人羊膜间充质干细胞移植对大鼠脊髓损伤后神经功能的影响

发布时间：2018-01-02 01:23

本文关键词：人羊膜间充质干细胞移植对大鼠脊髓损伤后神经功能的影响　出处：《遵义医学院》2009年硕士论文　论文类型：学位论文

【摘要】： 目的人羊膜间充质干细胞(hAMSCs)具有表型可塑性特征,作为再生医学的细胞资源已颇受重视。但目前尚未见有关hAMSCs体内移植治疗脊髓损伤的报道。本研究旨在探讨hAMSCs移植对大鼠脊髓损伤后神经功能恢复的影响,以评价hAMSCs在脊髓损伤修复中的应用价值。方法①采用胰酶-胶原酶两步消化法从足月分娩的人羊膜组织中分离hAMSCs,用流式细胞仪(FCM)和免疫荧光染色对其细胞表型进行鉴定。②雌性SD大鼠40只,制备脊髓全横断损伤模型,随机分为hAMSCs移植组和PBS对照组,每组20只,脊髓横断后立即以明胶海绵吸附10μl约2×10~5个hAMSCs植入脊髓两断端之间,对照组用明胶海绵吸附10μl PBS液同法植入。③术后每周按Basso Beatlie Bresnahan(BBB)评分标准测定各组大鼠神经功能,连续8周。④移植后第14 d各组随机处死3只大鼠,采用免疫荧光染色检测细胞移植区脊髓组织人细胞核抗原及神经细胞特异性蛋白MAP-2和GFAP的表达,以分析hAMSCs植活及分化情况。⑤术后第8周,采用免疫组化染色检测各组动物脊髓横断远端组织中神经纤丝(NF-200)的表达。结果①FCM分析结果显示,采用二酶法分离的hAMSCs不同程度表达CD29、CD44、CD71,其表达率分别为98.16%、50.96%和12.30%,而不表达造血细胞的表面标志CD34和CD45,HLA-DR亦为阴性;免疫荧光染色结果显示,hAMSCs表达间充质细胞标志波形蛋白,而不表达上皮细胞标志CK19。②免疫荧光染色显示,hAMSCs移植后第2周,大鼠脊髓移植区可见人细胞核抗原阳性细胞,但未检测到神经细胞特异性蛋白MAP-2和GFAP表达。③BBB评分结果表明,木后第2周开始直至第8周,hAMSCs移植组大鼠BBB分值从3.95±0.78升至7.67±0.90,均高于对照组(均P＜0.05)。④hAMSCs移植后8周,损伤脊髓远端组织中NF-200的表达强于对照组。结论hAMSCs移植能有助于大鼠脊髓损伤后的神经功能恢复,其机制可能与hAMSCs促进受损脊髓远端组织表达NF-200有关。
[Abstract]:Objective to study the phenotypic plasticity of human amniotic mesenchymal stem cells (hAMSCs). As a cell resource of regenerative medicine, however, there is no report on the treatment of spinal cord injury by in vivo transplantation of hAMSCs. The purpose of this study was to investigate the effect of hAMSCs transplantation on the nerve after spinal cord injury in rats. Effects of functional recovery. To evaluate the value of hAMSCs in spinal cord injury repair. Methods 1 hAMSCs was isolated from human amniotic membrane by trypsin collagenase two-step digestion. 40 female Spinal Cord Sprague-Dawley (SD) rats were identified by flow cytometry (FCM) and immunofluorescence staining. 20 rats in each group were randomly divided into hAMSCs transplantation group and PBS control group. After spinal cord transection, 10 渭 l of gelatin sponge was adsorbed into the spinal cord between the two ends of the spinal cord with about 2 脳 10 ~ 5 hAMSCs. In the control group, 10 渭 l PBS solution was absorbed by gelfoam and implanted weekly with Basso Beatlie Bresnahan. The neurological function of rats in each group was measured by scoring standard. Three rats in each group were randomly killed on the 14th day after transplantation for 8 weeks. The expression of human nuclear antigen (HNA) and neuron-specific proteins (MAP-2 and GFAP) in the spinal cord of the transplanted area were detected by immunofluorescence staining. The expression of NF-200 was detected by immunohistochemical staining at 8 weeks after transplantation and differentiation of hAMSCs. Results 1 the results of FCM analysis showed that hAMSCs isolated by two-enzyme method expressed CD29, CD4, CD41 and CD71 in different degrees, and the expression rates were 98.16%, respectively. 50.96% and 12.30%, while the surface markers of CD34 and CD45 were also negative. Immunofluorescence staining showed that hAMSCs expressed mesenchymal cell marker vimentin, but no epithelial cell marker CK19.2 immunofluorescence staining showed that hAMSCs were transplanted 2 weeks after transplantation. The positive cells of human nuclear antigen were found in the transplanted area of spinal cord of rats, but the expression of neuronal specific protein MAP-2 and GFAP were not detected. The BBB score of rats in the hAMSCs transplantation group increased from 3.95 卤0.78 to 7.67 卤0.90 from the 2nd week after wood transplantation to the 8th week. The expression of NF-200 in the injured distal spinal cord tissue was higher than that in the control group at 8 weeks after transplantation (P < 0.05). Conclusion hAMSCs transplantation can contribute to the recovery of neural function after spinal cord injury in rats, and the mechanism may be related to the promotion of NF-200 expression in the distal spinal cord by hAMSCs.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329.28

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