摘要
背景:研究表明,miR NA在脊髓的发育、脊髓可塑性和脊髓损伤后的病理改变中均发挥着至关重要的调节作用。目的:分析miR-136-5p调控白细胞介素17刺激小鼠星形胶质细胞对A20蛋白表达的影响。方法:体外培养C57BL/6小鼠星形胶质细胞并进行免疫荧光染色鉴定。用白细胞介素17(100μg/L)分别刺激小鼠星形胶质细胞0,3,6,12,24 h,行RT-PCR检测白细胞介素6和肿瘤坏死因子αmRNA的相对表达量,以确定白细胞介素17最佳刺激时间,用不同质量浓度的白细胞介素17(10,20,50,100,200μg/L)刺激6 h,行RT-PCR检测白细胞介素6和肿瘤坏死因子αmRNA的表达量以确定白细胞介素17最佳刺激浓度。采用白细胞介素17(50μg/L)刺激小鼠星形胶质细胞6 h后,行RT-PCR检测星形胶质细胞产生的miR-136-5p及A20 mRNA的表达含量,并用Western blot检测A20蛋白的表达水平。此外,构建miR-136-5p表达抑制(miR-136-5p-inhibition)的慢病毒表达载体,转染小鼠星形胶质细胞,再次行白细胞介素17刺激并检测miR-136-5p、A20 mRNA及A20蛋白表达水平。结果与结论:(1)白细胞介素17刺激小鼠星形胶质细胞6 h后,miR-136-5p-inhibition抑制组与空白组相比,miR-136-5p表达显著降低(P<0.05);(2)经白细胞介素17(50μg/L)刺激6 h后,各组内A20 mRNA及A20蛋白的表达较刺激前明显降低(P<0.05);miR-136-5p-inhibition抑制组的A20 mRNA及A20蛋白表达与空白组相比显著升高(P<0.05),空白对照组与转染阴性对照组比较蛋白相对表达量没有显著性差异(P>0.05);(3)结果提示,miR-136-5p对白细胞介素17刺激的星形胶质细胞表达A20蛋白可产生影响。
BACKGROUND： miRNA plays a critical regulatory role in the development and plasticity of spinal cord, and pathological changes after spinal cord injury. OBJECTIVE： To study the effect of miR-136-5p on the A20 expression in mouse astrocytes stimulated by interleukin-17 （IL-17）. METHODS： C57BL/6 mouse astrocytes were cultured in vitro, identified by immunofluorescence staining, and then stimulated by 100 μg/L IL-17 for 0, 3, 6, 12 and 24 hours, respectively. The relative mRNA expression levels of IL-6 and tumor necrosis factor-α were detected by RT-PCR to determine the optimal stimulation time of IL-17. The mouse astrocytes were respectively stimulated by 10, 20, 50, 100 and 200 μg/L IL-7 for 6 hours, and similarly, the relative mRNA expression levels of IL-6 and tumor necrosis factor-α were detected to determine the optimal concentration of IL-17. At 6 hours after IL-17 （50 μg/L） stimulation, the mRNA expression levels of miR-136-5p and A20 in mouse astrocytes were detected by RT- PCR, and the protein expression level of A20 was detected by western blot assay. In addition, the lentiviral expression vector （miR-136-5p-inhibition） was constructed and transfected into the mouse astrocytes that were also stimulated by IL-7 to detect the expression levels of miR-136-5p, A20 mRNA and A20 protein. RESULTS AND CONCLUSION： Compared with the blank control group, the expression level of miR-136-5p in the miR-136-5p-inhibition group was significantly decreased after 6-hour IL-17 stimulation （P 〈 0.05）. The expression levels of A20 mRNA and protein in each group were significantly decreased after 6-hour IL-17 （50 μg/L） stimulation （P 〈 0.05）. The expression levels of A20 mRNA and protein in the miR-136-5p-inhibition group were significantly higher than those in the blank control group （P 〈 0.05）, while there were no significant differences in the expression level of A20 protein between blank control and negative groups （P 〉 0.05）. To conclude, miR-136-5p makes certain effect on the expression of A20 protein in astrocytes after IL-17 stimulation.
作者
施雄智
宗少晖
何基琛
彭小明
高云兵
邓贵营
Shi Xiong-zhi Zong Shao-hui He Ji-chen Peng Xiao-ming Gao Yun-bing Deng Gui-ying(Department of Spine Osteopathia, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China)
出处
《中国组织工程研究》
CAS
北大核心
2017年第16期2587-2592,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金资助项目(81560351)~~
