1. 小鼠生后睾丸发育中TH1表达与分布的研究 2. SGT与CD99抗原相互作用在酵母中的验证
发布时间：2018-09-08 12:39
【摘要】： 目的：观察THl(trihydrophobinl)mRNA及THl蛋白在小鼠睾丸发育过程中的表达变化，探讨A-Raf的结合蛋白THl在睾丸发育过程中的生物学作用。 方法：提取小鼠生后睾丸发育过程中不同时间点的mRNA及蛋白，，首先用组织免疫共沉淀的方法验证THl和A-Raf在生理状态下也是共结合蛋白；用荧光PCR法检测发育睾丸mRNA水平的变化；Western Blotting检测THl在发育过程中蛋白的变化；通过免疫组织化学方法检测THl蛋白在小鼠睾丸切片中的时间和空间的表达样式。免疫荧光方法检测THl和A-Raf的细胞分布及共定位情况。 结果：(1)运用组织免疫共沉淀方法证明在小鼠睾丸中THl也作为A-Raf的结合伴侣而存在。 (2)荧光定量PCR试验结合统计分析结果显示，THlmRNA在睾丸发育过程中并不发生显著变化(P＞0.05)。 (3)Western Blotting结果显示THl蛋白在小鼠生后4周，6周睾丸中的表达量比小鼠生后1，2，8，10周睾丸中的蛋白表达量低(p＜0.05)。 (4)免疫组织化学的方法检测THl在睾丸中的分布，THl在生精小管中的分布发生变化，早期未成年的小鼠中，THl的阳性信号出现在初级精母细胞、支持细胞中；在成熟小鼠的睾丸切片，THl的阳性信号仅分布在精原细胞中。在小鼠生后的睾丸发育过程中THl蛋白均在睾丸的间质细胞(Leydig cells)中存在。 (5)为确定THl在Leydig细胞中的细胞内分布，我们提取大鼠原代睾丸间质细胞进行体外培养，用免疫荧光方法检测显示THl主要在胞浆中分布。 (6)小鼠睾丸间质细胞的肿瘤细胞(mLTC cells)与成熟小鼠睾丸组织切片的免疫荧光显示出THl和A-Raf在Leydig细胞中共定位。 结论：THl作为A-Raf在小鼠生后睾丸发育过程中的结合蛋白，在发育过程中THl蛋白在时间和空间的分布发生变化，由此推测THl可能在小鼠睾丸发育和精子的形成过程中发挥作用。 目的：证明SGT与CD99抗原在酵母中的相互作用。 方法：将质粒共转化EGY48酵母细胞，转移到缺少色氨酸、亮氨酸、尿嘧啶和组氨酸的选择性培养基，其中添加X-b-gal。能够生长和变蓝的克隆说明其中共转的质粒能够在酵母细胞相互结合。同时以pLexA-SGT和pB42AD，以及pLexA和pB42AD-CD99共转作为对照，观察酵母表型改变。将p53BD融合质粒和SV40 large T-antigen AD融合质粒共转，与BD、AD载体共转分别作为系统阳性、阴性对照。 结果：只有SGT和CD99共转化可激活LEU2+／LacZ+报告基因，且菌落生长和变蓝程度与阳性对照相当，显示二者在酵母内的相互作用。这也由二者的BD、AD载体相交换后共转化分析得到证实。 结论：SGT与CD99在酵母细胞中可以相互作用。
[Abstract]:Aim: to observe the expression of THl (trihydrophobinl) mRNA and THl protein in mouse testis and to explore the biological role of A-Raf binding protein THl in testicular development. Methods: mRNA and protein were extracted at different time points during postnatal testicular development in mice. The results of tissue immunoprecipitation were used to verify that THl and A-Raf were also co-binding proteins in physiological state, and the changes of mRNA level in testis were detected by fluorescence PCR method. Western Blotting was used to detect the changes of THl protein during development, and the expression pattern of THl protein in mouse testicular sections was detected by immunohistochemical method. The distribution and co-localization of THl and A-Raf were detected by immunofluorescence method. Results: (1) tissue immunoprecipitation method was used to prove that THl also existed as a binding companion of A-Raf in mouse testis. (2) fluorescence quantitative PCR assay combined with statistical analysis showed that THl was also present in mouse testis. There was no significant change during testicular development (P > 0. 05). (3) Western Blotting). The results showed that the expression of THl protein in testis of mice at 4 weeks and 6 weeks after birth was higher than that in testis of mice at 1 and 8 weeks after birth (P > 0. 05). (3) Western Blotting). The distribution of THl in testis and the distribution of THL in seminiferous tubules were detected by immunohistochemical method with low white expression (p < 0. 05). (4). In early immature mice, the positive signals of THl were found in primary spermatocytes and Sertoli cells, while in mature mouse testicular sections, the positive signals of THl were only distributed in spermatogonia. During the development of mouse testis, THl protein was found in (Leydig cells) of interstitial cells of testis. (5) in order to determine the distribution of THl in Leydig cells, We isolated rat primary testicular stromal cells and cultured them in vitro. Immunofluorescence method was used to detect the distribution of THl in cytoplasm. (6) (mLTC cells) of mouse testis stromal cells and sections of testicular tissue of mature mice showed that THl and A-Raf were co-located in Leydig cells. Conclusion as a binding protein of A-Raf in the development of mouse testis after birth, the distribution of THl protein changes in time and space during the development of mouse testis. It is suggested that THl may play an important role in the development of testis and spermatogenesis in mice. Objective: to demonstrate the interaction of SGT and CD99 antigen in yeast. Methods: plasmids were co-transformed into EGY48 yeast cells and transferred to selective medium lacking tryptophan, leucine, uracil and histidine, with the addition of X-b-gal. The clones which can grow and turn blue show that the co-transformed plasmids can bind to each other in yeast cells. The phenotypic changes of yeast were observed with pLexA-SGT and pB42AD, and pLexA and pB42AD-CD99 as control. P53BD fusion plasmid and SV40 large T-antigen AD fusion plasmid were co-transformed with BD,AD vector as system-positive and negative control respectively. Results: only the co-transformation of SGT and CD99 could activate the LEU2 / Lacz reporter gene, and the colony growth and the degree of blue change were similar to those of the positive control, indicating the interaction between the two genes in yeast. This was confirmed by the co-transformation analysis of the two BD,AD carriers after phase exchange. Conclusion: SGT and CD99 can interact with each other in yeast cells.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
本文编号：2230546
[Abstract]:Aim: to observe the expression of THl (trihydrophobinl) mRNA and THl protein in mouse testis and to explore the biological role of A-Raf binding protein THl in testicular development. Methods: mRNA and protein were extracted at different time points during postnatal testicular development in mice. The results of tissue immunoprecipitation were used to verify that THl and A-Raf were also co-binding proteins in physiological state, and the changes of mRNA level in testis were detected by fluorescence PCR method. Western Blotting was used to detect the changes of THl protein during development, and the expression pattern of THl protein in mouse testicular sections was detected by immunohistochemical method. The distribution and co-localization of THl and A-Raf were detected by immunofluorescence method. Results: (1) tissue immunoprecipitation method was used to prove that THl also existed as a binding companion of A-Raf in mouse testis. (2) fluorescence quantitative PCR assay combined with statistical analysis showed that THl was also present in mouse testis. There was no significant change during testicular development (P > 0. 05). (3) Western Blotting). The results showed that the expression of THl protein in testis of mice at 4 weeks and 6 weeks after birth was higher than that in testis of mice at 1 and 8 weeks after birth (P > 0. 05). (3) Western Blotting). The distribution of THl in testis and the distribution of THL in seminiferous tubules were detected by immunohistochemical method with low white expression (p < 0. 05). (4). In early immature mice, the positive signals of THl were found in primary spermatocytes and Sertoli cells, while in mature mouse testicular sections, the positive signals of THl were only distributed in spermatogonia. During the development of mouse testis, THl protein was found in (Leydig cells) of interstitial cells of testis. (5) in order to determine the distribution of THl in Leydig cells, We isolated rat primary testicular stromal cells and cultured them in vitro. Immunofluorescence method was used to detect the distribution of THl in cytoplasm. (6) (mLTC cells) of mouse testis stromal cells and sections of testicular tissue of mature mice showed that THl and A-Raf were co-located in Leydig cells. Conclusion as a binding protein of A-Raf in the development of mouse testis after birth, the distribution of THl protein changes in time and space during the development of mouse testis. It is suggested that THl may play an important role in the development of testis and spermatogenesis in mice. Objective: to demonstrate the interaction of SGT and CD99 antigen in yeast. Methods: plasmids were co-transformed into EGY48 yeast cells and transferred to selective medium lacking tryptophan, leucine, uracil and histidine, with the addition of X-b-gal. The clones which can grow and turn blue show that the co-transformed plasmids can bind to each other in yeast cells. The phenotypic changes of yeast were observed with pLexA-SGT and pB42AD, and pLexA and pB42AD-CD99 as control. P53BD fusion plasmid and SV40 large T-antigen AD fusion plasmid were co-transformed with BD,AD vector as system-positive and negative control respectively. Results: only the co-transformation of SGT and CD99 could activate the LEU2 / Lacz reporter gene, and the colony growth and the degree of blue change were similar to those of the positive control, indicating the interaction between the two genes in yeast. This was confirmed by the co-transformation analysis of the two BD,AD carriers after phase exchange. Conclusion: SGT and CD99 can interact with each other in yeast cells.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
【共引文献】
相关期刊论文 前3条
1 李雯,梁颜笑,刘国荣,林武周;不同抗原修复方法对肝癌组织中内源性生物素的影响及对策[J];中国组织化学与细胞化学杂志;2005年04期
2 赵继红,王晓文,尹有群;加热抗原修复对乳腺癌组织中内源性抗生物素蛋白结合物的影响及其对策[J];实用医技杂志;2005年07期
3 严超,朱正纲,于颖彦,计骏,燕敏,陈军,刘炳亚,尹浩然,林言箴;加热抗原修复对胃癌组织中内源性生物素样分子活性的影响[J];中国误诊学杂志;2004年02期
相关博士学位论文 前4条
1 刘伟成;新基因TH1的功能研究[D];复旦大学;2006年
2 杨延钟;Trihydrophobin 1相互作用蛋白以及Trihydrophobin 1与乳腺癌细胞化疗药物敏感性之间关系的研究[D];复旦大学;2007年
3 程春明;Trihydrophobin 1与PAK1的相互作用及其生物学功能的研究[D];复旦大学;2009年
4 邹惟莹;Trihydrophobin 1促进雄激素受体降解及其在乳腺癌进展过程中作用的研究[D];复旦大学;2010年
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