摘要
目的:探讨大鼠三叉神经节(TG)离体培养后降钙素基因相关肽(CGRP)表达增高的细胞内信号转导通路。方法:成年雄性SD大鼠TG在DMEM培养液离体培养12、24和48 h,应用免疫组化和实时定量PCR法分别检测CGRP阳性细胞数和mRNA表达水平变化。在培养液中分别加入一定浓度的炎症抑制剂地塞米松、转录水平特异性抑制剂放线菌素D、蛋白翻译水平特异性抑制剂环己酰胺,以及细胞内丝裂原激活的蛋白激酶(MAPK)信号转导系统ERK1/2、JNK、P38的3条信号通路上关键激酶的特异性阻断剂U0126、SP600125、SB239063与三叉神经节共同培养24 h,免疫组化和实时定量PCR法分别检测CGRP阳性反应细胞数和mRNA表达水平变化。结果:TG离体培养后12、24和48 h后CGRP阳性反应细胞数、平均吸光度和累积吸光度值均比新鲜组织明显增高,CGRP mRNA表达水平也明显增高。浓度为1×10^(-5)mol·L^(-1)的地塞米松、放线菌素D、环己酰胺以及U0126、SP600125分别与三叉神经节共同孵育24 h后,CGRP mRNA表达水平都显著降低。结论:TG离体培养后CGRP表达水平增高是通过细胞核内转录因子上调mRNA转录和蛋白翻译水平,是TG内非特异性炎症反应的结果,细胞内MAPK信号转导的ERK1/2通路和JNK信号通路参与了上调过程。
Aim： To explore the intracellular signaling pathways involved in the up-regulation of calcitonin gene-related peptide（CGRP） in rat trigeminal ganglion （TG） after organ culture. Methods： Immunohistochemistry and real-time polymerase chain reaction （real-time PCR） were used to detect the CGRP positive cells and CGRP mRNA expression levels in TG after 12 h, 24 h and 48 h incubation with DMEM culture. The specific transcriptional inhibitor actinomycin D, the translational inhibitor cyclohexamide, the extracellular signal- regulated kinase 1/2 （ERK1/2） inhibitor U0126, protein kinase 38 （P38） inhibitor SB239063 and Jun N-terminal kinase （JNK） inhibitor SP600125, and non-specific inflammation inhibitor dexamethasone were respectively co-incubated with TG for 24 h. Results： The number of CGRP-positive cells and the levels of mRNA expression in TG significantly increased after culture for 12 h, 24 h and 48 h, compared with fresh TG tissue. The 1 ×10^-5 mol.Ll concentration of dexamethasone, cyclohexamide, actinomycin D, U0126 and SP600125 could remarkably decrease the up-regulated CGRP mRNA independently. Conclusion： The increasing level of CGRP in TG after organ culture was regulated by up-regulation of mRNA transcription and translation. It would be a non-specific intracellular inflammation process. The intracellular ERK 1/2 and JNK signal transduction pathway of MAPK was involved in CGRP up-regulation.
出处
《中国临床神经科学》
2010年第2期113-118,共6页
Chinese Journal of Clinical Neurosciences
基金
国家自然科学基金资助项目(编号:30570631)
西安交通大学国际交流与合作重点项目(编号:062-10)
关键词
降钙素基因相关肽
三叉神经节
离体培养
信号转导
calcitonin gene-related peptide
trigeminal ganglion
organ culture
intracellularsignaling transduction
作者简介
罗国刚，男（1969-），汉族，陕西省宝鸡市人，博士，副主任医师，主要从事脑血管病和血管性头痛发生机制研究。
[通讯作者]吕社民，E-mail：1ushemin@mail．xjtu．edu．cn
