【作者】 秦海燕；

【导师】 王晶磊；

【作者基本信息】 南昌大学 ， 生理学， 2008， 硕士

【摘要】 目的:探索C-erbB₂蛋白在小鼠围着床期子宫组织中的表达与定位;研究小鼠子宫内膜上皮（endometrial epithelial cells EECs）细胞的分离、鉴定和体外培养技术;建立小鼠体外胚胎着床的模型和共培养体系;初步探讨c-erbB₂反义寡核苷酸（c-erbB₂ ASODN）对小鼠体外胚胎着床的影响。方法:1、用促超排法制作妊娠小鼠的动物模型;2、用免疫组化法观察C-erbB₂蛋白在围着床期小鼠子宫组织中的表达与定位;3、用分步酶消化法分离培养EECs,并用抗细胞角蛋白（CK-19）免疫细胞化学方法鉴定EECs;4、将已贴壁的EECs与获取的小鼠胚泡进行共培养以建立小鼠体外胚胎着床模型,分组观察比较胚泡在不同培养体系中的脱带率、粘附率、扩展率,确定理想的共培养体系;5、分组观察比较c-erbB₂ ASODN对小鼠体外胚胎着床的影响;6、用western blotting法检测各组EECs中C-erbB₂蛋白的表达情况。结果:1、免疫组化结果显示在未孕子宫和空白对照组子宫中未见C-erbB₂蛋白的表达,D1-3天子宫中C-erbB₂蛋白主要分布在子宫内膜腔上皮和腺上皮细胞胞膜上,子宫基质细胞中未见表达,D4-5天子宫中C-erbB₂蛋白除了表达在子宫内膜腔上皮和腺上皮细胞胞膜上,上皮下基质细胞膜上可见弱阳性表达,D6-8天的子宫中C-erbB₂蛋白主要分布在子宫内膜腔上皮和腺上皮细胞以及围绕着床位点的蜕膜细胞胞膜上,各组肌层均未见阳性表达;2、分步酶消化法成功分离培养EECs,用抗CK-19免疫细胞化学染色鉴定EECs,其阳性率达到90%以上;3、三组共培养组（无血清共培养、3%FBS共培养组、0.4%BSA共培养组）的脱带率、粘附率和扩展率明显高于胚泡单独培养组（P<0.05）,3%FBS共培养组和0.4%BSA共培养组的脱带率、粘附率和扩展率又明显高于无血清共培养组（P<0.05）,而3%FBS共培养组的脱带率、粘附率、扩展率和0.4%BSA共培养组相比,差别无显著性（P>0.05）;4、用c-erbB₂ ASODN干预着床模型结果显示:各组24小时脱带率差别无统计学意义（P>0.05）;5μmol/L c-erbB₂ ASODN组48小时粘附率与扩展率低于对照组,但差别无统计学意义（P>0.05）,10μmol/L c-erbB₂ ASODN组48小时粘附率与扩展率明显低于对照组,差别有统计学意义（P<0.05）,15、20μmol/L c-erbB₂ ASODN组48小时粘附率与扩展率显著低于对照组,差别有显著统计学意义（P<0.01）,而20μmol/L c-erbB₂ ODN组48小时的粘附率和扩展率与对照组相比无差别（P>0.05）; 5、Western Blotting法检测c-erbB₂ ASODN各组EECs中C-erbB₂蛋白表达结果显示:不同浓度的c-erbB₂ ASODN作用着床模型48小时后,5μmol/L c-erbB₂ ASODN组蛋白的表达低于对照组,但差别无统计学意义（P>0.05）, 10μmol/L c-erbB₂ ASODN组表达明显低于对照组,差别有统计学意义（P<0.05）,15、20μmol/L c-erbB₂ ASODN组蛋白的表达显著低于对照组,差别有显著统计学意义（P<0.01）;另外,20μmol/L c-erbB₂ ASODN作用24小时后C-erbB₂蛋白表达低于对照组（P<0.05）,20μmol/L c-erbB₂ ASODN作用48小时后C-erbB₂蛋白表达显著低于对照组（P<0.01）。结论:1、C-erbB₂蛋白在着床期小鼠子宫组织中表达有规律性变化;2、分步酶消化法是分离培养子宫内膜上皮细胞的有效方法;3、胚泡与EECs共培养是比较理想的着床模型,3%FBS和0.4%BSA都是比较理想的共培养体系;4、C-erbB₂蛋白能促进小鼠胚胎的体外着床。更多还原

【Abstract】 Objective:To investigate the expression and distribution of C-erbB₂ protein in the periimplantation mouse uterus; to research the separation, cultivation and identification technology of endometrial epithelial cells（EECs） in vitro; to build the mouse implantation model in vitro and find the ideal cultivational system of mouse implantation model; To preliminarily investigate the effection of c-erbB₂ ASODN on the mouse implantation model in vitro.Methods:1、The pro-ovulated method was used to make pregnant animal models; 2、Immunohistochemistry staining was used to examine the localization and expression of the C-erbB₂ protein in the peri-implantation uterus; 3、The EECs was divided from the Day 4 pregnant uterus by enzyme digestion, and the percentage of the EECs expressing CK-19 was more than 90%; 4、We used man-made glass pipes to inhale the mice blastocysts, and cultivated them with the EECs, in order to determine the ideal co-cultivated system then we divided them into 4 groups; 5、We used the contradict method to observe the effects of the c-erbB₂ ASODN on mouse implantation model in vitro, and divided them into 6 groups,after 24 hours’co-culture we recorded the hatching rate of these groups, after 48 hours’co-culture we recorded the adhesion rate and outgrowth rate of these groups; 6、The expression of C-erbB₂ albumen in the EECs was detected by Western Blotting.Results:1、Immunohistochemistry experiments showed that unique uterine cell-specific C-erbB₂ protein distribution. The nagetive control slice and non-pregnant uterus exhibited no signals. On day 1-3 unlike in the stromal cells, the C-erbB₂ protein was detected primarily in the EECs; on day 4-5 besides the EECs, the stromal cells expressed a little C-erbB₂ protein, on day 6-8 the EECs and the decidualized cells around the implanting blastocyst exhibited the accumulation of C-erbB₂ protein. 2、The percentage of the EECs expressing CK-19 was 90% more or less , which were isolated from the mouse day 4 pregnant uterus. 3、As shown in the blank the ratios of embryonic hatchment, attachment and outgrowth of the BSA group and the FBS group were obviously higher than the before two groups （P<0.05）, but the difference between the last two groups were no significant （P>0.05）;There was no difference in the ratios of embryonic hatchment in six groups; 4、The ratios of embryonic attachment and outgrowth between the c-erbB₂ ODN group and control group were no difference either（P>0.05）, the ratio of embryonic attachment and outgrowth in group including 5μmol/L c-erbB₂ ASODN is a little lower than control group（P>0.05）, the ratios of 48 hours’embryonic attachment and outgrown in groups including 10、15、20μmol/L c-erbB₂ ASODN were obviously lower than control group （P<0.05、P<0.01）; 5、The expression of C-erbB₂ protein in EECs was detected by Western blotting. the level of C-erbB₂ protein in groups including 5 c-erbB₂ ASODN was lower than control group, but the difference has no significance（ P>0.05）, and the level of C-erbB₂ protein in groups including 10 c-erbB₂ ASODN was lower than control group, the differnence has significance（P<0.05）, the level of C-erbB₂ protein in groups including 15、20μmol/L c-erbB₂ ASODN was lower than control group, the difference has big significance （P<0.01）; the level of C-erbB₂ protein in groups including 20 c-erbB₂ ASODN acting for 24 hours and 48 hours was lower than control group（P<0.05）.Conclusions:1、The expression of C-erbB₂ protein in the periimplantation mouse uterus changes regularly; 2、The method of enzyme digestion is a valid method to separate the EECs from the Day 4 pregnant mouse uterus; 3、The implantation models of blastocysts cultivated with EECs in medium including FBS and BSA are both the ideal implantation models; 4、C-erbB₂ protein could promote mouse embryo implantation in vitro.更多还原