小干扰RNA抑制瞬时感受器电位通道3表达对大鼠内脏高敏感的影响  

The effect of inhibition of transient receptor potential canonical-3 expression by siRNA on visceral hypersensitivity in rats

作　　者：杨静[1] 杨宏丽[1] 李坤[1] 雷晓斐[1] 李双玲[1] 徐昌青[1] 

机构地区：[1]山东省千佛山医院消化内科,济南250014

出　　处：《中华消化病与影像杂志（电子版）》2016年第6期255-260,共6页Chinese Journal of Digestion and Medical Imageology(Electronic Edition)

基　　金：国家自然科学基金青年科学基金项目(81200275);山东省自然科学基金(ZR2012HL20)

摘　　要：目的探讨鞘内注射经典瞬时感受器电位通道3（TRPC3）特异性小干扰RNA（siRNA）对大鼠内脏高敏感性的调节作用。方法 56只Sprague Dawley雄性乳鼠随机数字表法分成4组,每组14只,应用醋酸灌肠建立大鼠内脏高敏感模型,其中3组为模型组,出生后第8-21天予0.5%醋酸溶液0.3 ml灌肠,1组为对照组,出生后第8-21天予0.9%氯化钠注射液0.3 ml灌肠。出生后第8周进行试验,4组大鼠均予鞘内置管,每天鞘内注射1次,共干预3 d：对照＋空白组、模型＋空白组鞘内注射去核酸酶水（20μl/次）;模型＋错配siRNA组鞘内注射错配siRNA（20μg/20μl/次）;模型＋TRPC3-siRNA组鞘内注射TRPC3-siRNA（20μg/20μl/次）。注射结束1周后应用实时定量PCR及蛋白印迹法检测4组大鼠支配结肠的L5-S1节段背根神经节（DRG）神经元TRPC3 mRNA与蛋白表达水平;通过不同压力结直肠扩张（CRD）后腹壁收缩反射（AWR）评分及疼痛感觉阈值、最大容量阈值检测评估大鼠内脏敏感性。结果相较于对照＋空白组,模型＋空白组DRG的TRPC3 mRNA和蛋白表达均显著升高（P值分别为0.002、0.029）;相较于模型＋空白组,模型＋TRPC3-siRNA组DRG的TRPC3 mRNA与蛋白表达显著降低（P值分别为0.002、0.001）;模型＋错配siRNA组与模型＋空白组TRPC3表达差异无统计学意义（P〉0.05）。内脏敏感性检测结果提示不同压力CRD后,相较于对照＋空白组,模型＋空白组在40、60、80 mmHg AWR评分升高（P均〈0.01）,疼痛感觉阈值与最大容量感觉阈值下降（P均〈0.001）,提示内脏高敏感模型成功建立。相较模型＋空白组,模型＋TRPC3-siRNA组大鼠在60、80 mmHg压力扩张时AWR评分显著下降（P均〈0.01）,疼痛感觉阈值与最大容量感觉阈值显著升高（P值分别为0.007、0.023）。模型＋错配siRNA组与模型＋空白组比较AWR评分与感觉阈值差异无统计学意义（P〉0.05）。结论鞘内注射siRNA靶向阻断TObjective To investigate the regulating effect of intrathecal injection of specific siRNA targeting at transient receptor potential canonical 3（ TRPC3） on the visceral hypersensitivity in rats.Methods Fifty-six neonatal Sprague Dawley male rats were randomly divided into four groups with fourteen per group. Intracolonic infusion of acetic acid was used to set up visceral hypersensitive model. The model rats included three groups which received the infusion of 0. 3 ml 0. 5% acetic acid from 8-day-old to 21-day-old,while the rats in control group received an equal volume of saline. The four groups of rats were raised until 8-week-old and all inserted pipes intrathecally. The intrathecal injections were carried out one time per day and lasted for three days. Control ＋ Blank group and Model ＋ Blank group were both injected by RNAase free water（ 20 μlL per time）; Model ＋ Scrambled-siRNA group was injected by scrambled siRNA（ 20 μg /20 μlL per time）,; while Model ＋ TRPC3-siRNA group was injected by specific siRNA targeting at rat TRPC3（ 20 μg/20 μLl per time）. One week after intrathecal injections,real-time quantitative PCR and westernblotting were operated to investigate the mRNA and protein expression levels of TRPC3 in L5-S1 colonicafferent dorsal root ganglion（ DRG） neurons. Visceral responses to colorectal distension（ CRD） at gradient pressures were examined by（ abdominal withdrawal reflex） AWR scores,pain threshold and maximal tolerancethreshold to assess the visceral sensitivity in different groups of rats. Results Compared with Control ＋ Blank group,the expressions of TRPC3 at mRNA and protein levels in DRG neurons in Model ＋ Blank group were significantly increased（ P = 0. 002、,P = 0. 029）; Compared with Model ＋ Blank group,the expressions of TRPC3 at mRNA and protein levels in DRG neurons in Model ＋ TRPC3-siRNA group were significantly decreased（ P = 0. 002,P = 0. 001）; No significant difference in the expressions of mRNA and protein of TRPC3 was foun

关 键 词：TRPC阳离子通道 RNA 小分子干扰 内脏高敏感 

分 类 号：R57[医药卫生—消化系统]