硒缺乏对鸡睾丸的影响以及硒蛋白U对睾丸支持细胞的作用

发布时间：2023-04-01 08:52

　　硒(Se)对于维持精子的结构完整性,生精小管的发育发挥重要作用。硒缺乏与精子运动受损和生精小管变性有关。硒通过硒蛋白发挥其作用。大多数具有已知功能的硒蛋白都含有抗氧化和氧化还原调节特性。因此,Se缺乏会引起导致细胞损伤和/或死亡的氧化应激。Selenoprotein U(SelU)是一种新鉴定的蛋白质,在人类和鸡体内广泛表达。类似于硒蛋白的其他巯基依赖性氧化还原家族,SelU序列也具有类似折叠的甲状腺毒素;但是该功能仍然未知。因此,我们设计本研究以确定:(1)SelU在鸡睾丸支持细胞(SCs)自噬和凋亡中的作用(体外)(2)Se缺乏对鸡睾丸中硒蛋白表达的影响(3)硒缺乏对鸡睾丸氧化状态和组织病理学的影响(体内)(4)硒缺乏和SelU沉默对鸡SC中调节因子和连接相关基因表达的影响&体内。因此,对于体外实验,我们构建SCs培养模型以研究SelU在SC中的自噬,细胞凋亡,调节因子和连接相关基因中的作用。使用6周龄未成熟产蛋鸡分离原代SC,并在HEPES缓冲的F12/DMEM中培养。由于存在特征性形态学特征,利用显微鉴定SC。之后,用特异性SelU靶向siRNA转染细胞以建立SelU敲...

【文章页数】：100 页

【学位级别】：博士

【文章目录】：
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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