摘要
目的探讨促分裂原和应激激活的蛋白激酶1(mitogen-and stress-activated protein kinase 1,MSK1)在大鼠脊髓损伤后的表达变化及对脊髓损伤的修复作用。方法雄性SD大鼠120只(体质量220～250 g),取其中70只随机分为假手术组和脊髓损伤组(n=35),脊髓损伤组按照Allen法制作大鼠脊髓损伤模型,假手术组仅打开椎板不损伤脊髓;造模后8、12 h及1、2、3、5、7 d取材(n=5)行Western blot检测MSK1及增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达变化。另取20只SD大鼠同上法分组(n=10),于脊髓T10水平背侧深约0.8 mm处注射阴性对照慢病毒LV3NC稀释液,倒置荧光显微镜下观察1、3、5、7、14 d的转染效果,确定病毒最佳转染时间。将剩余30只SD大鼠随机分为单纯脊髓损伤组(A组)、脊髓损伤后转染阴性对照慢病毒LV3NC组(B组)和脊髓损伤后转染MSK1小干扰RNA(small interfering RNA,siRNA)组(C组),每组10只。于术后1、3、5、7、14 d行大鼠后肢功能BBB评分;于病毒最佳转染时间点取材行Western blot检测脊髓组织中MSK1及PCNA蛋白表达变化,并采用免疫荧光染色检测MSK1及PCNA在脊髓中的定位表达。结果 Western blot检测示假手术组各时间点脊髓组织中的MSK1及PCNA蛋白表达无明显变化。脊髓损伤组MSK1蛋白表达于伤后8 h开始逐渐下降,至伤后3 d降至最低,而后逐渐上升;PCNA蛋白表达与MSK1表达趋势相反。脊髓损伤组伤后1、2、3、5 d MSK1蛋白表达量显著低于假手术组,伤后8 h及1、2、3、5、7 d PCNA蛋白表达量显著高于假手术组,差异均有统计学意义(P<0.05)。脊髓损伤组及假手术组大鼠脊髓组织在慢病毒转染后各时间点均有较强的荧光表达,转染7 d内荧光强度与时间成正相关,7 d时达高峰。随术后时间延长,A、B、C组BBB评分均呈逐渐上升趋势,术后5、7、14 d C组BBB评分显著低于A、B组(P<0.05)。慢病毒转染7 d后,Western blot检测示C组MSK1蛋白表
Objective To investigate the expression changes and the repair effect of mitogen and stress-activated protein kinase 1（MSK1） on spinal cord injury（SCI） in rats. Methods One hundred and twenty male Sprague Dawley（SD） rats（weighing 220-250 g） were used for the study, 70 of them were randomly divided into sham-operation group and SCI group（n=35）, the rats in SCI group were given SCI according to Allen＇s method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day,2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen（PCNA） by Western blot assay. Another 20 SD rats were grouped by the same method as above（n=10）. In these rats, a negative control lentiviral LV3 NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3 NC injected after SCI, and group C with MSK1 small interfering RNA（siRNA） lentivirus injected after SCI, with 10 rats each group.The Basso, Beatlie, Bresnahan（BBB） score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins.Results Western blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from8 hours after injury to the lowest level at 3 da
作者
钟泽祥
周亦楠
冯思思
黄宇
陈宣维
ZHONG Zexiang;ZHOU Yinan;FENG Sisi;HUANG Yu;CHEN Xuanwei(Department of Spine Surgery, the First Affiliated Hospital, Fujian Medical University, Fuzhou Fujian, 350005, P.R.China;Department of Pathology and Pathophysiology, Fujian Medical University, Fuzhou Fujian, 350005, P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2018年第7期941-950,共10页
Chinese Journal of Reparative and Reconstructive Surgery
基金
福建省卫计委医学创新课题资助项目(2014-CX-22)
福建省自然科学基金资助项目(2015J01388)~~
关键词
慢病毒
小干扰RNA
促分裂原和应激活化蛋白激酶1
增殖细胞核抗原
脊髓损伤
大鼠
Lentivirus
small interfering RNA
mitogen- and stress-activated protein kinase 1
proliferating cellnuclear antigen
spinal cord injury
rat
