神经肽Y对原代大鼠心肌细胞线粒体结构及能量代谢的影响

发布时间：2018-06-04 11:20

本文选题：神经肽Y + 线粒体结构　；参考：《武汉大学》2016年博士论文

【摘要】：目的：采用不同剂量的NPY对原代培养的大鼠心肌细胞进行刺激,通过对心肌细胞活性、ATP含量、线粒体结构、线粒体膜电位、PGC-1α蛋白及mRNA进行检测,进而验证NPY对原代培养大鼠心肌细胞的影响,从细胞层面研究应激性心肌病的发病机理。方法：1.原代心肌细胞的培养与认定：利用联合酶消化法获得心肌细胞,通过差速贴壁法和添加BrdU纯化心肌细胞,根据心肌细胞自主搏动的特性,实时录像进行验证,根据特殊标记物通过免疫组织化学方法进行特异性检验。2.原代心肌细胞功能性检验：用NPY对原代培养的大鼠心肌细胞进行干预24小时后,用CCK-8试剂盒检测心肌细胞的活性改变并用ATP试剂盒检测ATP含量的变化。3.原代心肌细胞形态学检验：NPY对原代培养的大鼠心肌细胞进行干预后,用JC-1试剂盒检测线粒体膜电位的改变,荧光显微镜进行观察；利用透射电镜对干预后的原代大鼠心肌细胞线粒体结构的变化进行检测。4.原代心肌细胞分子学水平检验：NPY对原代培养的大鼠心肌细胞进行干预后,用Real-timePCR方法检测PGC-1α mRNA、Western blotting方法检钡PGC-1α蛋白的改变,并对相关钙离子通路(CaN.CaMKII、P38MARK)分别使用阻断剂(环孢素A.KN-93.SB203580)进行阻断实验,为进一步信号传导通路的研究提供参考。结果：1.经过纯化处理后的心肌细胞搏动良好,免疫组织化学标记物α-横纹肌肌动蛋白和心肌肌钙蛋白T呈阳性表达。2.经过不同剂量NPY刺激后,原代培养的大鼠心肌细胞活性在不同时间点均有下降,显示NPY对原代心肌细胞的活性有抑制作用,但与干预时间无显著相关性；不同剂量的NPY干预后,原代培养的心肌细胞ATP含量明显下降,呈剂量依赖性关系(P0.01)。3.经过不同剂量NPY刺激后,原代培养的大鼠心肌细胞膜电位受到抑制并呈剂量依赖性关系；电镜下,线粒体结构改变明显,包括线粒体肿胀、线粒体嵴的稀疏、融合、倒塌和破裂、线粒体内部密度的改变以及异质性物质的出现等。4.经过不同剂量NPY刺激后,原代培养的大鼠心肌细胞PGC-1α的mRNA和蛋白表达上调,呈剂量依赖性关系。选择性使用阻断剂阻断实验,其中P38MAPK途径(阻断剂SB203580)表现阳性。结论：NPY能抑制原代培养的大鼠心肌细胞的能量代谢并使其活性降低,其机制可能是通过影响PGC-1α的mRNA和蛋白表达,进而导致线粒体结构的破坏和膜电位的弥散障碍,抑制细胞的能量代谢。因此,使用NPY对原代培养的心肌细胞进行干预的研究结果可为应激性心肌病的动物模型提供参考依据。
[Abstract]:Aim: to stimulate primary cultured rat cardiomyocytes with different doses of NPY, and to detect the activity of ATP, mitochondrial structure, mitochondrial membrane potential, PGC-1 伪 protein and mRNA in primary cultured rat cardiomyocytes. Furthermore, the effect of NPY on primary cultured rat cardiomyocytes was verified, and the pathogenesis of stress cardiomyopathy was studied at the cellular level. Method 1: 1. Primary cardiomyocyte culture and identification: cardiomyocytes were obtained by combined enzyme digestion, purified by differential adherent method and added BrdU, and verified by real-time video recording according to the characteristics of cardiac myocyte pulsation. The specificity was tested by immunohistochemical method according to the special marker. Functional test of primary cardiomyocytes: after the primary cultured rat cardiomyocytes were treated with NPY for 24 hours, the activity of cardiomyocytes was detected by CCK-8 kit and the change of ATP content was detected by ATP kit. Primary cardiomyocyte morphology of primary cultured rat cardiomyocytes was examined by JC-1 assay. The changes of mitochondrial membrane potential were detected by JC-1 kit and observed by fluorescence microscope. The changes of mitochondria structure of primary rat cardiomyocytes after intervention were detected by transmission electron microscope (TEM). The molecular level of primary cardiomyocytes was assayed. After intervention of w / NPY on primary cultured rat cardiomyocytes, the changes of barium PGC-1 伪 protein were detected by PGC-1 伪 mRNAs and Western blotting. The blocking experiments were carried out on CaN.CaMKIIP38MARK by using cyclosporin A.KN-93.SB203580, which provided a reference for the further study of signal transduction pathway. The result is 1: 1. After purification, the cardiac myocytes had good pulsatility, and the immunohistochemical markers 伪 -rhabdomyactin and cardiac troponin T were positive. After stimulation with different doses of NPY, the activity of primary cultured rat cardiomyocytes decreased at different time points, indicating that NPY inhibited the activity of primary cardiomyocytes, but had no significant correlation with the time of intervention. The ATP content of primary cultured cardiomyocytes decreased in a dose-dependent manner. After different doses of NPY stimulation, the membrane potential of primary cultured rat myocardium was inhibited in a dose-dependent manner, and the mitochondrial structure changed obviously under electron microscope, including mitochondrial swelling, sparse mitochondrial ridge, and fusion. Collapse and rupture, changes in the density of mitochondria and the appearance of heterogeneous substances. 4. After different doses of NPY stimulation, the expression of mRNA and protein of PGC-1 伪 in primary cultured rat cardiomyocytes was up-regulated in a dose-dependent manner. The P38MAPK pathway (SB203580) was positive. Conclusion the effects of PGC-1 伪 on the expression of mRNA and protein in primary cultured rat cardiomyocytes may lead to the destruction of mitochondria structure and the diffusion of membrane potential, which may be due to the inhibition of energy metabolism and activity of primary cultured rat cardiomyocytes. Inhibit the energy metabolism of cells. Therefore, the results of intervention with NPY on primary cultured cardiomyocytes may provide reference for animal models of stress cardiomyopathy.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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