【作者】 孙艳艳；

【导师】 晁岚； 邓晓惠；

【作者基本信息】 山东大学 ， 妇产科学， 2015， 硕士

【摘要】 目的胚胎着床是生命开始的初始环节,包括定位、黏附、植入三个步骤,其调控机制复杂而精细。GRIM-19 (gene associated with retinoid-interferon-induced mortality-19)为干扰素/维甲酸联合应用诱导细胞凋亡相关基因(GRIMs)家族成员之一,是线粒体复合物I的一部分,在调节细胞凋亡和免疫反应等方面发挥重要作用。研究发现GRIM-19在早期胚胎发育中发挥重要作用,但目前其在胚胎着床中的作用未曾有过相关报道。本课题通过研究GRIM-19在小鼠围着床期子宫内膜的表达和定位情况,探讨GRIM-19在胚胎着床中的作用。在此基础上,通过体内和体外实验研究GRIM-19在着床过程中对细胞凋亡及免疫反应的影响,进一步探讨GRIM-19在胚胎着床中的可能作用机制。研究方法体内实验：收集早期妊娠第1-6天小鼠子宫组织,采用免疫组织化学法检测GRIM-19蛋白在子宫内膜上皮细胞中的表达及定位情况；收集早期妊娠、假孕和雌孕激素处理小鼠子宫组织,采用Western blotting和Real-time PCR检测GRIM-19蛋白和mRNA水平；原位末端标记技术(TUNEL)方法检测早期妊娠第1-6天小鼠子宫组织细胞凋亡情况。体外实验：采用pEGFP GRIM-19质粒GRIM-19-siRNA转染技术使RL95-2细胞株过表达或低表达GRIM-19,检测其对RL95-2-BeWo共培养体外着床模型球状体粘附率的影响,AnnexinV/PI双染色法检测细胞凋亡情况；采用Westernblotting和Real-time PCR检测转染后STAT3、TNF-α及IL-11蛋白和mRNA水平。统计学分析：采用SPSS19.0统计软件进行数据分析,结果用均数±标准差、中位数(第一个四分位数,第三个四分位数)或数字表示,组间差异采用独立样本资料的U检验,t检验或卡方检验。P<0.05为差异具有统计学意义。结果1. GRIM19在小鼠正常妊娠1-6天子宫内膜的腔上皮和腺上皮细胞均有表达,且表达具有时间特异性,GRIM-19蛋白和mRNA在妊娠D4天着床窗口期子宫内膜中表达水平显著低于妊娠D0-3及D5-6天,p<0.05。假孕小鼠D1-6天子宫内膜组织中GRIM-19蛋白和mRNA表达量无明显变化(p>0.05)。2.孕激素组GRIM19蛋白和mRNA水平较对照组表达无显著差异(p>0.05)但雌激素和雌+孕激素组表达明显降低(p<0.05)3.小鼠正常妊娠D4(着床窗口期)子宫内膜组织中细胞凋亡明显减少。4. pEGFP GRIM-19质粒和GRIM-19-siRNA转染RL95-2细胞后,GRIM-19过表达组球状体粘附率(37.8±2.13)明显低于对照组(84.2±1.56)及干扰组(79.8±1.72)(p<0.05)。5. GRIM-19过表达组与对照组和干扰组相比,细胞凋亡明显增加(p<0.05)6. GRIM-19过表达组,STAT3及IL-11蛋白和mRNA水平明显减低,TNF-α蛋白和mRNA水平明显升高。GRIM-19干扰组,STAT3及IL-11蛋白和mRNA水平明显升高,TNF-α蛋白和mRNA水平明显减低。结论GRIM-19在胚胎着床过程中发挥重要作用,GRIM-19可能通过与多种细胞因子的相互作用来调控细胞凋亡和免疫反应从而参与着床过程,有望成为新型抗生育技术的潜在靶分子。更多还原

【Abstract】 Background & AimEmbryo implantation is the basis for the beginning of life, including positioning, adhesion, implantation, whose regulatory mechanisms is complex and sophisticated. GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) is a member of GRIMs family,part of the mitochondrial complex Ⅰ, which play important role in cell apoptosis and immunity. Some studies have shown that GRIM-19 play important part in early embryonic development, but it remains unclear what role it would play in the embryo implantation. This experiment studies the expression and location of GRIM-19 in mouse endometrium in early pregnancyto explore its function in embryo implantation.On this basis, we study the influence of GRIM-19 on cell apoptosis and immunity by experiment in vivo and in vitro to further explore the mechanism acting on embryo implantation.MethodsIn vivo study:We detected the expression of GRIM-19 in the pregnant uterus of mice by Immunohistochemistry. We studied the expression of GRIM-19 in mouse uterus by RT-PCR and Western blot with early pregnancy、hormone treatment and pseudopregnancy models. TUNEL method was used to detect apoptosis in uterine tissues of pregnant mice.In vitro study:An optimized model of R95-2 spheroids and Bewo monolayer co-culture model was employed to study the effects of GRIM-19 in the embryo-endometrial epithelium attachment; Apoptosis was assayed by flow cytometry, Western blotting and real-time PCR was used to detect the mRNA and protein level of STAT3, TNF-α and IL-Ⅱ.Statistical analysis:Data are given as mean ± standard division (SD) of the results from three or four different samples in each item of the experiment. The cells from each sample were separately evaluated. Differences between two groups were measured by the Student’s t-test. A p value less than 0.05 was defined statistically significant difference.Results1. The immunohistochemical positive staining for GRIM-19 was distributed in the luminal epithelium and glandular epithelium in pregnant mice. GRIM-19 decreased to the lowest level at pregnancy day4. The expression of the GRIM-19 protein and mRNA levels reached the bottom on day 4 of pregnant mice, but, no significantly difference in the uterus of pseudopregnant mice on D1 to D6.2.The expression of GRIM-19 inEstrogen-treated group was decreased, butprogestin-treated group showed no significant difference.3. The ratio of apoptotic cells on D4 was significantly lower than the other days (P <0.05).4. Overexpression of GRIM-19 reducedthe adhesion rate of RL95-2-BeWo co-culture spheroid.5.Flow cytometric analysis the showed that overexpression of GRIM-19 increased apoptosis.6. The mRNA and protein levels of STAT3 and IL-11 inGRIM-19-overexpression group were significantly lower than the control. However, the mRNA and protein levels of TNF-a were observed increased. In GRIM-19-silencegroup, the mRNA and protein levels of STAT3 and IL-11 increased, the expression of TNF-a was decreased.ConclusionGRIM-19 plays an important role in embryo implantation. GRIM-19 plays a role in endometrial receptivity, cell proliferation, apoptosis and immune tolerance, interacting with a variety of cytokines, will likely become a new potential target molecule anti-fertility technology.更多还原