Piezo2在肠易激综合征内脏高敏感发生中的作用研究

The abdominal pain or discomfort of irritable bowel syndrome(IBS) harasses millions of patients. Researches demonstrate that visceral hypersensitivity is significantly correlated with the abdominal symptoms of IBS patients, but the mechanisms underlying visceral hypersensitivity are still elusive. Piezo2 is a newly-discovered mechanosensor which has strong expression in both colon and dorsal root ganglion(DRG) and participates in the mechanosensory transduction of DRG neurons. Based on the biological characteristics of Piezo2 as above, we propose the hypothesis that Piezo2 is involved in the generation of visceral hypersensitivity of IBS for the first time.The present research includes three parts and plans to apply the techniques of genetic engineering, immunohistochemistry and electro- physiology et al. Firstly we will study on the physiological function of Piezo2 in the DRG neurons innervating colon and then try to clarify the upstream neuro- transmitters and the downstream signal transduction molecules involved in the activation of Piezo2. Secondly we will set up animal model to imitate the visceral hypersensitivity of IBS patients and compare the differences of the expression of Piezo2 between groups. We will specifically knock down the expression of Piezo2 by injecting the SiRNA targeting at Piezo2 intrathecally and investigate whether the visceral hypersensitivity could be inhibited thereby.Thirdly we will collect the colonic mucosal biopsies from IBS patients and controls to investigate the correlations between the expression level of Piezo2, the abdominal symptom scores and the visceral sensitivity. In brief, the present research will explore the role of Piezo2 in the mechanism underlying the visceral hypersensitivity of IBS from different perspectives and try to provide new pharmacological target for the treatment of IBS.

IBS腹痛或腹部不适症状困扰着数以百万计的患者，研究显示内脏高敏感与IBS患者腹部症状密切相关，但其发生机制尚不明确。Piezo2是最新发现的机械感受器，鉴于其在结肠和DRG均有高表达，并参与神经元机械刺激后的信号转导，申请者首次提出Piezo2参与IBS内脏高敏感发生这一假说。本课题拟用基因工程学、免疫组化、电生理学等方法，首先探讨Piezo2在支配结肠的DRG神经元的生理作用以及参与Piezo2激活的神经递质和下游信号通路分子；然后建立动物模型模拟IBS内脏高敏感状态，通过鞘内注射SiRNA阻断Piezo2表达，观察内脏高敏感是否得到抑制；临床研究部分收取IBS患者和对照者的结肠黏膜标本，研究Piezo2的表达水平与IBS腹部症状评分和内脏敏感性的相关性。本课题在国内外首次探讨Piezo2在IBS内脏高敏感发生的作用，将进一步丰富对内脏高敏感发生机制的认识并为IBS临床防治提供新靶点。

内脏高敏感是IBS患者腹部症状的主要病理生理机制，本研究探讨Piezo2分子在肠易激综合征内脏高敏感调节中的作用及可能机制。1. 构建及筛选Piezo2特异性shRNA腺病毒：针对大鼠piezo2 mRNA序列设计多条shRNA序列并连接载体，构建干扰腺病毒，检测病毒滴度及活力。不同shRNA腺病毒感染后测定piezo2抑制水平，筛选特异性shRNA。2.制备纯化抗大鼠Piezo2抗体：选取目的基因合成并表达纯化，制备免疫原免疫小鼠，采集血清并纯化抗体、免疫印迹验证特异性。3.建立内脏高敏感模型：出生第8-21天乳鼠用0.5%醋酸溶液0.3mL灌肠，对照组用生理盐水0.3mL灌肠，9周龄时行AWR评分和痛阈值测定，并测定腹外斜肌放电频次及曲线下面积。模型组60mmHg压力时腹外斜肌放电次数显著升高，在40、60mm、80Hg压力时曲线下面积显著增加，在20、30、40、50、60mmHg压力时AWR评分显著升高，痛阈值降低，提示造模成功。4.鞘内注射Piezo2 shRNA腺病毒对大鼠内脏敏感性的作用：对照组和模型组均分3组，分别鞘内注射shRNA腺病毒、对照腺病毒或者去核酸酶盐水，结果证实模型+shRNA组30mmHg压力时AWR评分降低，痛阈值升高，在20、40、60mm、80Hg压力时EMG曲线下面积减少，提示鞘内注射Piezo2 shRNA腺病毒可有效改善大鼠内脏敏感性。5.Piezo2 mRNA表达：实时定量PCR测定结肠及L6-S2 DRG Piezo2表达，证实对照+空白组与模型+空白组结肠及DRG Piezo2表达无显著性差异，鞘内注射Piezo2 shRNA可有效降低DRG Piezo2 mRNA表达。6.Piezo2蛋白表达：证实相较于对照组，模型组DRG Piezo2、Epac1、PKCϵ表达显著性增加，鞘内注射Piezo2 shRNA可抑制对照组及模型组DRG Piezo2、PKCϵ蛋白表达，并抑制模型组Epac1蛋白表达，Piezo2可能通过Epac1-PKCϵ途径调节内脏敏感性。7.临床研究证实IBS-D患者内脏敏感性异常，表现为初始感觉阈值、疼痛阈值、直肠顺应性较对照组下降，IBS-D患者肠粘膜Piezo2 表达增加，与患者内脏高敏感相关。本研究证实Piezo2在IBS内脏高敏感发生中有重要作用，为寻找IBS治疗的靶点提供了新的方案。