脊髓缺血再灌注损伤对大鼠CaMKIV基因及蛋白表达的影响

Effect of ischemia-reperfusion injury on expression of CaMKIV gene and protein in spinal cord of rats

摘要:

目的 分析大鼠脊髓缺血再灌注损伤（SCII）后钙调蛋白依赖性蛋白激酶4（CaMKIV）基因 及蛋白表达的变化。方法 雄性SD 大鼠84 只，按随机数字表法分为假手术组、SCII 0 d 组、SCII 1 d 组、 SCII 3 d 组、SCII 7 d 组、SCII 14 d 组、SCII 28 d 组，每组12 只。假手术组仅暴露肾动脉下腹主动脉，不阻 断。SCII 各组采用Naslund 法阻断腹主动脉，复制SCII 模型。采用免疫组织化学法、Western blot、实时荧光 定量聚合酶链反应（qRT-PCR）检测各组大鼠CaMKIV 蛋白及基因的表达，BBB 评分评价大鼠后肢运动功 能。结果 免疫组织化学法结果显示，假手术组CaMKIV 蛋白表达很少，SCII 组CaMKIV 蛋白高表达于神经 元的细胞浆和细胞核；在伤后3 d，CaMKIV 阳性细胞数最多，且高于假手术组（P <0.05）。Western blot 结果 提示，CaMKIV 蛋白表达在再灌注0 d 即高于假手术组（P <0.05），于再灌注3 d 达峰值后逐渐降低；至28 d， CaMKIV 蛋白表达量与假手术组比较，差异无统计学意义（P >0.05）。qRT-PCR 结果表明，CaMKIV 基因表 达在再灌注0 d 开始增加，与假手术组比较，差异有统计学意义（P <0.05），于3 d 达峰值后降低，至28 d 仍 高于假手术组（P <0.05）。BBB 评分显示，SCII 各组大鼠后肢功能随再灌注时间延长有所改善，但至28 d 时 仍差于假手术组。结论 SCII 早期可快速诱导CaMKIV 基因表达上调和蛋白合成，CaMKIV 在SCII 中可能发 挥重要作用。

Abstract:

Objective To observe the change law of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV ) gene and protein in spinal cord ischemia-reperfusion injury (SCII) rats. Methods Using random number table, 84 male Sprague-Dawleys rats were randomly and equally divided into sham group, SCII 0 day group, SCII 1 day group, SCII 3 days group, SCII 7 days group, SCII 14 days group, and SCII 28 days group. The rats of the sham group were only subjected to exposure of the abdominal aorta below renal artery but without occlusion; in the SCII groups, the SCII model was established by blocking abdominal aorta using Naslund method. Immunohistochemistry, Western blot and qRT-PCR technique were used to detect CaMKIV protein and gene expressions respectively. BBB score was utilized to evaluate movement function of rat hindlimbs. Results According to the results of immunohistochemistry, CaMKIV protein was expressed less in the sham group, while was highly expressed in neuronal cytoplasm and nuclei in the SCII groups, and the number of CaMKIV-positive cells was maximal on the 3rd day after injury and obviously larger than that of the sham group, the difference was statistically significant (P < 0.05). Western blot results suggested that CaMKIV protein expression in the SCII 0 day group was significantly higher than that in the sham group (P < 0.05), reached the peak value in the SCII 3 day group and then reduced, on the 28th day after reperfusion the expression of CaMKIV protein had no significant difference from that in the sham group (P > 0.05). qRT-PCR showed CaMKIV gene expression began to increase from the day of reperfusion, and compared with the sham group the difference was statistically significant (P < 0.05), and then reduced from 3 days after reperfusion, but was still higher than that of the sham group till the 28th day after reperfusion (P < 0.05). BBB score showed that the hindlimb function of the rats in each SCII group was improved with the prolongation of reperfusion time, but was still worse than that of the sham group till the 28th day. Conclusions Early-stage ischemia-reperfusion injury of spinal cord can rapidly induce CaMKIV gene expression up-regulation and protein synthesis. CaMKIV may play an important role in spinal cord ischemia-reperfusion injury.