慢性偏头痛模型的建立及相关区域Cav2．1通道的表达

Establishment of chronic inflammatory migraine models and expression of P/Q type calcium channel Cav2.1

目的探索慢性偏头痛模型的建立方法并研究Cav2．1通道与偏头痛的关系。方法18只C57BL／6小鼠按随机数字表法分成对照组、模型组及假手术组．每组6只。模型组采用炎性汤反复间断刺激硬脑膜，对照组则给予间质液刺激。给药8次后，观察小鼠烦躁情况（如挠头次数）等行为学变化：免疫组化染色检测小鼠三叉神经脊束核尾侧亚核c-fos及中脑导水管周围灰质降钙素基因相关肽（CGRP）表达；采用荧光定量PCR、Westernblotting检测小鼠皮层和三叉神经节、三叉神经颈复合体Cav2．1通道mRNA及蛋白的表达。结果末次给药后观察发现，模型组2h内挠头次数[（497±78）次]明显高于对照组[（117±34）次]，差异有统计学意义（P〈0．05）。免疫组化染色检测发现，模型组C-FOS阳性表达细胞数（220±20）明显高于对照组（30±4），差异有统计学意义（P〈0．05）；模型组CGRP表达平均吸光度值（13．00±0．01）明显高于对照组（4．50±0．02），差异均有统计学意义（P〈0．05）。荧光定量PCR、Western blotting检测发现，模型组三叉神经节、三叉神经颈复合体Cav2．1通道mRNA相对表达量分别是对照组的（1．60±0．17）倍、（1．42±0．15）倍，差异均有统计学意义（P〈0．05）；模型组皮层和三叉神经节、三叉神经颈复合体Cav2．1通道蛋白条带灰度值较对照组分别增加（1．50±0．02）倍与（1．30±0．01）倍，差异均有统计学意义（P〈0．05）。结论反复炎性刺激小鼠硬脑膜可作为慢性偏头痛模型建立的一种可行方案；Cav2．1通道可能通过表达上调来调控偏头痛的发生。

Objective To explore the establishment of models of chronic inflammatory migraine and preliminary study on the relationship between P/Q type calcium channel Cav2.1 and migraine. Methods Eighteen C57BL/6 mice were randomly divided into control group, model group and sham-operated group （n=6）. In model group, the dura mater of mice were consciously stimulated by inflammatory soup, and in the control group, interstitial fluid was given. Eight times after the treatment, behavioral observation of irritable condition of mice （scratching frequency changes） was performed; cal staining was employed to detect the c-los expression in the trigeminal nucleus caudalis and calcitonin gene related peptide （CGRP） expression in the eriaqueductal gray aqueduct. Fluorogenic quantitative PCR and Western blotting were used to detect the P/Q-type calcium channel Cav2.1 mRNA and protein expressions. Results Behavioral results showed that the scratching frequency of the model group within 2 h of final administration （[497±78] times） was significantly higher than that of the control group （[117±34] times, （P〈0.05）. Number of neurons with c-los gene positive expression in the model group （220±20） was significantly larger than that in the control group （30±4, P〈0.05）. The mean density of CGRP expression （13.00±0.01） was significantly higher than that in the control group （4.50±0.02, P〈0.05）. The relative mRNA expressions of Car2.1 gene in the trigeminal nerve and trigeminocervical complex of model group increased （1.60±0.17） and （1.42±0.15） times as compared with those in the control group （P〈0.05）; the gray values of Cav2.1 protein in the cortex and trigeminocervical complex of model group were increased （1.50±0.02） and （1.30±0.01） times as compared with those in the control group （P〈0.05）. Conclusion Repeated inflammatory stimulation of the dura mater can provide experimental basis for chronic migraine models, and P/Q type calcium channel Car2.1 may regulate the occurrence of migraine via up-regulated expression.