山东大学学报 (医学版) ›› 2024, Vol. 62 ›› Issue (6): 17-29.doi: 10.6040/j.issn.1671-7554.0.2024.0163
• 基础医学 • 上一篇
卫嘉晟1,杨保仲2,魏伟1,薛亚婷1,崔臣龙1,方俊1
WEI Jiacheng1, YANG Baozhong2, WEI Wei1, XUE Yating1, CUI Chenlong1, FANG Jun1
摘要: 目的 探讨microRNA-210-3p(miR-210-3p)与10-11易位蛋白2(ten-eleven translocation 2,TET2)在完全弗氏佐剂(complete freunds adjuvant, CFA)诱导的大鼠炎性疼痛模型中的作用及其相互调控机制。 方法 通过生物信息学方法和双荧光素酶实验,分析并确定大鼠miR-210-3p中可以靶向调节的基因。实验中的质粒和miR-210-3p共转染组合分为pmirGLO+mimics NC组、pmirGLO+mimics-miR-210-3p组、TET2-WT-pmirGLO+mimics-NC组、TET2-WT-pmirGLO+mimics-miR-210-3p组、TET2-MT-pmirGLO+mimics-NC组和TET2-MT-pmirGLO+mimics-miR-210-3p组;60只大鼠按随机数字表法分为正常对照(normal control, CON)组(n=20)、CFA组(n=20)、CFA+腺相关病毒载体阴性对照(adeno-associated virus negative control, AAV NC)组(n=10)、CFA+AAV miR-210-3p抑制剂(adeno-associated virus miR-210-3p inhibitor, AAVi)组(n=10)。通过在大鼠左后足底部皮下注入CFA的方式建立大鼠炎性疼痛模型;通过尾静脉注入miR-210-3p inhibitor的AAV建立干预模型;观察并测量大鼠行为学;采用RT-qPCR法检测miR-210-3p的表达量;采用Western blotting法和免疫荧光染色法检测L4~L6腰膨大节段脊髓中TET2蛋白的表达水平及荧光强度的变化;采用免疫荧光染色法观察TET2蛋白在大鼠脊髓中的细胞表达定位。 结果 生物信息学方法发现,TET2基因3'UTR区域存在与miR-210-3p的结合位点;双荧光素酶报告基因实验证实了miR-210-3p与TET2基因之间存在结合位点,呈负向调控关系;注射CFA显著减小了大鼠的机械缩足反射阈值(paw withdrawal mechanical threshold, PWMT)和热缩足潜伏期(paw thermal withdrawal latency, PTWL)(P<0.05);CFA组大鼠脊髓腰膨大中miR-210-3p的表达水平明显上调,伴随着TET2的表达水平降低(P<0.05);免疫荧光结果显示,TET2蛋白主要和神经元细胞存在共定位:CFA组大鼠脊髓内TET2蛋白表达水平降低(P<0.05);经过AAVi干预后,CFA+AAVi组大鼠在各个时间的PWMT和PTWL较CFA+AAV NC组大鼠升高(P<0.05);CFA+AAVi组大鼠脊髓组织中TET2蛋白表达较CFA+AAV NC组升高(P<0.05)。 结论 miR-210-3p可以抑制TET2蛋白表达,通过抑制miR-210-3p在炎性疼痛大鼠中的表达可以有效减轻炎性疼痛。
中图分类号:
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