硒(Se)對于維持精子的結(jié)構(gòu)完整性,生精小管的發(fā)育發(fā)揮重要作用。硒缺乏與精子運(yùn)動(dòng)受損和生精小管變性有關(guān)。硒通過硒蛋白發(fā)揮其作用。大多數(shù)具有已知功能的硒蛋白都含有抗氧化和氧化還原調(diào)節(jié)特性。因此,Se缺乏會(huì)引起導(dǎo)致細(xì)胞損傷和/或死亡的氧化應(yīng)激。Selenoprotein U(SelU)是一種新鑒定的蛋白質(zhì),在人類和雞體內(nèi)廣泛表達(dá)。類似于硒蛋白的其他巰基依賴性氧化還原家族,SelU序列也具有類似折疊的甲狀腺毒素;但是該功能仍然未知。因此,我們設(shè)計(jì)本研究以確定:(1)SelU在雞睪丸支持細(xì)胞(SCs)自噬和凋亡中的作用(體外)(2)Se缺乏對雞睪丸中硒蛋白表達(dá)的影響(3)硒缺乏對雞睪丸氧化狀態(tài)和組織病理學(xué)的影響(體內(nèi))(4)硒缺乏和SelU沉默對雞SC中調(diào)節(jié)因子和連接相關(guān)基因表達(dá)的影響&體內(nèi)。因此,對于體外實(shí)驗(yàn),我們構(gòu)建SCs培養(yǎng)模型以研究SelU在SC中的自噬,細(xì)胞凋亡,調(diào)節(jié)因子和連接相關(guān)基因中的作用。使用6周齡未成熟產(chǎn)蛋雞分離原代SC,并在HEPES緩沖的F12/DMEM中培養(yǎng)。由于存在特征性形態(tài)學(xué)特征,利用顯微鑒定SC。之后,用特異性SelU靶向siRNA轉(zhuǎn)染細(xì)胞以建立SelU敲...

【文章頁數(shù)】：100 頁

【學(xué)位級別】：博士

【文章目錄】：
摘要
Abstract
1 Introduction
    1.1 Chicken as experimental model
    1.2 Selenium
        1.2.1 Form and distribution
        1.2.2 Se deficiency and disease
        1.2.3 Selenoproteins
        1.2.4 Expression pattern of Selenoproteins in Se deficiency
        1.2.5 Selenoproteins knockdown and/or over-expression
        1.2.6 Autophagy and Apoptosis
    1.3 Se and Male Reproduction
        1.3.1 Selenium role in Testes
        1.3.2 Oxidative stress in Testes
        1.3.3 Functional importance of Sertoli cells
        1.3.4 Selenoprotein U and its role in reproduction
    1.4 Objectives of Research
2 Material and methods
    2.1 In-vitro Experiment
        2.1.1 Sertoli Cell culture
        2.1.2 si RNA transfection of Sertoli cells
        2.1.3 Total RNA Extraction
        2.1.4 Primers Design
        2.1.5 Quantitative Real-time PCR
        2.1.6 Western Blotting
        2.1.7 Electron Microscopy
    2.2 In-vivo Experiment
        2.2.1 Birds and experimental design
        2.2.2 Total RNA Extraction
        2.2.3 Primers Design
        2.2.4 Quantitative Real-time PCR
        2.2.5 Measurement of Oxidative stress
        2.2.6 Hematoxylin and Eosin Staining
    2.3 Statistical analysis
3 Results
    3.1 In-vitro Experiment
        3.1.1 Establishment of Sel U knockdown model
        3.1.2 mRNA expression of autophagy and cyto-skeleton genes in SCs
        3.1.3 mRNA expression of apoptosis genes in SCs
        3.1.4 mRNA expression of PI3K-Akt signaling pathway genes in SCs
        3.1.5 mRNA expression of Regulatory factors in SCs
        3.1.6 mRNA expressions of Junction associated genes in SCs
        3.1.7 Protein expression of autophagy genes in SCs
        3.1.8 Protein expression of apoptosis genes in SCs
        3.1.9 Protein expression of PI3K-Akt signaling pathway genes in SCs
        3.1.10 Ultrastructure analysis
    3.2 In-vivo Experiment
        3.2.1 mRNA expressions of selenoproteins in Testis tissues
        3.2.2 mRNA expression of PI3K-Akt and MEK-ERK pathway genes in Testis tissues
        3.2.3 mRNA expression of Regulatory factors in Testis tissues
        3.2.4 mRNA expression of Junction associated genes in Testis tissues
        3.2.5 Measurement of oxidative stress in Testis tissues
        3.2.6 Histo-pathology of Testis tissues
4 Discussion
    4.1 Role of Selenoprotein U in autophagy and apoptosis in chicken Sertoli cells (in-vitro)
    4.2 Effect of Se deficiency on the expression of selenoproteins in chicken testis (in-vivo)
    4.3 Effect of Se deficiency on the oxidative status and histopathology in chicken testis (in-vivo)
    4.4 Effect of Se deficiency and Sel U silencing on the expression of Regulatory factors andJunction associated genes in chicken Sertoli cells (in-vitro and in-vivo)
5 Conclusion
Acknowledgement
References
Papers Published in the period of PhD Study



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