P27kip1及相关分子Jab1在大鼠损伤脊髓中的表达变化及意义

The P27kip1/Jab1 expression and its role in injured spinal cord of rats

中文关键词:  脊髓损伤  P27kip1  Jab1  胶质瘢痕

英文关键词:Spinal cord injury  P27kip1  Jab1  Glial scar

中文摘要:

  【摘要】 目的：观察大鼠脊髓损伤过程中P27kip1及相关分子Jab1在脊髓中的表达及细胞定位情况，探讨其在脊髓损伤后胶质细胞增生及胶质瘢痕生成中的作用。方法：取56只雄性SD大鼠，随机分为实验组与对照组，其中实验组按脊髓损伤时间分为6h、12h、1d、3d、5d、7d、14d组，实验组按照Allen′s法制作SD大鼠脊髓损伤模型，对照组则仅打开椎板，不损伤脊髓。首先利用Western Blot观察P27kip1及相关分子Jab1在脊髓损伤后的表达水平变化，然后利用免疫组化观察脊髓损伤后3d时的P27kip1及5d时Jab1的阳性细胞表达变化，最后利用免疫荧光双标观察脊髓损伤后5d时Jab1的细胞定位变化。结果：Western Blot结果显示P27kip1在脊髓损伤后6h、12h、1d、3d、5d、7d、14d组的相对灰度值分别是1.00±0.10、0.82±0.05、0.27±0.03、0.34±0.03、0.25±0.02、0.28±0.03、0.57±0.05，其中1d、3d、5d、7d、14d组与对照组（0.98±0.08）相比均明显降低（P<0.05），而Jab1的6h、12h、1d、3d、5d、7d、14d组的相对灰度值分别是0.58±0.08、0.53±0.04、0.89±0.11、1.06±0.24、1.22±0.26、0.85±0.16、0.25±0.03，其中6h、12h、1d、3d、5d、7d组与对照组（0.19±0.04）相比明显升高（P<0.05），P27kip1于脊髓损伤后表达下降，Jab1在脊髓损伤后表达上升。3d时实验组P27kip1免疫组化阳性细胞数为130.8±29.8个，与对照组（406.8±56.6个）相比减少（P<0.05）；5d时实验组Jab1免疫组化阳性细胞数为383.5±68.8个，与对照组（123.5±42.6个）相比增多（P<0.05），从形态学上进一步证明了这种变化。在免疫荧光双标实验中，5d时实验组Jab1与GFAP双阳性细胞数量为383.3±39.1个，较对照组（114.3±35.6个）明显增多（P<0.05）。结论：在大鼠脊髓损伤中，Jab1与P27kip1存在相互作用，Jab1参与脊髓损伤后P27kip1的降解，这可能与脊髓损伤后星形胶质细胞的过度增殖及胶质瘢痕的生成相关。

英文摘要:

  【Abstract】 Objectives: To investigate the expression and distribution of P27kip1 and Jab1 in SD rat models with spinal cord injury(SCI), to explore their functions in the process of spinal cord injury and proliferation of astrocytes followed immediately. Methods: 56 male Sprague-Dawley(SD) rats were randomly divided into two groups: sham group(simple vertebra excision) and spinal cord injury group. And the rats in spinal cord injury group were divided into 6h, 12h, 1d, 3d, 5d, 7d, 14d groups according to the length of time of injury. The adult SD rat SCI models were established by using improved Allen′s method, T9 level of spinal cord was chosen to induce injury. First of all, the temporal expression of P27kip and Jab1 were detected by Western Blot. Then, the P27kip1 positive cell number in 3d group and Jab1 positive cell number in 5d were counted in immunohistochemistry. At the last, the change of cellular localization of Jab1 in 5d group was observed by immunofluorescence. Results: The Western Blot result showed the relative grey value of P27kip1 in 6h, 12h, 1d, 3d, 5d, 7d, 14d groups was 1.00±0.10, 0.82±0.05, 0.27±0.03, 0.34±0.03, 0.25±0.02, 0.28±0.03, 0.57±0.05 respectively, and the 1d, 3d, 5d, 7d, 14d group had obviously lower value than sham group(P<0.05). On the contrary, the relative grey value of Jab1 in 6h, 12h, 1d, 3d, 5d, 7d, 14d groups was 0.58±0.08, 0.53±0.04, 0.89±0.11, 1.06±0.24, 1.22±0.26, 0.85±0.16, 0.25±0.03 respectively, and the 6h, 12h, 1d, 3d, 5d, 7d groups had higher value than sham group(P<0.05). And the positive cell number in 3d spinal cord injury and P27kip1 group was 130.8±29.8, which was lower than sham group(406.8±56.6)(P<0.05). The positive cell number of 5d spinal cord injury and Jab1 group was 383.5±68.8, which was higher than sham group(123.5±42.6)(P<0.05). Moreover, double labeling immunofluorescence of 5d Jab1 group presented that the Jab1 and GFAP double positive cell number was significantly more than that of the sham group(spinal cord injury group: 383.3±39.1, sham group: 114.3±35.6, P<0.05). Conclusions: The up regulation of Jab1 is related to the degradation of P27kip1 in spinal cord injury of rats, which may be involved in the excessive proliferation of astrocytes.