本文選題：豬　切入點：斯鈣素　出處：《揚州大學》2017年碩士論文　論文類型：學位論文

【摘要】：斯鈣素(Stanniocalcin,STC)是最早在魚類中發(fā)現一種糖蛋白激素,起調節(jié)鈣/磷平衡的作用。血清鈣升高可以引起斯鈣尼氏小體釋放STC-1,鈣離子流通過魚鰓、腸來保持血液中鈣離子濃度的穩(wěn)定。近年來在人和其它哺乳動物中也發(fā)現STC,先后分別命名為STC-1和STC-2。這兩種基因在大部分組織中廣泛表達,它們主要通過自分泌/旁分泌方式來調節(jié)動物機體多個生理功能,其中STC-1基因主要功能是通過調控鈣/磷平衡、調控體內骨代謝以及通過線粒體中解偶聯蛋白阻礙三磷酸腺苷酸(ATP)正常產生,從而影響生長發(fā)育。本研究通過構建STC-1基因過表達載體和設計合成的STC-1 siRNA載體分別上調和下調STC-1基因的表達,在細胞水平上檢測STC-1基因對線粒體功能的影響。通過巴馬豬和大白豬兩種不同體型豬中STC-1基因及其與細胞代謝、生長發(fā)育相關基因表達水平分析,研究這些基因的表達特征。通過對上述基因生理功能和其調控機制的分析以及鑒定影響豬體型發(fā)育的新通路,為初步闡明不同體型豬發(fā)育差異的遺傳學機制提供了理論基礎。主要研究內容和結果如下:1.豬STC-1基因對細胞線粒體功能的影響本研究根據豬肌肉斯鈣素(STC)mRNA序列設計了 3對小干擾RNA(Smallinterfering RNA;siRNA)、陰性對照siRNA。轉染PK15細胞(豬腎細胞)后,通過熒光顯微鏡觀察熒光判斷轉染效率和基因表達效率,利用QRT-PCR與Western-blot測定細胞內siRNA基因沉默后STC-1 siRNA表達水平與STC-1蛋白的表達量。結果表明,成功設計了豬STC-1 siRNA引物序列,在細胞水平對該基因沉默效率進行了檢測,證明設計的STC-1 siRNA均能對STC-1進行基因沉默,其中STC1-132基因沉默效率最好。將篩選出的STC-1 siRNA沉默效果好的基因(siSTC組)與構建的STC-1基因過表達載體(STC組)轉染PK15細胞后,通過Westernblot分別檢測STC組與siSTC組的STC-1蛋白、線粒體相關蛋白及乙�；谋磉_量。結果顯示,STC-1基因過表達或沉默能顯著上調或下調PMP70、OPA、DRP、mfn的表達水平。利用流式細胞儀分別檢測各組細胞凋亡、線粒體膜電位、活性氧(ROS)、ATP等相關理化指標,以及用透射電鏡觀察各組細胞線粒體結構和形態(tài)的變化。結果顯示,STC-1基因過表達和基因沉默后都可以降低細胞凋亡率和活性氧生成,顯著提高細胞膜電位,大幅度降低細胞內ATP的生成,同時也可影響線粒體的形態(tài)結構和數量。說明STC-1基因表達水平的變化和線粒體功能密切相關,該基因在維持線粒體正常功能方面可能發(fā)揮一定的作用。2.豬STC-1基因及其信號通路相關蛋白在不同體型豬中表達模式檢測本研究分別采集巴馬豬和大白豬的肺臟、腎臟、軟骨、肝臟、脾臟、肌肉、心臟等組織,運用Western blot分別對大小體型豬中多組織的STC-1蛋白與線粒體融合蛋白OPA、Mfn、DRP以及線粒體能量代謝蛋白PMP70表達進行檢測。并對STC-1基因進行表觀遺傳學檢測。結果表明,大小體型豬多組織尤其是骨骼肌、心肌及軟骨組織中的STC-1蛋白與線粒體融合蛋白OPA、Mfn、DRP以及線粒體能量代謝蛋白PMP70的表達有顯著差異。上述蛋白在巴馬豬多組織中顯著高于大白豬,初步說明STC-1基因及其信號通路基因可能與豬的體型發(fā)育有一定關聯。
[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.

【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.2

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