本文選題：超極化激活環(huán)核苷酸門控陽離子通道 + 蛋白基因產(chǎn)物9.5��； 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 胃腸道運(yùn)動的調(diào)節(jié),不僅受植物神經(jīng)的支配,亦受相對獨(dú)立的腸神經(jīng)系統(tǒng)(enteric nervous system,ENS)的調(diào)控。但至今為止,ENS的對胃腸的具體調(diào)控機(jī)制尚不完全清楚。進(jìn)一步闡明ENS對胃腸運(yùn)動的調(diào)控機(jī)制則對胃腸動力障礙性疾病的臨床治療具有重要意義。胃腸動力起搏是以胃腸道平滑肌節(jié)律性電活動—慢波活動作為基礎(chǔ)。胃腸道運(yùn)動包含著非常復(fù)雜的過程,最重要的部分是細(xì)胞膜電活動變化引起的平滑肌細(xì)胞(Smooth muscle cell ,SMC)的收縮,而細(xì)胞膜電活動的變化是受多種因素的調(diào)控。超極化激活的環(huán)化核苷酸門控通道(Hyperpolarization activated cyclic nucleotide gated channel, HCN)基因家族是特種離子流(funny current, If )形成的分子基礎(chǔ),它來自于一個(gè)非選擇性陽離子電壓門控通道家族,現(xiàn)已證明HCN是調(diào)節(jié)慢波節(jié)律起搏的一個(gè)重要通道。目前對HCN的研究尚限制在腦干之呼吸中樞對呼吸節(jié)律以及心臟竇房結(jié)對心臟節(jié)律調(diào)節(jié)方面。而對于HCN對胃腸運(yùn)動功能的調(diào)節(jié)目前尚屬空白。本實(shí)驗(yàn)是在借鑒HCN在腦干之呼吸中樞對呼吸節(jié)律、心臟竇房結(jié)對心臟節(jié)律調(diào)節(jié),結(jié)合胃腸道起搏基礎(chǔ)亦為慢波起搏的特點(diǎn),通過①免疫熒光雙標(biāo)記技術(shù),驗(yàn)證HCN2在胃腸道神經(jīng)上的分布以及與神經(jīng)遞質(zhì)的共存情況。②在上述研究基礎(chǔ)上,采用電生理技術(shù),分別觀察離體腸段平滑肌條給予HCN2激動劑或拮抗劑前后,腸平滑肌肌條蠕動波之波幅和頻率的變化。通過上述研究,確立HCN2在胃腸道動力調(diào)節(jié)方面的作用角色和地位,為胃腸動力起搏及胃腸動力調(diào)節(jié)機(jī)制的研究拓展新的方向。 方法:①采用免疫熒光組織化學(xué)雙標(biāo)記方法,對大鼠胃組織進(jìn)行PGP9.5與HCN2的雙標(biāo)記;HCN2分別與SP、CGRP以及VIP進(jìn)行雙標(biāo)記。②分離取得大鼠空腸離體平滑肌肌條,分別觀察在給予HCN2受體激動劑CAMP和拮抗劑MDL前、后空腸平滑肌條收縮幅度、頻率的變化。③對大鼠空腸離體平滑肌肌條分別給予HCN通道激動劑CAMP和拮抗劑MDL,通過酶聯(lián)免疫法檢測灌流液中給藥前后SP和VIP含量的變化情況。 結(jié)果: 1. HCN2是以串珠樣結(jié)構(gòu)分布于胃黏膜間神經(jīng)纖維末梢。HCN2與SP、CGRP和VIP在胃黏膜和平滑肌間隙中存在雙標(biāo)記。 2.正常狀態(tài)下,平滑肌肌條孵育1h后出現(xiàn)穩(wěn)定的收縮活動;給予HCN2受體激動劑CAMP后,大鼠空腸平滑肌收縮頻率和振幅均與給予激動劑前顯增加(P0.05);而給予HCN2受體阻斷劑MDL后,大鼠空腸收縮頻率和振幅與給予阻斷劑前顯著降低(P0.05)。 3.給予HCN2受體激動劑CAMP后,環(huán)形肌和縱形肌中的SP含量均顯著增加(P0.05);VIP含量顯著減少(P0.05);而給予HCN2受體阻斷劑MDL后,環(huán)形肌和縱形肌中的SP含量均顯著減少(P0.05);VIP含量顯著增加(P0.05)。 結(jié)論:發(fā)現(xiàn)HCN2在胃組織中是存在于胃腸道神經(jīng)纖維末梢的周圍,且并與神經(jīng)遞質(zhì)有共存現(xiàn)象。HCN2受體功能的變化可以對大鼠空腸平滑肌的運(yùn)動起正向調(diào)節(jié)作用,其對灌流液中SP含量起正向調(diào)節(jié)作用,而對灌流液中VIP含量起負(fù)向調(diào)節(jié)作用,與SP及VIP對胃腸道的調(diào)節(jié)相符,提示HCN2可能在胃腸道的動力調(diào)節(jié)方面起著較為重要的作用。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R333

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