1.中國雜交奶水牛A型精原細(xì)胞的分離、鑒定和純化從睪丸細(xì)胞中分離A型精原細(xì)胞部分是本實(shí)驗(yàn)的必要組成部分,因此本實(shí)驗(yàn)有相當(dāng)一部分是用來研究水牛犢牛（3-6月齡）A型精原細(xì)胞的分離、鑒定和純化。組織學(xué)和DBA免疫組化研究結(jié)果表明5月齡時A型精原細(xì)胞數(shù)量達(dá)到最大值。一種改進(jìn)的睪丸組織分離細(xì)胞方法是剪碎之后研磨,然后再用膠原酶、透明質(zhì)酸酶和脫氧核糖核酸酶消化兩次。利用層粘連蛋白、多聚賴氨酸和明膠微分度可以顯著影響A型精原細(xì)胞的純度（P<0.05）。在這些細(xì)胞外基質(zhì)分子（ECMs）中,層粘連蛋白和明膠效果很好,純度分別達(dá)到39.38±1.21%and32.15±1.60%。另外,利用層粘連蛋白和明膠混合后細(xì)胞分離液離心效果最好,可以達(dá)到>90%的純度。另外,細(xì)胞的活力并沒有因?yàn)榻M分不同而受到影響（P>0.05）。2.A型精原細(xì)胞的低溫儲藏A型精原細(xì)胞可以持續(xù)產(chǎn)生雄性配子。低溫儲藏是保存精原細(xì)胞的重要方法,但是改善低溫保存過程以免影響活力和減少的活性氧（ROS）的損害是一個迫切解決的問題。本研究的目的是利用�；撬幔�2-aminoethanesulfonic acid）作為抗氧化劑... 

【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校

【文章頁數(shù)】：75 頁

【學(xué)位級別】：博士

【文章目錄】：
ABSTRACT
摘要
1. Review of Literature
    1.1 Preamble
    1.2 Testis
    1.3 Spermatogenesis
    1.4 Stages of Spermatogenesis
        1.4.1 Mitosis
        1.4.2 Meiosis
        1.4.3 Spermiogenesis
    1.5 In vivo spermatogenesis
    1.6 In vitro spermatogenesis
    1.7 Comparison of spermatogenesis in rodents and bovine
    1.8 Strategies for isolation of spermatogonial stem cells
        1.8.1 Mechanical Isolation
        1.8.2 Enzymatic Isolation
    1.9 Purification/Enrichment of spermatogonial stem cells
        1.9.1 Laminin Selection
        1.9.2 Flow Cytometry
        1.9.3 Percoll density gradient separation
        1.9.4 Morphology based enrichment
    1.10 Identification of Spermatogonia
        1.10.1 Cluster Designation 34
        1.10.2 Podocalyxin
        1.10.3 Caudal type homebox 2
        1.10.4 Zinc finger protein 42
2. Isolation,identification and enrichment of type A spermatogonia from the testis of Chinese crossbred buffaloes（Swamp×River）
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 Testes collection and tissue preparation
        2.2.2 Testicular Histology
        2.2.3 Cell viability and yield
        2.2.4 Immunohistochemistry and Immunocytochemistry
        2.2.5 Experimental Design
        2.2.6 Statistical Analysis
    2.3 Results
        2.3.1 Testicular histology
        2.3.2 Identification of spermatogonia in the testis and after isolation
        2.3.3 Application of erythrocyte lysis buffer solution
        2.3.4 Selection of isolation strategy
        2.3.5 Application of ECM molecules for the enrichment of type A spermatogonia
    2.4 Discussion
3. Cryopreservation of Type A spermatogonia in Chinese crossbred buffaloes （Swamp ×River）
    3.1 Introduction
    3.2 Material and methods
        3.2.1 Testes collection and tissue preparation
        3.2.2 Histology and immunohistochemistry
        3.2.3 Isolation of Type A spermatogonia
        3.2.4 Cell viability
        3.2.5 Cryopreservation and thawing
        3.2.6 Analysis of oxidative stress parameters
        3.2.7 Statistical Analysis
    3.3 Results
        3.3.1 Testicular histology and immunohistochemistry
        3.3.2 Effect of taurine on viability after thawing
        3.3.3 Analysis of oxidative stress parameters
    3.4 Discussion
4. Expression analysis of spermatogonia specific markers in Chinese crossbred buffalo（Swamp x River）
    4.1 Introduction
    4.2 Materials and methods
        4.2.1 Testes collection and tissue preparation
        4.2.2 Isolation of spermatogonial stem cells（SSCs）
        4.2.3 Immunohistochemistry of testis tissues
        4.2.4 RNA extraction and cDNA synthesis
        4.2.5 Polymerase Chain Reaction,cloning and sequencing of CD34,PODXL,CDX2 and REX1
        4.2.6 Bioinformatic Analyses
        4.2.7 Quantitative real-time PCR
        4.2.8 Western-Blot Analysis
        4.2.9 Statistical analysis
    4.3 Results
        4.3.1 CD34,PODXL,REX1 and CDX2 mRNA is expressed in testis of prepubertal and adult buffalo bulls
        4.3.2 Spermatogonia express CD34,PODXL,REX1 and CDX2
        4.3.3 Quantitative real-time PCR
        4.3.4 Western blotting
    4.4 Discussion
5. Conclusions and Future Directions
References
Curriculum Vitea
ACKNOWLEDGEMENTS



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