大鼠脊髓损伤模型的改良及伤后再生基因-2蛋白的表达变化

摘要

目的:制备改良的大鼠脊髓损伤(SCI)动物模型,并探讨再生基因(Reg)-2蛋白在SCI后的表达规律.方法:采用36只SD大鼠.参考Allen法,使用自制打击装置致大鼠T13段脊髓中度损伤.以行为联合评分法(CBS)评定模型的可靠性.免疫印迹法和免疫组织化学(SABC)法对正常对照组、伤后第1天、第2天、第3天、第5天和第7天的大鼠脊髓组织中的Reg-2蛋白进行检测.结果:SCI后大鼠一般情况符合临床损伤特点,且稳定性强；各损伤组大鼠神经功能联合评分均呈明显下降趋势,与正常对照组比较差异具有显著性意义(P＜0.05)；在正常对照组大鼠脊髓神经元内有微量的Reg-2蛋白表达(阳性细胞数为17.3±2.6,Reg-2相对表达量为0.038±0.007).SCI后1天,大鼠脊髓内Reg-2表达的免疫阳性细胞随着损伤时间的推移逐渐增多,至伤后第7天仍呈高水平表达(阳性细胞数为90.0±3.6,相对表达量为0.694±0.018),各组间比较差异具有显著性意义(P＜ 0.05).伤后3天内,Reg-2免疫阳性细胞以后角神经元为主,而伤后7天以前角神经元和胶质细胞为主.结论:本实验装置制作的大鼠SCI模型稳定、可靠；SCI后Reg-2蛋白表达开始升高,对受损神经起保护和修复作用.%Objective: To design and produce a kind of modified rat model of spinal cord injury(SCI), and explore the expression pattern and effects of regenerating gene (Reg) -2 protein after SCI in rats.rnMethod: Thirty-six SD rats were subjected to moderate SCI by modified Allen's crush method with a self-designed experimental device at T13 level of spinal cord. Combined behavioral score(CBS) was used to assess the reliability of model. Western-blot and immunohistochemical techniques (SABC) were used to detect the expression of Reg-2 in rats of different groups (normal control, 1, 2, 3, 5 and 7d post-injury).rnResult: After operation the general health status of rat model was accorded with the clinical characteristics of SCI in human, and was with high-stability. The CBS in model groups declined with the time of injury, and compared with the normal control the difference was statistically significant (P<0.05). In normal control group, the number of Reg-2-positive cells and relative expression of Reg-2 in spinal cord were 17.3 ± 2.6 and 0.038 ± 0.007. At Id post-injury the level of Reg-2 elevated and reached a high level at 7d post-injury (the number of Reg-2-positive cells and relative expression of Reg-2 were 90.0 ± 3.6 and 0.694 ± 0.018), the difference compared with model groups were statistically significant (P < 0.05). In the early phase of injury (in 3d post-injury), neurons　in dorsal horn of spinal cord were accounted for the most part of Reg-2-positive cells. But the main Reg-2-positive cells were neurons of spinal anterior horn and gliocytes in the later phase of injury.rnConclusion: The modified rat model prepared with our self-designed experimental device was with high-stability and higher reliability, and the experimental results suggested that SCI could stimulate the Reg-2 expression, which might contribute to maintenance of nerve cell survival and repair of damaged neural tissues after SCI.