摘要
目的探讨细胞周期素依赖激酶抑制剂olomouc ine对脊髓损伤后轴突再生微环境的影响及意义。方法建立脊髓半切损伤模型,实验大鼠随机分为假手术组、损伤对照组和olomouc ine干预组,采用W estern印迹分析脊髓损伤后细胞周期相关蛋白的表达;应用免疫荧光技术检测损伤区域胶质纤维酸性蛋白(GFAP)、硫酸软骨素蛋白多糖(CSPG)以及生长相关蛋白43(GAP-43)的表达;采用改良的Gale联合评分法对大鼠瘫痪后肢进行运动功能评估。结果假手术组细胞周期素(cyc lin)A、B1、E和增殖细胞核抗原以及GFAP、CSPG和GAP-43的表达较弱;脊髓损伤后cyc linA、cyc linB1、cyc linE和细胞周期素(PCNA)等细胞周期相关蛋白的表达显著增高,星形胶质细胞明显活化增殖,GFAP和CSPG的表达明显增强(均P<0.01),GAP-43的表达高于假手术组(P<0.05);给予olomouc ine干预后,细胞周期相关蛋白的表达显著下调,损伤区域星形胶质细胞的增殖和胶质瘢痕形成受到抑制,胶质瘢痕产生的CSPG的表达明显减少,GAP-43的表达显著增高,瘫痪后肢的运动功能明显改善(均P<0.05)。结论olomouc ine可通过调控细胞周期,抑制脊髓损伤后胶质细胞的活化增殖和胶质瘢痕的形成,减少抑制因子CSPG的表达以及上调有利于轴突再生的GAP-43的表达,改善损伤后轴突再生微环境最终促进瘫痪后肢的运动功能恢复。
Objective To investigate the effects of olomoucine, a cyclin dependent protein kinase （CDK） inhibitor, on the microenvironment of axonal regeneration after spinal cord injury （SCI）. Methods Forty-five SD rats were randomly divided into 3 equal groups： SCI group undergoing SCI by hemisection technique and peritoneal injection of dimethyl sulfoxide （DMSO） solution 30 min after the SCI, SCI ＋ olomoucine （SCI ＋ Olo） group undergoing SCI by hemisection technique and peritoneal injection of olomoucine solution 30 vain after the SCI, and sham operation group undergoing sham operation and peritoneal injection of DMSO solution 30 min after the operation. Three days after the operation the injured spinal cord segments of 5 rats from each group were taken out. Western blotting was used to detect the expression of the cell cycle related proteins, cyclin A, cyclin B, cyclin E, and proliferating cell nuclear antigen （PCNA）. Immunofluorescence （IF） staining was used to detect the expression of glial fibriliary acidic protein （GFAP）, growth associated protein-43 （GAP-43） and chondroitin sulphate proteoglycan （CSPG）. Four weeks after the operation specimens of the injured spinal cord segment 15 mm in length were taken out from 5 rats in each group to undergo histological examination. The locomotion function of the hindlimbs was determined by modified Gale combined behavioral scoring （SBS） 1 day and 1, 2, 4, 6, and 8 weeks after the operation. Results Western blotting 3 days after the operation showed that the expressions of cyclin A, cyclin B, cyclin E, and PCNA were very weak in the sham operation group, were significantly increased in the SCI group , and were significantly down - regulated in the SCI ＋ Olo group compared with those of the SCI group. IF staining showed that the number of astrocytes was small and the expressions of GFAP, CSPG, and GAP-43 were weak in the sham operation group; in the SCI group the astrocytic proliferation and glial scar was obvious, and the expressions of GFAP, CSPG, and GAP-43 were significantly increased compared with those of the sham operation group （ all P 〈 0. 05 ） ; and the astrocytic proliferation was significantly weaker and no obvious glial scar could be seen, and the expressions of GFAP and CSPG were weaker in the SCI ＋ Olo group in comparison with the SCI group, however, the GAP-43 expression of the sham operation group was significantly increased compared with that of the sham operation group （P 〈 0. 05）. The hindlimbs of the SCI ＋ Olo group and sham operation group were paralyzed without significant difference in the CBS values between these 2 groups, however, two weeks after the operation, the locomotion function scores at different time points of the SCI ＋ Olo group were all significantly improved in comparison with that of the SCI group （ all P 〈 0. 05 ）. Conclusion Olomoucine promotes the recovery of the locomotion function of the paralyzed hindlimbs, probably through microenvironmental improvement of axonal regeneration by inhibiting the glial scar formation and CSPG secretion as well as upregulating the GAP-43 expression.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第13期901-905,共5页
National Medical Journal of China
基金
国家自然科学基金重点资助项目(30230140)
