糖尿病大鼠胃窦平滑肌细胞Ryanodine受体表达变化的研究

龙庆林,房殿春

(第三军医大学附属西南医院全军消化研究所,重庆 400038)

糖尿病大鼠胃窦平滑肌细胞Ryanodine受体表达变化的研究

龙庆林,房殿春

(第三军医大学附属西南医院全军消化研究所,重庆 400038)

目的探讨糖尿病大鼠胃窦平滑肌Ryanodine受体(RyR)的改变及其意义。方法Wistar大鼠50只,随机分为糖尿病组和对照组,用链尿佐菌素建立大鼠糖尿病模型,3个月后测定胃排空时间,用激光共聚焦方法测定胃窦平滑肌细胞内Ca2+浓度,Western blot法检测胃窦平滑肌组织RyR蛋白变化。结果与对照组大鼠相比,糖尿病组大鼠胃排空时间明显延长(P＜0.05),平滑肌细胞内 Ca2+浓度明显增加(P＜0.05),而胃窦平滑肌组织 RyR蛋白表达明显减少(P＜0.05)。结论糖尿病大鼠胃窦平滑肌RyR受损,从而导致细胞内Ca2+信号调控异常致使平滑肌功能异常是糖尿病胃肠道功能障碍重要因素之一。

糖尿病;平滑肌;兰尼碱

Ca2+在平滑肌收缩反应的调节中发挥关键的作用,但在糖尿病情况下,细胞内的Ca2+代谢存在明显异常,这种异常有可能导致糖尿病患者胃运动功能障碍。Ryanodine受体(RyR)是位于细胞的内质网和肌质网膜上的钙释放通道,具有重要的生理功能。本研究旨在探讨糖尿病大鼠胃窦平滑肌细胞内RyR表达变化及其对平滑肌功能的影响。

1 材料与方法

1.1 糖尿病模型建立 健康Wistar大鼠50只,2月龄,体质量100～160 g,雌雄各半,随机分为糖尿病组(30只)和对照组(20只)。糖尿病组大鼠用链尿佐菌素(STZ)建立糖尿病模型;对照组注射生理盐水。每2周测血糖1次。糖尿病模型建立后,糖尿病组大鼠血糖明显升高,体质量减轻。

1.2 胃排空时间测定 将2%的美兰溶液胃内灌注,30 min后处死大鼠,将胃内残留物冲洗并收集离心,取上清液用分光光度计测定吸光度,求出其与对照组均值的百分比即为各样本的胃内色素相对残留率。

1.3 Western blot检测胃窦平滑肌RyR蛋白表达 提取胃平滑肌组织总蛋白,电泳后转至硝酸纤维素膜,用脱脂奶粉封闭,加入抗体,4℃孵育过夜,加ECL化学发光试剂显影。

1.4 平滑肌细胞内Ca2+浓度检测 制备游离平滑肌细胞,滴1滴于载玻片上,细胞贴片后,弃培养皿内的培养液,取Fluo-3/AM探针37℃避光孵育后,用激光共聚焦显微镜检测。

2 结 果

2.1 两组大鼠胃排完时间测定 糖尿病组大鼠胃内色素相对残留率为(134.38±21.25)%,而对照组大鼠胃内色素相对残留率仅为(100.00±16.46)%,两组比较差异有统计学意义(P＜0.05)。

2.2 两组大鼠胃窦平滑肌组织RyR蛋白的表达 对照组大鼠胃窦平滑肌组织内RyR蛋白表达(746.51±62.02)明显高于糖尿病组大鼠(423.58±42.96),两组比较差异有统计学意义(P＜0.05),见图 1。

2.3 两组大鼠胃窦平滑肌细胞Ca2+测定 对照组大鼠胃窦平滑肌细胞Ca2+荧光强度为57.2±4.9,见封 3图 2,糖尿病组大鼠胃窦平滑肌细胞Ca2+荧光强度为57.2±4.9,见封3图3,两组比较差异有统计学意义(P＜0.05)。

图1 胃窦平滑肌组织RyR蛋白表达

3 讨 论

胃运动功能障碍是糖尿病常见的一种慢性并发症,但目前对糖尿病胃肠运动障碍的发生机制了解甚少。大量研究表明糖尿病胃运动功能障碍与平滑肌功能异常密切相关。Ca2+是调节细胞生命过程的第二信使,平滑肌的收缩及增殖均与细胞内的Ca2+密不可分,糖尿病患者常伴有细胞内Ca2+增加。有研究显示在STZ糖尿病大鼠心肌组织中细胞内Ca2+浓度明显增加,心肌超微结构明显异常,细胞内Ca2+降低后,心肌结构异常减轻[1-5]。本研究测定糖尿病胃运动功能障碍大鼠胃窦平滑肌细胞内Ca2+变化,发现糖尿病组大鼠胃窦平滑肌细胞内游离Ca2+浓度明显高于对照组,表明糖尿病大鼠胃窦平滑肌细胞内Ca2+浓度升高,导致平滑肌结构损害,从而影响平滑肌的收缩、舒张功能;除此之外,细胞内Ca2+浓度增加可直接影响平滑肌的收缩、舒张功能。但糖尿病大鼠胃窦平滑肌细胞内游离Ca2+浓度增加机制尚不清楚。

RyR是位于细胞的内质网和肌浆网膜上的钙释放通道,它能迅速地将Ca2+从内质网和肌浆网中释放出来,从而发挥一系列的生理功能[6-9]。由于编码RyR的基因发生突变或者其他病理因素,引起该蛋白的功能或结构发生改变,往往导致严重的疾病[10-12]。由于RyR数量降低,导致Ca2+信号缺陷,Ca2+瞬态幅度降低以及动力学变缓,从而导致细胞内Ca2+异常,进而致使心肌异常收缩。胃肠道 RyR也有重要的生理作用,它参与了平滑肌细胞的收缩-兴奋耦联,在胃电产生中发挥着至关重要的作用[13-16]。因此,胃肠道平滑肌RyR异常也必然会导致胃肠疾病的发生,如结肠平滑肌RyR基因突变后会导致内质网Ca2+的释放减少,致使便秘发生。在本实验中,利用Western blot方法测定RyR在胃窦平滑肌组织中表达,发现糖尿病组大鼠胃窦平滑肌组织RyR表达明显低于对照组,提示糖尿病时胃肠道平滑肌细胞RyR病变可能是糖尿病胃肠运动功能障碍的重要原因。糖尿病时,由于高血糖等原因导致平滑肌细胞内RyR受损、功能降低,从而导致细胞内Ca2+信号调控异常致使平滑肌功能异常是胃肠道功能障碍重要因素之一。

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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)

·综 述·