猪斯钙素-1基因对线粒体功能的影响及其信号通路相关基因在不同体型猪表达模式检测

发布时间：2018-03-18 19:19

本文选题：猪　切入点：斯钙素　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：斯钙素(Stanniocalcin,STC)是最早在鱼类中发现一种糖蛋白激素,起调节钙/磷平衡的作用。血清钙升高可以引起斯钙尼氏小体释放STC-1,钙离子流通过鱼鳃、肠来保持血液中钙离子浓度的稳定。近年来在人和其它哺乳动物中也发现STC,先后分别命名为STC-1和STC-2。这两种基因在大部分组织中广泛表达,它们主要通过自分泌/旁分泌方式来调节动物机体多个生理功能,其中STC-1基因主要功能是通过调控钙/磷平衡、调控体内骨代谢以及通过线粒体中解偶联蛋白阻碍三磷酸腺苷酸(ATP)正常产生,从而影响生长发育。本研究通过构建STC-1基因过表达载体和设计合成的STC-1 siRNA载体分别上调和下调STC-1基因的表达,在细胞水平上检测STC-1基因对线粒体功能的影响。通过巴马猪和大白猪两种不同体型猪中STC-1基因及其与细胞代谢、生长发育相关基因表达水平分析,研究这些基因的表达特征。通过对上述基因生理功能和其调控机制的分析以及鉴定影响猪体型发育的新通路,为初步阐明不同体型猪发育差异的遗传学机制提供了理论基础。主要研究内容和结果如下:1.猪STC-1基因对细胞线粒体功能的影响本研究根据猪肌肉斯钙素(STC)mRNA序列设计了 3对小干扰RNA(Smallinterfering RNA;siRNA)、阴性对照siRNA。转染PK15细胞(猪肾细胞)后,通过荧光显微镜观察荧光判断转染效率和基因表达效率,利用QRT-PCR与Western-blot测定细胞内siRNA基因沉默后STC-1 siRNA表达水平与STC-1蛋白的表达量。结果表明,成功设计了猪STC-1 siRNA引物序列,在细胞水平对该基因沉默效率进行了检测,证明设计的STC-1 siRNA均能对STC-1进行基因沉默,其中STC1-132基因沉默效率最好。将筛选出的STC-1 siRNA沉默效果好的基因(siSTC组)与构建的STC-1基因过表达载体(STC组)转染PK15细胞后,通过Westernblot分别检测STC组与siSTC组的STC-1蛋白、线粒体相关蛋白及乙酰化的表达量。结果显示,STC-1基因过表达或沉默能显著上调或下调PMP70、OPA、DRP、mfn的表达水平。利用流式细胞仪分别检测各组细胞凋亡、线粒体膜电位、活性氧(ROS)、ATP等相关理化指标,以及用透射电镜观察各组细胞线粒体结构和形态的变化。结果显示,STC-1基因过表达和基因沉默后都可以降低细胞凋亡率和活性氧生成,显著提高细胞膜电位,大幅度降低细胞内ATP的生成,同时也可影响线粒体的形态结构和数量。说明STC-1基因表达水平的变化和线粒体功能密切相关,该基因在维持线粒体正常功能方面可能发挥一定的作用。2.猪STC-1基因及其信号通路相关蛋白在不同体型猪中表达模式检测本研究分别采集巴马猪和大白猪的肺脏、肾脏、软骨、肝脏、脾脏、肌肉、心脏等组织,运用Western blot分别对大小体型猪中多组织的STC-1蛋白与线粒体融合蛋白OPA、Mfn、DRP以及线粒体能量代谢蛋白PMP70表达进行检测。并对STC-1基因进行表观遗传学检测。结果表明,大小体型猪多组织尤其是骨骼肌、心肌及软骨组织中的STC-1蛋白与线粒体融合蛋白OPA、Mfn、DRP以及线粒体能量代谢蛋白PMP70的表达有显著差异。上述蛋白在巴马猪多组织中显著高于大白猪,初步说明STC-1基因及其信号通路基因可能与猪的体型发育有一定关联。
[Abstract]:Stanniocalcin-1 (Stanniocalcin, STC) is a glycoprotein hormone found early in fish, regulates calcium and phosphate homeostasis. Elevated serum calcium can cause J Nis body to release STC-1, calcium ion flow through the gills, intestine to maintain the blood calcium concentration stability. In recent years in humans and other mammals also found in STC, respectively named the extensive expression of these two genes STC-1 and STC-2. in most organizations, they mainly through autocrine / paracrine manner to regulate multiple physiological functions of the animal body, the main function of the STC-1 gene is through the regulation of calcium / phosphorus balance, metabolism of bone and mitochondrial uncoupling protein in the regulation block three AMP (ATP) in normal production, thus affecting the growth and development. This study constructed STC-1 gene expression vector and siRNA vector synthesis design of STC-1 and downregulation of STC-1 respectively. Gene expression detection effect of STC-1 gene on the mitochondrial function at the cellular level. The Bama pigs and large white pigs in two different type STC-1 gene and cell metabolism, the expression level of genes related to growth and development, expression of these genes. The new pathway analysis of the gene physiological function and its regulation mechanism the identification and effect of pig development, provides a theoretical basis for clarify the genetic mechanism of different type of pig development differences. The main research contents and results are as follows: 1. of porcine STC-1 gene on mitochondrial function of the influence according to the study of pig muscle stanniocalcin-1 (STC) mRNA sequence designed 3 pairs of small interfering RNA (Smallinterfering RNA; siRNA), negative control siRNA. transfected PK15 cells (pig kidney cells), by fluorescence microscopy fluorescence to determine transfection efficiency and gene expression efficiency by QRT- PCR and Western-blot determination of expression level and STC-1 protein STC-1 in siRNA cells after siRNA gene silencing. The results showed that the successful design of the porcine STC-1 siRNA primer sequence, at the cellular level to detect the gene silencing efficiency, proved that the design of the STC-1 siRNA are able to STC-1 gene silencing, STC1-132 gene silencing efficiency best siRNA. STC-1 silencing screened good genes (siSTC group) and STC-1 gene over expression vector (group STC) were transfected into PK15 cells, STC group and siSTC group of STC-1 protein were detected by Westernblot, the expression of mitochondrial proteins and acetylated. The results showed that overexpression of STC-1 or silencing can significantly up-regulated or down regulated PMP70, OPA, DRP, the expression level of MFN. The cell apoptosis was detected by flow cytometry, mitochondrial membrane potential and reactive oxygen species (ROS), ATP and other related physical and chemical The index, and to observe the changes of mitochondrial structure and morphology of each group by transmission electron microscopy. The results showed that STC-1 gene overexpression and gene silencing can reduce cell apoptosis and ROS generation, significantly increased the cell membrane potential, decrease intracellular ATP generation, but also can affect the morphology and number of mitochondria. That the expression level of STC-1 gene changes associated with mitochondrial dysfunction, may play a role in the.2. of porcine STC-1 gene and its signal pathway related proteins in the different expression of lung, pattern detection in this study were collected in Bama pigs and large white pigs in the kidney, liver, spleen, muscle, cartilage, the gene in maintaining normal mitochondrial the function of the heart, and other organizations, the use of Western blot and STC-1 protein respectively on mitochondrial organization size in pigs OPA fusion protein Mfn, DRP and mitochondria To detect expression of PMP70 protein metabolism. STC-1 gene was analyzed by epigenetic detection. The results show that the size of pig tissues especially skeletal muscle, STC-1 protein and myocardial mitochondria and cartilage in OPA fusion protein Mfn, DRP and PMP70 protein expression of mitochondrial energy metabolism are significantly different. The protein in Bama the pig tissues was significantly higher than that of largewhite, indicated that the STC-1 gene and its signal transduction genes may be associated with pig body development has a certain relevance.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.2

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