1. 小鼠生后睪丸發(fā)育中TH1表達(dá)與分布的研究 2. SGT與CD99抗原相互作用在酵母中的驗(yàn)證
發(fā)布時(shí)間：2018-09-08 12:39
【摘要】： 目的：觀察THl(trihydrophobinl)mRNA及THl蛋白在小鼠睪丸發(fā)育過(guò)程中的表達(dá)變化，探討A-Raf的結(jié)合蛋白THl在睪丸發(fā)育過(guò)程中的生物學(xué)作用。 方法：提取小鼠生后睪丸發(fā)育過(guò)程中不同時(shí)間點(diǎn)的mRNA及蛋白，，首先用組織免疫共沉淀的方法驗(yàn)證THl和A-Raf在生理狀態(tài)下也是共結(jié)合蛋白；用熒光PCR法檢測(cè)發(fā)育睪丸mRNA水平的變化；Western Blotting檢測(cè)THl在發(fā)育過(guò)程中蛋白的變化；通過(guò)免疫組織化學(xué)方法檢測(cè)THl蛋白在小鼠睪丸切片中的時(shí)間和空間的表達(dá)樣式。免疫熒光方法檢測(cè)THl和A-Raf的細(xì)胞分布及共定位情況。 結(jié)果：(1)運(yùn)用組織免疫共沉淀方法證明在小鼠睪丸中THl也作為A-Raf的結(jié)合伴侶而存在。 (2)熒光定量PCR試驗(yàn)結(jié)合統(tǒng)計(jì)分析結(jié)果顯示，THlmRNA在睪丸發(fā)育過(guò)程中并不發(fā)生顯著變化(P＞0.05)。 (3)Western Blotting結(jié)果顯示THl蛋白在小鼠生后4周，6周睪丸中的表達(dá)量比小鼠生后1，2，8，10周睪丸中的蛋白表達(dá)量低(p＜0.05)。 (4)免疫組織化學(xué)的方法檢測(cè)THl在睪丸中的分布，THl在生精小管中的分布發(fā)生變化，早期未成年的小鼠中，THl的陽(yáng)性信號(hào)出現(xiàn)在初級(jí)精母細(xì)胞、支持細(xì)胞中；在成熟小鼠的睪丸切片，THl的陽(yáng)性信號(hào)僅分布在精原細(xì)胞中。在小鼠生后的睪丸發(fā)育過(guò)程中THl蛋白均在睪丸的間質(zhì)細(xì)胞(Leydig cells)中存在。 (5)為確定THl在Leydig細(xì)胞中的細(xì)胞內(nèi)分布，我們提取大鼠原代睪丸間質(zhì)細(xì)胞進(jìn)行體外培養(yǎng)，用免疫熒光方法檢測(cè)顯示THl主要在胞漿中分布。 (6)小鼠睪丸間質(zhì)細(xì)胞的腫瘤細(xì)胞(mLTC cells)與成熟小鼠睪丸組織切片的免疫熒光顯示出THl和A-Raf在Leydig細(xì)胞中共定位。 結(jié)論：THl作為A-Raf在小鼠生后睪丸發(fā)育過(guò)程中的結(jié)合蛋白，在發(fā)育過(guò)程中THl蛋白在時(shí)間和空間的分布發(fā)生變化，由此推測(cè)THl可能在小鼠睪丸發(fā)育和精子的形成過(guò)程中發(fā)揮作用。 目的：證明SGT與CD99抗原在酵母中的相互作用。 方法：將質(zhì)粒共轉(zhuǎn)化EGY48酵母細(xì)胞，轉(zhuǎn)移到缺少色氨酸、亮氨酸、尿嘧啶和組氨酸的選擇性培養(yǎng)基，其中添加X(jué)-b-gal。能夠生長(zhǎng)和變藍(lán)的克隆說(shuō)明其中共轉(zhuǎn)的質(zhì)粒能夠在酵母細(xì)胞相互結(jié)合。同時(shí)以pLexA-SGT和pB42AD，以及pLexA和pB42AD-CD99共轉(zhuǎn)作為對(duì)照，觀察酵母表型改變。將p53BD融合質(zhì)粒和SV40 large T-antigen AD融合質(zhì)粒共轉(zhuǎn)，與BD、AD載體共轉(zhuǎn)分別作為系統(tǒng)陽(yáng)性、陰性對(duì)照。 結(jié)果：只有SGT和CD99共轉(zhuǎn)化可激活LEU2+／LacZ+報(bào)告基因，且菌落生長(zhǎng)和變藍(lán)程度與陽(yáng)性對(duì)照相當(dāng)，顯示二者在酵母內(nèi)的相互作用。這也由二者的BD、AD載體相交換后共轉(zhuǎn)化分析得到證實(shí)。 結(jié)論：SGT與CD99在酵母細(xì)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.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346
本文編號(hào)：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.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346
【共引文獻(xiàn)】
相關(guān)期刊論文 前3條
1 李雯,梁顏笑,劉國(guó)榮,林武周;不同抗原修復(fù)方法對(duì)肝癌組織中內(nèi)源性生物素的影響及對(duì)策[J];中國(guó)組織化學(xué)與細(xì)胞化學(xué)雜志;2005年04期
2 趙繼紅,王曉文,尹有群;加熱抗原修復(fù)對(duì)乳腺癌組織中內(nèi)源性抗生物素蛋白結(jié)合物的影響及其對(duì)策[J];實(shí)用醫(yī)技雜志;2005年07期
3 嚴(yán)超,朱正綱,于穎彥,計(jì)駿,燕敏,陳軍,劉炳亞,尹浩然,林言箴;加熱抗原修復(fù)對(duì)胃癌組織中內(nèi)源性生物素樣分子活性的影響[J];中國(guó)誤診學(xué)雜志;2004年02期
相關(guān)博士學(xué)位論文 前4條
1 劉偉成;新基因TH1的功能研究[D];復(fù)旦大學(xué);2006年
2 楊延鐘;Trihydrophobin 1相互作用蛋白以及Trihydrophobin 1與乳腺癌細(xì)胞化療藥物敏感性之間關(guān)系的研究[D];復(fù)旦大學(xué);2007年
3 程春明;Trihydrophobin 1與PAK1的相互作用及其生物學(xué)功能的研究[D];復(fù)旦大學(xué);2009年
4 鄒惟瑩;Trihydrophobin 1促進(jìn)雄激素受體降解及其在乳腺癌進(jìn)展過(guò)程中作用的研究[D];復(fù)旦大學(xué);2010年
本文編號(hào)：2230546
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2230546.html
最近更新
教材專著
