Zhangfei蛋白在小鼠发情周期卵巢及子宫中的表达
发布时间：2018-12-07 19:31
【摘要】：Zhangfei属于具有碱性亮氨酸拉链bZIP结构的转录因子,它参与调节内质网应激和未折叠蛋白反应,并在胚胎发育、人类疾病过程等方面也发挥着重要作用。至今,国内外对Zhangfei蛋白的研究甚少,在生殖内分泌领域的作用未见报道。本实验在抗Zhangfei单克隆抗体大量制备的基础上,采用超敏感的免疫组织化学方法和蛋白免疫印迹技术对小鼠在发情周期中卵巢和子宫中的Zhangfei蛋白表达进行了研究。主要结果如下: 1采用切胶电洗脱的方法对Zhangfei融合蛋白进行纯化,可以获得纯度较高的Zhangfei融合蛋白,浓度达2.002 mg/mL;通过培养杂交瘤细胞和小鼠腹水诱生法对抗Zhangfei单抗进行制备,ELISA方法检测杂交瘤细胞培养上清中单抗的效价为1∶1000,腹水单抗的效价为1∶2×10~5。 2 Zhangfei在发情周期各阶段小鼠卵巢的卵母细胞胞浆中均有表达。原始卵泡和初级卵泡的颗粒细胞中未见Zhangfei蛋白阳性染色,次级卵泡阶段的颗粒细胞出现Zhangfei蛋白的阳性染色;成熟卵泡的颗粒细胞中Zhangfei蛋白的阳性染色更加明显。此外,在整个发情周期的黄体中均有Zhangfei蛋白阳性分布,而在凋亡的颗粒细胞和各级卵泡的卵泡膜细胞中无表达。从发情前期,发情期,发情后期到间情期,Zhangfei蛋白的表达逐渐增强,推论其可能与卵巢中的类固醇激素有关,对卵巢的组织学变化和协调卵泡成熟过程有重要的生殖生理调控作用。 3发情各阶段小鼠子宫均见Zhangfei蛋白的表达,主要分布于内膜腔上皮和腺上皮细胞中,其他部位无表达。其中,Zhangfei蛋白在间情期表达较强,发情期表达较弱,暗示它的表达可能受到卵巢类固醇激素的调控,参与子宫周期性变化的调节。 综上所述,本试验在制备抗Zhangfei单克隆抗体的基础上,利用免疫组化技术和蛋白免疫印迹技术对在发情周期不同阶段小鼠卵巢和子宫中的Zhangfei蛋白表达分布进行研究,为进一步研究Zhangfei蛋白在生殖内分泌领域的作用奠定了基础。
[Abstract]:Zhangfei is a transcription factor with basic leucine zipper bZIP structure. It plays an important role in regulating endoplasmic reticulum stress and unfolded protein reaction, and also plays an important role in embryonic development and human disease process. Up to now, there are few studies on Zhangfei protein at home and abroad, and the role of Zhangfei protein in the field of reproductive endocrine has not been reported. Based on the preparation of monoclonal antibodies against Zhangfei, the expression of Zhangfei protein in ovary and uterus of mice during estrous cycle was studied by ultrasensitive immunohistochemical method and Western blotting technique. The main results are as follows: 1 Zhangfei fusion protein with high purity can be obtained by electroelution of Zhangfei with a concentration of 2.002 mg/mL;. Zhangfei monoclonal antibody was prepared by culture of hybridoma cells and mice ascites. The titer of monoclonal antibody in the supernatant of hybridoma cells detected by ELISA method was 1: 1000, and the titer of monoclonal antibody in ascites was 1:2 脳 10 ~ (5). 2 Zhangfei was expressed in oocyte cytoplasm of mouse ovary at all stages of estrous cycle. No positive staining of Zhangfei protein was found in granulosa cells of primordial follicles and primary follicles, but the positive staining of Zhangfei protein was found in granulosa cells of secondary follicles, especially in granulosa cells of mature follicles. In addition, there was a positive distribution of Zhangfei protein in the corpus luteum of the whole estrous cycle, but not in the apoptotic granulosa cells and follicular membrane cells. From early estrus, late estrus to interoestrus, the expression of Zhangfei protein increased gradually, inferred that the expression of Zhangfei protein might be related to steroid hormones in ovary. It plays an important role in regulating ovarian histology and coordinating follicular maturation. (3) the expression of Zhangfei protein was found in the uterus of all stages of estrus, mainly in the epithelial cells of endometrium and glandular epithelium, but not in other parts. Among them, the expression of Zhangfei protein was strong in the interoestrous stage and weak in the estrus stage, suggesting that the expression of Zhangfei protein might be regulated by ovarian steroid hormone and participate in the regulation of the periodic changes of uterus. In conclusion, on the basis of the preparation of monoclonal antibodies against Zhangfei, the expression and distribution of Zhangfei protein in ovary and uterus of mice at different stages of estrous cycle were studied by immunohistochemistry and Western blotting. It lays a foundation for further study on the role of Zhangfei protein in reproductive endocrine field.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R339.2
本文编号：2367722
[Abstract]:Zhangfei is a transcription factor with basic leucine zipper bZIP structure. It plays an important role in regulating endoplasmic reticulum stress and unfolded protein reaction, and also plays an important role in embryonic development and human disease process. Up to now, there are few studies on Zhangfei protein at home and abroad, and the role of Zhangfei protein in the field of reproductive endocrine has not been reported. Based on the preparation of monoclonal antibodies against Zhangfei, the expression of Zhangfei protein in ovary and uterus of mice during estrous cycle was studied by ultrasensitive immunohistochemical method and Western blotting technique. The main results are as follows: 1 Zhangfei fusion protein with high purity can be obtained by electroelution of Zhangfei with a concentration of 2.002 mg/mL;. Zhangfei monoclonal antibody was prepared by culture of hybridoma cells and mice ascites. The titer of monoclonal antibody in the supernatant of hybridoma cells detected by ELISA method was 1: 1000, and the titer of monoclonal antibody in ascites was 1:2 脳 10 ~ (5). 2 Zhangfei was expressed in oocyte cytoplasm of mouse ovary at all stages of estrous cycle. No positive staining of Zhangfei protein was found in granulosa cells of primordial follicles and primary follicles, but the positive staining of Zhangfei protein was found in granulosa cells of secondary follicles, especially in granulosa cells of mature follicles. In addition, there was a positive distribution of Zhangfei protein in the corpus luteum of the whole estrous cycle, but not in the apoptotic granulosa cells and follicular membrane cells. From early estrus, late estrus to interoestrus, the expression of Zhangfei protein increased gradually, inferred that the expression of Zhangfei protein might be related to steroid hormones in ovary. It plays an important role in regulating ovarian histology and coordinating follicular maturation. (3) the expression of Zhangfei protein was found in the uterus of all stages of estrus, mainly in the epithelial cells of endometrium and glandular epithelium, but not in other parts. Among them, the expression of Zhangfei protein was strong in the interoestrous stage and weak in the estrus stage, suggesting that the expression of Zhangfei protein might be regulated by ovarian steroid hormone and participate in the regulation of the periodic changes of uterus. In conclusion, on the basis of the preparation of monoclonal antibodies against Zhangfei, the expression and distribution of Zhangfei protein in ovary and uterus of mice at different stages of estrous cycle were studied by immunohistochemistry and Western blotting. It lays a foundation for further study on the role of Zhangfei protein in reproductive endocrine field.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R339.2
【引证文献】
相关硕士学位论文 前1条
1 陈风雷;小鼠Zhangfei基因过表达和shRNA干扰慢病毒载体的构建和鉴定[D];西北农林科技大学;2013年
，本文编号：2367722
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