ATP敏感性钾通道在小鼠胚胎心脏发育过程的发育依赖性变化及功能

发布时间：2018-05-03 00:34

本文选题：ATP敏感性钾通道 + 急性缺血缺氧　；参考：《华中科技大学》2012年博士论文

【摘要】：目的：心脏缺血缺氧是目前临床上死亡率最高的病因之一，心脏在发生缺血缺氧后会产生预调制和慢性缺氧适应这两种保护性作用, K_(ATP)被认为是唯一参与该过程的离子通道。已经发现哺乳动物胚胎对缺氧的耐受性明显高于成年动物，这可能和离子通道的发育依赖性变化有关。因此本文旨在探讨K_(ATP)通道随小鼠胚胎心脏发育特性,以及该通道在生理和缺血缺氧状态下的功能变化。方法：分离早晚期小鼠胚胎心室，胶原酶B消化得到单个跳动心室肌细胞。以pinacidil作为K_(ATP)的特异性开放剂，glibenclamide作为膜K_(ATP)特异性阻断剂，电流钳记录该通道在生理状态下功能的发育依赖性变化，电压钳记录K_(ATP)的电生理学特点。以glibenclamide或5-HD分别作为膜K_(ATP)或线粒体K_(ATP)的阻断剂，电流钳记录该通道在急性缺血缺氧状态下发挥的作用。采用RT-PCR和Western blot研究构成K_(ATP)的亚单位Kir6.1、Kir6.2、SUR1及SUR2在心室肌细胞膜和线粒体上表达的发育依赖性变化。结果：生理状态下，，K_(ATP)特异性开放剂pinacidil使早晚期心室肌细胞动作电位时程缩短，晚期心室肌细胞膜电位发生超极化，而对早期心室肌细胞膜电位水平没有影响。K_(ATP)特异性阻断剂则对早晚期心室肌细胞动作电位无明显作用。电压钳数据表明随胚胎心脏发育，心室肌细胞K_(ATP)电流密度并没有发生显著改变。急性缺血缺氧时，早期心室肌细胞停跳率达62.5%，同时伴随有动作电位时程的缩短，晚期心室肌细胞则没有发现停跳，但同时伴随有动作电位时程的缩短及膜的超极化。急性缺血缺氧同时应用阻断剂阻断膜K_(ATP)后，早期心室肌动作电位时程反而延长，停跳率下降，晚期心室肌细胞膜反而去极化，42.8%细胞发生停跳。急性缺血缺氧同时应用阻断剂阻断线粒体K_(ATP)后，早晚期心室肌细胞对急性缺血缺氧的反应并无明显改变。RT-PCR及Western blot结果显示Kir6.1、SUR1及SUR2均随发育依赖性表达增加，而Kir6.2表达无明显差异。细胞膜蛋白检测结果显示膜K_(ATP)上无SUR1的表达，Kir6.1及SUR2的表达呈发育依赖性增加，Kir6.2表达无明显差异。对线粒体蛋白检测结果显示线粒体K_(ATP)上无SUR2的表达，Kir6.1、Kir6.2及SUR1均在线粒体上表达，但各亚单位随胚胎发育并无明显变化。免疫荧光染色发现SUR1在细胞膜上并不表达，其余各亚单位在早晚期心室肌细胞膜均有表达。结论：随着胚胎心脏的发育，膜K_(ATP)在急性缺血缺氧过程中发挥明显不同的作用，线粒体K_(ATP)则在急性缺血缺氧过程中并未起到明显作用。组成膜K_(ATP)亚单位包括Kir6.1、Kir6.2及SUR2，且Kir6.1和SUR2的表达呈发育依赖性增加。组成线粒体K_(ATP)的亚单位包括Kir6.1、Kir6.2和SUR1，各亚单位的表达随发育并未发生明显变化。胸腺肽β4是一种能促进细胞迁移、存活和分化的细胞内小分子多肽。然而，它在小鼠胚胎干细胞增殖和向心血管系统分化中的作用仍未被研究。因此我们阐述了胸腺肽β4对小鼠胚胎干细胞增殖和分化的作用。方法：小鼠胚胎干细胞增殖和分化过程中的目的基因通过RT-PCR, real-time PCR检测，通过Western blotting和流式细胞术检测目的蛋白的表达水平。运用膜片钳技术研究胚胎干细胞分化的心肌细胞的电生理特性。结果：研究发现胸腺肽β4呈部分浓度依赖性的抑制了D3系小鼠胚胎干细胞增殖，同时下调了细胞周期调控基因c-myc,c-fos及c-jun的表达。对信号通路的研究发现胸腺肽β4抑制了STAT3和Akt的磷酸化，促进RAS和磷酸化ERK的表达，而对BMP2/BMP4和下游蛋白smad均无作用。胸腺肽β4使得Wnt3和Wnt11的表达明显下降，同时伴随着Tcf3表达的上调，β-catenin的表达则不变。在胚胎干细胞分化过程中，胸腺肽β4对a-MHC, PECAM, a-SMA的表达并无影响，并且由胚胎干细胞分化而来的心肌细胞其电生理特性和对激素的调节均不受胸腺肽β4影响。结论：胸腺肽β4通过激活STAT3, Akt, ERK和Wnt等途径抑制小鼠胚胎干细胞增殖。然而，对小鼠胚胎干细胞向心血管系统的分化并无影响，也不影响胚胎干细胞源性的心肌细胞的成熟。
[Abstract]:Purpose :

Cardiac ischemia and hypoxia is one of the most common causes of death in clinic . Both protective effects of premodulation and chronic hypoxia can be generated after ischemia and hypoxia . K _ ( ATP ) has been found to be the only ion channel involved in the process . It has been found that the tolerance of mammalian embryo to hypoxia is significantly higher than that of adult animals , which may be related to the development - dependent changes of ion channels .

Method :

In the early and late stage mouse embryo ventricle , collagenase B was digested to obtain single beat ventricular myocytes . As a specific openers of K _ ( ATP ) , the current clamp recorded the electrophysiological characteristics of K _ ( ATP ) . The development - dependent changes of Kir6.1 , Kir6.2 , SUR1 and SUR2 in ventricular muscle cell membrane and mitochondria were studied by RT - PCR and Western blot .

At the same time , there was no significant difference in the expression of Kir6.1 , SUR1 and SUR2 . The results showed that there was no significant difference in the expression of Kir6.1 , SUR1 and SUR2 in the early stage ventricular myocytes . The results of RT - PCR and Western blot showed that the expression of SUR1 , Kir6.1 , Kir6.2 and SUR1 were not significantly different in the early stage .

The membrane K _ ( ATP ) plays a distinct role in the course of acute ischemia and hypoxia , and mitochondria K _ ( ATP ) plays an obvious role in the course of acute ischemia and hypoxia . The subunits of the membrane K _ ( ATP ) include Kir6.1 , Kir6.2 and SUR2 , and the expression of Kir6.1 and SUR2 increases . The subunits of the constituent mitochondria K _ ( ATP ) include Kir6.1 , Kir6.2 and SUR1 , and the expression of the subunits does not change significantly with development .

thymosin beta4 is a small molecule polypeptide capable of promoting cell migration , survival and differentiation .

Method :

The target genes in the proliferation and differentiation of mouse embryonic stem cells were detected by RT - PCR , real - time PCR and Western blotting and flow cytometry .

Results :

It was found that thymosin beta4 inhibited the proliferation of mouse embryonic stem cells and downregulated the expression of c - myc , c - fos and c - jun .

Conclusion :

However , the differentiation of mouse embryonic stem cells into the cardiovascular system is not affected , and the maturation of cardiomyocytes derived from embryonic stem cells is not affected .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R33

【共引文献】