HCN2调控胃肠运动机制作用的研究

发布时间：2018-07-07 21:13

本文选题：超极化激活环核苷酸门控阳离子通道 + 蛋白基因产物9.5　；参考：《第四军医大学》2010年硕士论文

【摘要】： 胃肠道运动的调节,不仅受植物神经的支配,亦受相对独立的肠神经系统(enteric nervous system,ENS)的调控。但至今为止,ENS的对胃肠的具体调控机制尚不完全清楚。进一步阐明ENS对胃肠运动的调控机制则对胃肠动力障碍性疾病的临床治疗具有重要意义。胃肠动力起搏是以胃肠道平滑肌节律性电活动—慢波活动作为基础。胃肠道运动包含着非常复杂的过程,最重要的部分是细胞膜电活动变化引起的平滑肌细胞(Smooth muscle cell ,SMC)的收缩,而细胞膜电活动的变化是受多种因素的调控。超极化激活的环化核苷酸门控通道(Hyperpolarization activated cyclic nucleotide gated channel, HCN)基因家族是特种离子流(funny current, If )形成的分子基础,它来自于一个非选择性阳离子电压门控通道家族,现已证明HCN是调节慢波节律起搏的一个重要通道。目前对HCN的研究尚限制在脑干之呼吸中枢对呼吸节律以及心脏窦房结对心脏节律调节方面。而对于HCN对胃肠运动功能的调节目前尚属空白。本实验是在借鉴HCN在脑干之呼吸中枢对呼吸节律、心脏窦房结对心脏节律调节,结合胃肠道起搏基础亦为慢波起搏的特点,通过①免疫荧光双标记技术,验证HCN2在胃肠道神经上的分布以及与神经递质的共存情况。②在上述研究基础上,采用电生理技术,分别观察离体肠段平滑肌条给予HCN2激动剂或拮抗剂前后,肠平滑肌肌条蠕动波之波幅和频率的变化。通过上述研究,确立HCN2在胃肠道动力调节方面的作用角色和地位,为胃肠动力起搏及胃肠动力调节机制的研究拓展新的方向。方法:①采用免疫荧光组织化学双标记方法,对大鼠胃组织进行PGP9.5与HCN2的双标记;HCN2分别与SP、CGRP以及VIP进行双标记。②分离取得大鼠空肠离体平滑肌肌条,分别观察在给予HCN2受体激动剂CAMP和拮抗剂MDL前、后空肠平滑肌条收缩幅度、频率的变化。③对大鼠空肠离体平滑肌肌条分别给予HCN通道激动剂CAMP和拮抗剂MDL,通过酶联免疫法检测灌流液中给药前后SP和VIP含量的变化情况。结果: 1. HCN2是以串珠样结构分布于胃黏膜间神经纤维末梢。HCN2与SP、CGRP和VIP在胃黏膜和平滑肌间隙中存在双标记。 2.正常状态下,平滑肌肌条孵育1h后出现稳定的收缩活动;给予HCN2受体激动剂CAMP后,大鼠空肠平滑肌收缩频率和振幅均与给予激动剂前显增加(P0.05);而给予HCN2受体阻断剂MDL后,大鼠空肠收缩频率和振幅与给予阻断剂前显著降低(P0.05)。 3.给予HCN2受体激动剂CAMP后,环形肌和纵形肌中的SP含量均显著增加(P0.05);VIP含量显著减少(P0.05);而给予HCN2受体阻断剂MDL后,环形肌和纵形肌中的SP含量均显著减少(P0.05);VIP含量显著增加(P0.05)。结论:发现HCN2在胃组织中是存在于胃肠道神经纤维末梢的周围,且并与神经递质有共存现象。HCN2受体功能的变化可以对大鼠空肠平滑肌的运动起正向调节作用,其对灌流液中SP含量起正向调节作用,而对灌流液中VIP含量起负向调节作用,与SP及VIP对胃肠道的调节相符,提示HCN2可能在胃肠道的动力调节方面起着较为重要的作用。
[Abstract]:The regulation of gastrointestinal motility is not only controlled by the autonomic nervous system, but also regulated by the relatively independent enteric nervous system (ENS). But up to now, the specific regulatory mechanism of ENS on the gastrointestinal tract is not completely clear. Further clarifies the clinical treatment of gastrointestinal motility by the regulation mechanism of ENS on gastrointestinal motility. The gastrointestinal motility is based on the smooth muscle activity of the gastrointestinal tract - slow wave activity. The gastrointestinal movement contains a very complex process. The most important part is the contraction of the Smooth muscle cell (SMC) caused by the changes in the electrical activity of the cell membrane, and the changes in the electrical activity of the cell membrane are varied. Regulation of factors. The hyperpolarized cyclic nucleotide gate gated channel (Hyperpolarization activated cyclic nucleotide gated channel, HCN) gene family is the molecular basis for the formation of special ionic flow (funny current, If). It comes from a non selective cation electrogated gated channel family, which has now proved that HCN is the regulation of slow wave rhythm. The current research on HCN is limited to the respiratory rhythm of the brain stem and the regulation of the rhythm of the heart in the heart sinoatrial chamber. The regulation of HCN's regulation of gastrointestinal motility is still a blank. This experiment is based on the respiratory rhythm of HCN in the brain stem and the heart node in the heart sinoatrial node. Regulation, combined with the characteristics of the pacing of the gastrointestinal pacing, the distribution of HCN2 on the gastrointestinal tract and the coexistence of neurotransmitters in the gastrointestinal tract were verified by the immunofluorescence double labeling technique. 2. On the basis of the above study, electrophysiological techniques were used to observe the isolated intestinal smooth muscle strips before HCN2 agonists or antagonists were given respectively. The changes in the amplitude and frequency of the peristaltic wave of the muscle strips of the intestinal smooth muscle after the study were used to establish the role and status of HCN2 in the dynamic regulation of gastrointestinal tract, and to develop new directions for the study of gastrointestinal motility and gastrointestinal motility.
Methods: (1) double labeling of PGP9.5 and HCN2 in rat gastric tissue was carried out by immunofluorescence histochemical double labeling; HCN2 was labeled with SP, CGRP and VIP respectively. The isolated muscle strips of rat jejunum isolated from the jejunum were isolated, and the contraction amplitude of the smooth muscle strips of the jejunum was observed before the HCN2 receptor agonist CAMP and antagonist MDL were given, respectively. HCN channel agonist CAMP and antagonist MDL were given to the isolated smooth muscle strips of the rat jejunum, and the changes of the content of SP and VIP before and after the administration of the perfusion fluid were detected by enzyme immunoassay.
Result:
1. HCN2 was distributed in the gastric mucosal nerve fibers at.HCN2 and SP, CGRP and VIP in the gastric mucosa and smooth muscle spaces.
2. normal state, smooth muscle strips incubated for 1h after incubation, and after giving HCN2 receptor agonist CAMP, the contractile frequency and amplitude of the jejunum smooth muscle increased significantly with the agonist (P0.05), but after the HCN2 receptor blocker MDL, the frequency and amplitude of the jejunum contraction in the rat decreased significantly (P0.05).
3. after the HCN2 receptor agonist CAMP was given, the SP content in the ring muscle and the longitudinal muscle increased significantly (P0.05), and the content of VIP decreased significantly (P0.05), while HCN2 receptor blocker MDL, the SP content in the circular and longitudinal muscles decreased significantly (P0.05), and VIP content was significantly increased (P0.05).
Conclusion: it is found that HCN2 exists around the nerve fiber end of the gastrointestinal tract in the gastric tissue, and the change of.HCN2 receptor function can regulate the movement of the jejunum smooth muscle in the rat, which regulates the content of SP in the perfusion fluid and regulates the VIP content in the perfusion fluid negatively. It is consistent with the regulation of SP and VIP on gastrointestinal tract, suggesting that HCN2 may play a more important role in gastrointestinal motility regulation.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R333

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