PEI介导的小鼠生精细胞基因转染及其在NYD-SP12基因功能研究中的应用

发布时间：2018-10-20 12:00

【摘要】：对生精细胞特异表达基因的功能界定将有利于揭示生精过程的生物学机理。由于体外细胞学研究方式的困难,研究者们积极地探索着体内基因转染方法。然而以往大部分的基因转染方法都倾向于转染Sertoli细胞而非生精细胞。多聚乙酰亚胺(polyethylenimine,PEI)是新近发现的一种非病毒阳离子转染介导剂,本研究探索了一个新的简便而有效的体内基因转染方法:PEI介导的在体睾丸基因转染。许多研究表明PEI有着比传统的脂质体更好的转染特性,并且已被广泛应用于许多研究中,但还未在睾丸组织的转基因研究中被研究过。本研究通过活体小鼠睾九输出管注射技术将PEI/DNA(pEGFP-NYD-SP12或pEGFP质粒)复合物导入生精小管,三天后冰冻切片,立即在荧光镜下观察绿色荧光蛋白的表达,判断转染状况。结果表明目的基因通过PEI的介导可以实现生精细胞的转染,而Sertoli细胞无明显被转染迹象,且被转染细胞大多为精母细胞。对于Sertoli细胞的阴性转染结果,可能是由于Sertoli未被转染或转染后基因表达效率很低,没有被检测到,这还需进一步的实验去验证。将本研究前期克隆的新基因NYD-SP12通过以上方式转入生精细胞。NYD-SP12基因是睾九特异表达基因,小鼠中原位杂交实验表明其表达只限于生精上皮而不在间质细胞中表达。在前期的体外细胞学实验中证明NYS-SP12基因定位于高尔基体,因此我们推断该基因可能参与了精子顶体的形成。本实验
[Abstract]:The function definition of spermatogenic cell specific expression gene will be helpful to reveal the biological mechanism of spermatogenesis. Due to the difficulty of in vitro cytological research, researchers are actively exploring in vivo gene transfection methods. However, most of the previous gene transfection methods tend to transfect Sertoli cells rather than spermatogenic cells. Polyimide (polyethylenimine,PEI) is a newly discovered non-viral cationic transfection mediator. In this study, a new simple and effective method for gene transfection in vivo, PEI mediated gene transfection of testis in vivo, was explored. Many studies have shown that PEI has better transfection properties than traditional liposomes, and has been widely used in many studies, but has not been studied in the study of testicular tissue transgenic. In this study, PEI/DNA (pEGFP-NYD-SP12 or pEGFP plasmid) complexes were introduced into seminiferous tubules by injection of pEGFP-NYD-SP12 or pEGFP plasmid into seminiferous tubules. After 3 days, the expression of green fluorescent protein (GFP) was observed under fluorescence microscope, and the transfection status was evaluated. The results showed that the target gene could be transfected into spermatogenic cells by PEI, but the Sertoli cells showed no evidence of transfection, and most of the transfected cells were spermatocytes. The result of negative transfection of Sertoli cells may be due to the low efficiency of gene expression after Sertoli has not been transfected or transfected, which needs further experiments to verify. NYD-SP12, a new gene cloned in this study, was transferred into spermatogenic cells in the above way. NYD-SP12 gene is a specific gene expressed in testis. In situ hybridization in mice showed that the expression of NYD-SP12 gene was limited to spermatogenic epithelium but not in interstitial cells. In vitro cytological experiments showed that the NYS-SP12 gene was located in Golgi body, so we infer that the gene may be involved in the formation of acrosome in spermatozoa. This experiment
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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