摘要
目的研究胞浆泛素蛋白连接酶2(Kip1 ubiquitylation-promoting complex 2,KPC2)在大鼠脊髓损伤(spinal cord injury,SCI)过程中的蛋白表达及细胞定位情况,探讨其在SCI修复过程中的生物学功能。方法将成年SD大鼠随机分为2组,对照组7只仅行单纯T9椎板全切除术,实验组49只采用改良Allen法制作T9节段脊髓撞击损伤模型,实验组于伤后6、12 h及1、3、5、7、14 d分别取7只大鼠进行以下检测。采用Western blot检测p27kip1、KPC2、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和细胞周期蛋白A(Cyclin A)在SCI前后的蛋白表达变化,免疫组织化学染色观察KPC2在SCI后的大体定位及表达,免疫荧光双标记染色观察KPC2在SCI过程中与神经元特异性核蛋白(neuronal nuclei,Neu N)、神经胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和PCNA的共定位情况。细胞水平采用体外培养星形胶质细胞增殖模型,Western blot检测KPC2、P27kip1、PCNA表达;免疫共沉淀分析KPC2、KPC1和p27kip1之间在细胞增殖过程中的相互作用。结果 Western blot结果示SCI后3 d,p27kip1显著下调,伴随KPC2、Cyclin A、PCNA表达明显增加。免疫组织化学染色示KPC2阳性信号广泛分布,包括脊髓灰质和白质,实验组KPC2阳性细胞数显著高于对照组(t=10.982,P=0.000)。免疫荧光双标记染色示在脊髓灰质,对照组和实验组KPC2与Neu N双标记阳性细胞数分别为(0.43±0.53)、(0.57±0.53)个/视野,比较差异无统计学意义(t=0.548,P=0.604);在脊髓白质,对照组和实验组KPC2与GFAP双标记阳性细胞数分别为(3.86±0.90)、(0.71±0.49)个/视野,差异有统计学意义(t=7.778,P=0.000);对照组和实验组KPC2与PCNA标记的星形胶质细胞共定位明显,阳性细胞数分别为(0.57±0.53)、(5.57±1.13)个/视野,差异有统计学意义(t=8.101,P=0.000)。体外培养并模拟星形胶质细胞增殖,提取细胞蛋白行Western blot示PCNA和KPC2蛋白表达在血清刺激增殖后即开始增加,24 h达峰值,而p27kip1表达则逐渐减少。免疫共沉淀示KPC2可沉淀p27kip1、KPC1,p27kip1也可沉淀KPC2、KPC1,且在刺激后相互作用明显增加。结论 SCI后KPC2参与介导的p27kip1表达下调,KPC2与SCI后星形胶质细胞的增殖相关。
Objective To explore the biological functions of Kipl ubiquitylation-promoting complex 2 （KPC2） in the repair process of spinal cord injury （SCI） by studying the expression and cellular localization of KPC2 in rat SCI models. Methods Fifty-six adult Sprague-Dawley rats were randomly divided into 2 groups： in the control group （n=7）, simple % laminectomy was performed; in the experimental group （n=49）, the SCI model was established at T9, 7 rats were used to detect follow indexs at 6 hours, 12 hours, 1 day, 3 days, 5 days, 7 days, and 14 days after SCI. Western blot analysis was used to detect the protein expressions of P27~pl, KPC2, CyclinA and proliferating cell nuclear antigen （PCNA） after SCI. Immunohistochemistry was used to observed the cellular localization of KPC2 after SCI, double- labeling immunofluorescence staining to observe the co-localization of KPC2 with neuronal nuclei （NeuN）, glial fibrillary acidic protein （GFAP） and PCNA. In vitro astrocytes proliferation model was used to further validate these results, Western blot to detect KPC2, p27kipl, and PCNA expressions. The interaction of p27~p~, KPC1, and KPC2 in cell proliferation was analyzed by co-immunoprecipitation. Results The Western blot analysis showed a significant down- regulation of p27~pl and a concomitant up-regulation of KPC2, CyclinA, and PCNA after SCI. Immunohistochemistry staining revealed a wide distribution of KPC2 positive signals in the gray matter and white matter of the spinal cord. The number of KPC2 positive cells in the experimental group was significantly higher than that in the control group （t= 10.982, P=0.000）. Double-labeling immunofluorescence staining revealed the number of KPC2/NeuN co-expression cells in the gray matter of spinal cord was （0.43＋0.53）/visual field in the control group and （0.57＋0.53）/visual field in the experimental group, showing no significant difference （t=0.548, P=0.604）; in the white matter of spinal cord, the number of KPC2/PCNA co-expression cells was （3.86＋0.90）/visual field in the control group and （0.71＋0.49）/visual field in the experimental group, showing significant difference （t=7.778, P=0.000）. And then, the number of KPC2/PCNA co- expression cells were （0.57＋0.53）/visual field in the control group and （5.57_＋ 1.13）/visual field in the experimental group, showing significant difference （t=8.101, P=0.000）. Concomitantly, there was a similar kinetic in proliferating astrocytes in vitro. The Western blot analysis showed a significant down-regulation of p27~pl and a concomitant up-regulation of KPC2 and PCNA after serum stimulated. Co-immunoprecipitation demonstrated increased interactions between p27ups, KPC 1, and KPC2 after stimulation. Conclusion The up-regulated expression of KPC2 after SCI is related to the down- regulation of p27ups, this event may be involved in the proliferation of astrocytes after SCI.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2015年第8期978-985,共8页
Chinese Journal of Reparative and Reconstructive Surgery
