糖尿病大鼠胃竇平滑肌細(xì)胞Ryanodine受體表達(dá)變化的研究

龍慶林,房殿春

(第三軍醫(yī)大學(xué)附屬西南醫(yī)院全軍消化研究所,重慶 400038)

糖尿病大鼠胃竇平滑肌細(xì)胞Ryanodine受體表達(dá)變化的研究

龍慶林,房殿春

(第三軍醫(yī)大學(xué)附屬西南醫(yī)院全軍消化研究所,重慶 400038)

目的探討糖尿病大鼠胃竇平滑肌Ryanodine受體(RyR)的改變及其意義。方法Wistar大鼠50只,隨機(jī)分為糖尿病組和對(duì)照組,用鏈尿佐菌素建立大鼠糖尿病模型,3個(gè)月后測(cè)定胃排空時(shí)間,用激光共聚焦方法測(cè)定胃竇平滑肌細(xì)胞內(nèi)Ca2+濃度,Western blot法檢測(cè)胃竇平滑肌組織RyR蛋白變化。結(jié)果與對(duì)照組大鼠相比,糖尿病組大鼠胃排空時(shí)間明顯延長(P＜0.05),平滑肌細(xì)胞內(nèi) Ca2+濃度明顯增加(P＜0.05),而胃竇平滑肌組織 RyR蛋白表達(dá)明顯減少(P＜0.05)。結(jié)論糖尿病大鼠胃竇平滑肌RyR受損,從而導(dǎo)致細(xì)胞內(nèi)Ca2+信號(hào)調(diào)控異常致使平滑肌功能異常是糖尿病胃腸道功能障礙重要因素之一。

糖尿病;平滑肌;蘭尼堿

Ca2+在平滑肌收縮反應(yīng)的調(diào)節(jié)中發(fā)揮關(guān)鍵的作用,但在糖尿病情況下,細(xì)胞內(nèi)的Ca2+代謝存在明顯異常,這種異常有可能導(dǎo)致糖尿病患者胃運(yùn)動(dòng)功能障礙。Ryanodine受體(RyR)是位于細(xì)胞的內(nèi)質(zhì)網(wǎng)和肌質(zhì)網(wǎng)膜上的鈣釋放通道,具有重要的生理功能。本研究旨在探討糖尿病大鼠胃竇平滑肌細(xì)胞內(nèi)RyR表達(dá)變化及其對(duì)平滑肌功能的影響。

1 材料與方法

1.1 糖尿病模型建立 健康Wistar大鼠50只,2月齡,體質(zhì)量100～160 g,雌雄各半,隨機(jī)分為糖尿病組(30只)和對(duì)照組(20只)。糖尿病組大鼠用鏈尿佐菌素(STZ)建立糖尿病模型;對(duì)照組注射生理鹽水。每2周測(cè)血糖1次。糖尿病模型建立后,糖尿病組大鼠血糖明顯升高,體質(zhì)量減輕。

1.2 胃排空時(shí)間測(cè)定 將2%的美蘭溶液胃內(nèi)灌注,30 min后處死大鼠,將胃內(nèi)殘留物沖洗并收集離心,取上清液用分光光度計(jì)測(cè)定吸光度,求出其與對(duì)照組均值的百分比即為各樣本的胃內(nèi)色素相對(duì)殘留率。

1.3 Western blot檢測(cè)胃竇平滑肌RyR蛋白表達(dá) 提取胃平滑肌組織總蛋白,電泳后轉(zhuǎn)至硝酸纖維素膜,用脫脂奶粉封閉,加入抗體,4℃孵育過夜,加ECL化學(xué)發(fā)光試劑顯影。

1.4 平滑肌細(xì)胞內(nèi)Ca2+濃度檢測(cè) 制備游離平滑肌細(xì)胞,滴1滴于載玻片上,細(xì)胞貼片后,棄培養(yǎng)皿內(nèi)的培養(yǎng)液,取Fluo-3/AM探針37℃避光孵育后,用激光共聚焦顯微鏡檢測(cè)。

2 結(jié) 果

2.1 兩組大鼠胃排完時(shí)間測(cè)定 糖尿病組大鼠胃內(nèi)色素相對(duì)殘留率為(134.38±21.25)%,而對(duì)照組大鼠胃內(nèi)色素相對(duì)殘留率僅為(100.00±16.46)%,兩組比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。

2.2 兩組大鼠胃竇平滑肌組織RyR蛋白的表達(dá) 對(duì)照組大鼠胃竇平滑肌組織內(nèi)RyR蛋白表達(dá)(746.51±62.02)明顯高于糖尿病組大鼠(423.58±42.96),兩組比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.05),見圖 1。

2.3 兩組大鼠胃竇平滑肌細(xì)胞Ca2+測(cè)定 對(duì)照組大鼠胃竇平滑肌細(xì)胞Ca2+熒光強(qiáng)度為57.2±4.9,見封 3圖 2,糖尿病組大鼠胃竇平滑肌細(xì)胞Ca2+熒光強(qiáng)度為57.2±4.9,見封3圖3,兩組比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。

圖1 胃竇平滑肌組織RyR蛋白表達(dá)

3 討 論

胃運(yùn)動(dòng)功能障礙是糖尿病常見的一種慢性并發(fā)癥,但目前對(duì)糖尿病胃腸運(yùn)動(dòng)障礙的發(fā)生機(jī)制了解甚少。大量研究表明糖尿病胃運(yùn)動(dòng)功能障礙與平滑肌功能異常密切相關(guān)。Ca2+是調(diào)節(jié)細(xì)胞生命過程的第二信使,平滑肌的收縮及增殖均與細(xì)胞內(nèi)的Ca2+密不可分,糖尿病患者常伴有細(xì)胞內(nèi)Ca2+增加。有研究顯示在STZ糖尿病大鼠心肌組織中細(xì)胞內(nèi)Ca2+濃度明顯增加,心肌超微結(jié)構(gòu)明顯異常,細(xì)胞內(nèi)Ca2+降低后,心肌結(jié)構(gòu)異常減輕[1-5]。本研究測(cè)定糖尿病胃運(yùn)動(dòng)功能障礙大鼠胃竇平滑肌細(xì)胞內(nèi)Ca2+變化,發(fā)現(xiàn)糖尿病組大鼠胃竇平滑肌細(xì)胞內(nèi)游離Ca2+濃度明顯高于對(duì)照組,表明糖尿病大鼠胃竇平滑肌細(xì)胞內(nèi)Ca2+濃度升高,導(dǎo)致平滑肌結(jié)構(gòu)損害,從而影響平滑肌的收縮、舒張功能;除此之外,細(xì)胞內(nèi)Ca2+濃度增加可直接影響平滑肌的收縮、舒張功能。但糖尿病大鼠胃竇平滑肌細(xì)胞內(nèi)游離Ca2+濃度增加機(jī)制尚不清楚。

RyR是位于細(xì)胞的內(nèi)質(zhì)網(wǎng)和肌漿網(wǎng)膜上的鈣釋放通道,它能迅速地將Ca2+從內(nèi)質(zhì)網(wǎng)和肌漿網(wǎng)中釋放出來,從而發(fā)揮一系列的生理功能[6-9]。由于編碼RyR的基因發(fā)生突變或者其他病理因素,引起該蛋白的功能或結(jié)構(gòu)發(fā)生改變,往往導(dǎo)致嚴(yán)重的疾病[10-12]。由于RyR數(shù)量降低,導(dǎo)致Ca2+信號(hào)缺陷,Ca2+瞬態(tài)幅度降低以及動(dòng)力學(xué)變緩,從而導(dǎo)致細(xì)胞內(nèi)Ca2+異常,進(jìn)而致使心肌異常收縮。胃腸道 RyR也有重要的生理作用,它參與了平滑肌細(xì)胞的收縮-興奮耦聯(lián),在胃電產(chǎn)生中發(fā)揮著至關(guān)重要的作用[13-16]。因此,胃腸道平滑肌RyR異常也必然會(huì)導(dǎo)致胃腸疾病的發(fā)生,如結(jié)腸平滑肌RyR基因突變后會(huì)導(dǎo)致內(nèi)質(zhì)網(wǎng)Ca2+的釋放減少,致使便秘發(fā)生。在本實(shí)驗(yàn)中,利用Western blot方法測(cè)定RyR在胃竇平滑肌組織中表達(dá),發(fā)現(xiàn)糖尿病組大鼠胃竇平滑肌組織RyR表達(dá)明顯低于對(duì)照組,提示糖尿病時(shí)胃腸道平滑肌細(xì)胞RyR病變可能是糖尿病胃腸運(yùn)動(dòng)功能障礙的重要原因。糖尿病時(shí),由于高血糖等原因?qū)е缕交〖?xì)胞內(nèi)RyR受損、功能降低,從而導(dǎo)致細(xì)胞內(nèi)Ca2+信號(hào)調(diào)控異常致使平滑肌功能異常是胃腸道功能障礙重要因素之一。

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Study of the Ryanodine receptor in smooth muscle cells in gastric antrum of diabetic rats

Long Qinglin,Fang Dianchun
(Research Center of Gastroenterology,Southwest Hospital,Third Military Medical University,Chongqing 40038,China)

ObjectiveTo study the change of ryanodine receptor(RyR)in gastric antrum of diabetic rat.Methods50 rats were randomly divided into diabetic group and control group.Gastric empty time was determined 3 months after diabetes reproduced in rats,the content of cytosolic Ca2+in smooth muscle cells was measured by laser scanning confocal microscopy and the expressions of RyR in smooth muscle of diabetic rats were measured with Western blot method.ResultsIn diabetic rats,gastric emptying was significantly delayed(P＜0.05).Compared the normal rats,the content of cytosolic Ca2+in smooth muscle cells from the antrum of diabetic rats was increased and the expression of RyR in muscular layer in diabetic rats was significantly down-regulated(P＜0.05).ConclusionDown-regulation of RyR induces abnormality of cytosolic Ca2+in gastric antral smooth muscle in diabetic rats and then leads to gastric motility disorder.

diabetes mellitus;smooth muscle;ryanodine

10.3969/j.issn.1671-8348.2011.06.006

A

1671-8348(2011)06-0538-02

2010-06-09

2010-08-22)

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