小鼠子宫内膜上皮细胞体外培养方法比较

为探讨适用于小鼠子宫内膜上皮细胞的培养方法,对胰蛋白酶消化法、胰蛋白酶消化+刮取法、Ⅰ型胶原酶消化法、Ⅰ型胶原酶消化法+刮取法、子宫翻转组织块培养法5种方法进行了比较研究.结果表明,胰蛋白酶消化法获得的上皮细胞数量稀少,活力低,生长速度缓慢;刮取法+胰蛋白酶消化法、胶原酶消化法、刮取法+Ⅰ型胶原酶消化法获得的细胞数量较单纯胰蛋白酶消化法多,但达不到研究所需的要求;子宫翻转组织块培养法获得的上皮细胞活力强,生长速度快,纯度高,比较适宜小鼠子宫内膜上皮细胞的原代培养.     

小鼠胚泡着床期LongSAGE文库中双标签的制备研究

研究探讨小鼠胚胎着床期LongSAGE文库构建过程中总RNA提取、cDNA合成及双标签(Ditags)制备方法,优化PCR扩增中双标签PCR的最佳模板稀释度及扩增循环次数.     

小鼠胚泡着床期LongSAGE文库中双标签的制备研究

研究探讨小鼠胚胎着床期LongSAGE文库构建过程中总RNA提取、cDNA合成及双标签(D itags)制备方法,优化PCR扩增中双标签PCR的最佳模板稀释度及扩增循环次数。     

小鼠胚泡植入不同时期子宫内膜组织细胞蛋白质组研究

利用固相pH梯度双向凝胶电泳（2D-PAGE）分别分离妊娠第3天（3 d）、第5天（5 d）、第7天（7 d）小鼠子宫内膜的总蛋白质;考马斯亮蓝染色、PDQuest 2DE软件分析获得3个不同孕期的蛋白质表达谱;图像分析以5 d的图谱为参考,3 d、7 d与其匹配率分别为68%、61%.在等电点PI 3-10、分子量14.4 KDa-116.0 KDa范围内分离得3 d、5 d、7 d小鼠子宫内膜蛋白点大约分别为713个、736个、673个.选取差异蛋白质点进行MEDLI-TOF质谱分析,查询数据库,获取有意义的蛋白点,分别为载脂蛋白A-I、膜联蛋白A1、谷光甘肽转移酶、丝氨酸蛋白酶抑制剂、烯醇化酶.研究结果表明,在胚泡植入窗口期（5 d）,小鼠子宫内膜合成蛋白质的种类和数量明显增加,以适应胚泡植入.子宫内膜表达的蛋白质在调控子宫基质降解和子宫内膜的局部免疫反应方面中扮演着重要角色.     

小鼠胚泡植入不同时期子宫内膜组织细胞蛋白质组研究

利用固相pH梯度双向凝胶电泳(2D-PAGE)分别分离妊娠第3天(3 d)、第5天(5 d)、第7天(7 d)小鼠子宫内膜的总蛋白质;考马斯亮蓝染色、PDQuest 2DE软件分析获得3个不同孕期的蛋白质表达谱;图像分析以5 d的图谱为参考,3 d、7 d与其匹配率分别为68%、61%.在等电点PI 3～10、分子量14.4 KDa～116.0 KDa范围内分离得3 d、5 d、7 d小鼠子宫内膜蛋白点大约分别为713个、736个、673个.选取差异蛋白质点进行MEDLI-TOF质谱分析.查询数据库,获取有意义的蛋白点,分别为载脂蛋白A-I、膜联蛋白A1、谷光甘肽转移酶、丝氨酸蛋白酶抑制剂、烯醇化酶.研究结果表明,在胚泡植入窗口期(5 d),小鼠子宫内膜合成蛋白质的种类和数量明显增加,以适应胚泡植入.子宫内膜表达的蛋白质在调控子宫基质降解和子宫内膜的局部免疫反应方面中扮演着重要角色.     

瘦素在妊娠小鼠子宫着床点与非着床点中的表达研究

目的:探讨瘦素在妊娠小鼠子宫着床点与非着床点中的表达规律.方法:性成熟雌性NIH小鼠与成熟雄鼠自然交配,在妊娠5,6 d分别脱臼处死小鼠取其子宫,然后进行以下实验:1.分别提取总RNA,检测胚胎着床点和非着床点子宫瘦素mRNA表达规律;2.分别进行石蜡包埋,切片,然后进行免疫组化试验,检测瘦素蛋白的表达规律.结果:妊娠5,6 d小鼠子宫着床点与非着床点中均有瘦素mRNA和蛋白表达,蛋白主要定位于腔上皮和腺上皮,着床点mRNA水平比非着床点显著增强.结论:瘦素在着床点与非着床点子宫组织中的表达差异提示瘦素在胚胎着床中的生理作用.     

瘦素在妊娠小鼠子宫着床点与非着床点中的表达研究

目的:探讨瘦素在妊娠小鼠子宫着床点与非着床点中的表达规律。方法:性成熟雌性NIH小鼠与成熟雄鼠自然交配,在妊娠5,6 d分别脱臼处死小鼠取其子宫,然后进行以下实验:1.分别提取总RNA,检测胚胎着床点和非着床点子宫瘦素mRNA表达规律;2.分别进行石蜡包埋,切片,然后进行免疫组化试验,检测瘦素蛋白的表达规律。结果:妊娠5,6 d小鼠子宫着床点与非着床点中均有瘦素mRNA和蛋白表达,蛋白主要定位于腔上皮和腺上皮,着床点mRNA水平比非着床点显著增强。结论:瘦素在着床点与非着床点子宫组织中的表达差异提示瘦素在胚胎着床中的生理作用。     

ICR小鼠着床障碍模型的建立

为建立ICR小鼠胚泡着床障碍模型,本研究用米非司酮和吲哚美辛对妊娠ICR雌鼠进行抗着床处理,观察其对胚泡着床及子宫内膜的影响。实验分为米非司酮组和吲哚美辛组,每组设对照组。于妊娠第4天分别注射0.3、0.6、0.9、1.2 g.L-1的米非司酮0.1 mL.mouse-1,及0.12、0.13、0.14、0.15 mL吲哚美辛溶液,对照组注射二者相应的溶剂丙二醇和麻油。妊娠第5天检查子宫内膜对台盼蓝的反应点数;妊娠第8天观察胚泡着床情况,统计子宫重量,制作切片,检测子宫内膜形态结构的变化。结果表明,0.09 mg米非司酮组和0.13 mL吲哚美辛组的子宫重量、内膜容受性、发育及蜕膜化均受到明显的抑制。结果表明,米非司酮和吲哚美辛可成功建立ICR着床障碍模型,用于相关研究。     

牛磺酸对昆明小鼠早期胚胎体外发育和着床的影响

本研究主要探讨了牛磺酸对昆明(KM)小鼠体外胚胎发育及对胚胎着床的作用。在mKSOM(modified potassium simplex optimized medium)胚胎体外培养液中分别添加不同浓度牛磺酸,培养24、72 h后分别统计4-细胞率、囊胚率、囊胚总细胞数;采用子宫角注射的方法,研究牛磺酸转运抑制剂β-丙氨酸对孕D9胚胎着床数的影响。结果表明,添加10 mmol/L牛磺酸培养液组的胚胎4-细胞率高于对照组(P<0.05),囊胚率与囊胚总细胞数明显高于对照组(P<0.01)。子宫角注射β-丙氨酸(20 μg/mL)显著降低D9着床的胚胎数。本研究初步明确了牛磺酸对小鼠体外胚胎发育及对胚胎着床的影响,为进一步研究胚胎发育和着床提供参考。     

热应激小鼠胚胎着床模型的建立

模拟夏季高温环境对孕鼠的应激作用,建立热应激影响昆明白小鼠胚胎着床的模型。试验设为对照组(23℃±2℃)、40℃和42℃应激组(小鼠妊娠1～4d,分别应激2h),比较观察妊娠5、6、7d小鼠胚胎着床数及子宫状况和产仔数。结果表明,与对照组相比40℃和42℃应激组妊娠5d(11.00±2.65、10.00±2.12vs15.00±1.58,P0.05)、6d(11.40±3.21、10.60±2.30vs 15.00±0.71,P0.05)、7d(11.00±2.55、11.00±1.58vs 15.60±1.67,P0.05)胚胎着床率显著下降,产仔数(8.60±1.14、8.40±1.82vs 11.40±1.82,P0.05)也显著下降;且相比对照组其着床位点均较少且分布失衡。40℃和42℃应激组之间无显著差异,以42℃应激组影响相对较大。可见,胚胎着床前孕鼠连续4d40℃、42℃应激2h,可显著影响小鼠的繁殖能力。42℃可作为影响小鼠胚胎着床的热应激模型。     

反刍动物的胚胎着床与妊娠识别过程的研究进展

反刍动物的胚胎着床与妊娠识别是个复杂的生理过程，这一过程受多种细胞因子和生殖激素调控。文中叙述了各种细胞因子和生殖激素对妊娠建立和维持的调控作用。     

A Novel Three-Dimensional Mouse Embryonic Implantation Model In Vitro

To regenerate three-dimensional endometrium in vitro as a novel model for studying the mechanism of implantation of embryos, the luminal epithelial cells and stromal cells of the rabbit uterus were separated and cultured in vitro. The type Ⅰ mouse tail collagen was used as scaffolding material. The stromal cells were inoculated in the type I mouse tail collagen, and the luminal epithelial cells were inoculated on the type i mouse tall collagen to regenerate the endometrium in vitro. The regenerated endometrium was cultured in DMEM-F/12 media containing 100 nmol L^-1 progesterone, 10 nM β-estradiol, and 10% fetal bovine serum （FBS） for 3 d. The media were then replaced with CZB containing 100 nM progesterone, 10 nmol L-1 β-estradiol, and 10% FBS, and the mouse blastulas were co-cultured with it. The results of scanning electronic micrography showed that the epithelial cells on the surface of the reconstructed endometrium were covered with numerous slender microvilli and some epithelial cells protruded pinopodes. After culturing for 12 h with the mouse blastula, the shedding, attachment, and implantation of the blastula were observed. The blastula can escape from zona pellucida and attach to the three-dimensional endometrium and is then implanted into it. This study showed that the reconstructed three-dimensional endometrium can serve as a robust embryo implantation model in vitro.     

选择素及其配体在哺乳动物胚胎着床过程中的作用

选择素是一种非免疫来源的多价糖类结合蛋白,为细胞粘附分子(celladhesionmolecules,cAMs)超家族的一员,选择素家族有3个结构类似的成员:E-选择素、L-选择素、P-选择素,它们在炎症发生时可介导白细胞与血管内皮细胞之间、白细胞与白细胞及白细胞与血小板之间的粘附。近年来研究发现,选择素及其配体还在精卵识别、滋养层细胞和子宫内膜的相互识别、滋养层细胞侵入、胎盘形成等过程中发挥重要作用。在此,主要对E-选择素、L-选择素及它们的配体在哺乳动物胚胎着床过程中发挥的作用及其表达调节作一综述。     

小鼠囊胚及孵化胚胎细胞计数方法的探讨

实验以ICR小鼠体外培养的囊胚及孵化胚胎为材料,比较两种方法对小鼠胚胎细胞计数的效果。方法1为采用0.5%的柠檬酸钠低渗溶液和姬姆萨染液对囊胚及孵化胚进行压片,且不同时期胚胎低渗和染色处理时间相同;方法2染色液为苏木精染液,且不同时期胚胎低渗和染色处理时间不同。结果表明,方法2小鼠囊胚及孵化胚胎细胞球轮廓清晰,细胞间隙明显可见,其观察率分别为94.7%和96.8%;而方法1胚胎细胞球间隙不明显,一些细胞轮廓模糊,其观察率分别为70.5%和69.6%。小鼠囊胚及孵化胚胎采用压片计数法,以方法2显著好于方法1(P<0.05)。     

小鼠乳腺上皮细胞的体外培养

采用I型、II型胶原酶和胰蛋白酶混合（1：1：1）消化乳腺组织,并用差速贴壁法纯化培养乳腺上皮细胞,倒置显微镜下观察细胞的形态,并用角蛋白18免疫荧光染色的方法对细胞进行鉴定。结果表明：改良型混合酶消化法能获得较多的细胞团,且12h后细胞团绝大多数已贴在细胞瓶壁,72h后细胞团完全铺开呈单层融合,细胞呈典型的铺路石样。免疫荧光鉴定结果显示,角蛋白18呈阳性反应。因此,应用改良型混合酶消化法能在短时间内获得大量较纯的乳腺上皮细胞。     

Expression of Eph-Ephrin A Molecules in Endometrium During Swine Embryo Implantation Examined Using Real-Time RT-PCR

Erythropoietin-producing hepatocellular receptor and its membrane-bound ligands (Eph-Ephrin) system could regulate some mammalian blastocyst attachment and spreading. In order to investigate the involvement of the Eph-Ephrin system in swine embryo attachment, mRNA expression of Eph-Ephrin molecules in endometrium was examined by real-time RT-PCR during embryo implantation in pigs. The results indicated that mRNA expressions of Eph A5, A7 and Ephrin A5 all continually increased from pregnancy day 13 to 24. Ephrin A3 mRNA expression significantly increased from day 13 to 18 and decreased from day 18 to 24, and the expression was the lowest on pregnancy day 13 and the highest on day 18. However, Ephrin A4 mRNA expression was the lowest on pregnancy day 18 and the highest on day 24, and the expression decreased from day 13 to 18 and increased from day 18 to 24. Furthermore, mRNA expressions of Eph A5 and A7 were both found in other tissues, such as brain, muscle, intestine, stomach, etc. These findings suggest that the Eph-Ephrin system may play an important role in regulating the contact between blastocysts and endometrium during swine embryo implantation.     

胚胎着床过程中MMPs对子宫内膜重塑的作用研究进展

胚胎着床是一种细胞侵袭和转移的正常生理过程,涉及细胞外基质的降解和重塑、胎盘绒毛血管的发生、子宫螺旋动脉的重建等,在基质金属蛋白酶、组织金属蛋白酶抑制剂以及其他许多因子的调控下,对子宫内膜细胞外基质进行有序地降解和重塑。对MMPs参与胚胎着床中子宫内膜重塑过程中的作用及其引起细胞外基质的变化情况进行了综述。     

Lipopolysaccharide-binding Protein Involved in Process of Mouse Embryo Implantation and Decidualization of Endometrial Stromal Cells

Lipopolysaccharide-binding protein (LBP) functions as an acute phase protein and plays a role in the innate immune response to bacterial challenge.To investigate the uterine expression of LBP during peri-implantation in mice,in situ hybridization and immunohistochemical staining were used to detect the mRNA and protein expression of LBP in mouse uteri in the early pregnancy,pseudopregnancy,artificial decidualization and hormone-treated mice.The results showed that LBP was expressed in uterine luminal epithelium (LE) and glandular epithelium (GE) during days 1-4 of pregnancy.During days 5-8,LBP was weakly expressed in the decidual cells around the embryo on the 5th day of pregnancy (implantation occurred),then gradually increased,LBP was strongly expressed in the decidual zone on the 8th day of pregnancy.The expression of LBP in pseudopregnancy was similar with pregnancy on days 1-4.In artificial decidualization mice,LBP was observed in uterine LE and GE in the control horn,whereas LBP expression was significantly higher in decidua of mouse uteri under artificial decidualization.In hormone-treated mice,the expression of LBP wasup-regulated by 17β-estradiol (E_2) and progesterone (P_4).In addition,the cultured mouse endometrial stromal cells (mESCs) were induced for in vitro decidualization with 10 nmol·L~(-1) E_2 and 1 μmol·L~(-1) P_4.Real-time PCR results showed that LBP mRNA expression was highly induced in mESCs after decidual stimulus.In vivo and in vitro experiments showed that LBP was expressed in the decidual cells,indicating that LBP involved in decidualization of mouse uteri.