acellular spinal cord scaffolds.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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