目的 研究炎症性内脏高敏大鼠背根神经节(DRG)基因表达的变化及电压门控钙离子通道(VGCC)在炎症性内脏高敏感性中的作用.方法 雄性清洁级SD大鼠180只,体重200～300 g,三硝基苯磺酸(TNBS)模型组(n=90)缓慢注入2.0%TNBS,剂量为100 mg/kg;正常对照组(n=90)仅灌注等量0.9%氯化钠溶液.造模后第4天,大鼠的cDNA表达谱基因芯片检测大鼠1.6S2节段DRG基因表达谱变化.通过实时定量逆转录聚合酶链反应(RT-PCR)和Western印迹检测进行验证.胞内钙检测及全细胞膜片钳记录分析胞内钙及电压门控钙电流变化.另取SD大鼠54只,均分为6组,分别予以TNBS造模及脊髓鞘内给予特异性钙通道阻滞剂,观察大鼠内脏敏感性的变化.腹部撤离反射(AWR)检测大鼠内脏敏感性.结果 TNBS模型组L6～s2节段DRG172个基因的表达出现显著变化,包括离子通道、膜受体及胞内第二信使等.其中,L-型钙通道(Cav 1.2)和R-型钙通道(Cav2.3)显著上调,RT-PCR及Western印迹检测结果均验证了芯片结果.胞内钙检测显示,TNBS模型组结肠特异感觉神经元的静息胞内钙含量与正常对照组相比差异无统计学意义(P＞0.05);而胞内钙瞬变量较正常对照组显著增加(P＜0.05).全细胞膜片钳记录显示TNBS模型组结肠初级感觉神经元L-型和R-型钙电流较正常对照组明显增加(P＜0.05).脊髓鞘内给予尼莫地平和SNX-482后可显著降低炎症性内脏高敏大鼠的内脏敏感性(P＜0.05).结论 Cav1.2和Cav2.3表达的上调在炎症性内脏高敏中起了非常重要的作用,很可能是炎症性内脏高敏治疗的潜在靶点.%Objective To screen the difference of gene expression in dorsal root ganglia (DRG)of inflammatory visceral hypersensitivity rats and to explore the role of voltage gated calcium channel (VGCC) in inflammatory visceral hypersensitivity. Methods Total 180 male Sprague-Dawley rats were in this study,the weight varied from 200 gram to 300 gram. 2,4,6-trinitrobenzenesulfonic acid (TNBS) model group was maken by 2. 0% TNBS slowly injection,the dosage was 100mg per kilogram. The normal control group was only injected with same volume of 0. 9% sodium chloride solution. After the model had been maken for four days,gene expression profiles of L6-S2 DRGs were tested by rat cDNA microarray chips. And the result was verified by RT-PCR and Western blot. The changes of intracellular Ca2+ and the voltage gated calcium currents were recorded by patch-clamp.The special Ca2+ channel blockers were given by intrathecal injection,and then the changes of visceral sensitivity were observed. The visceral sensitivity was measured by abdominal withdrawal reflex (AWR). Results There were significant changes of 172 genes expression in L6-S2 DRGs of TNBS model rats,which included Ca2+ channel,membrane receptor and intracellular second messenger. Of those,L-type Ca2+ channel (Cav1. 2) and R-type Ca2+ channel (Cav2. 3) were significantly up-regulated. The results of gene microarray chips were further confirmed by RT-PCR and Western blot.The intracellular Ca2+ testing indicated that there was no statistical significant of resting intracellular Ca2+ in colonic special sensory neuron between TNBS group and normal control group (P＞0. 05);while the evoked transients [Ca2+] significantly increased compared with normal control group (P＜0. 05). The whole cell patch clamp recording showed that the L-type and R-type calcium current were significantly increased in colonic primary sensory neurons of TNBS group compared with normal control group (P＜0. 05). The inflammatory visceral hypersensitivity was significantly reduced by intrathecal injection of nimodipine and SNX-482 (P＜0. 05). Conclusion The up-regulation of Cav1. 2and Cav2. 3 play an important role in inflammatory visceral hyperalgesia,which may be the possible potential therapeutic targets for visceral inflammatory hyperalgesia.
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