奶牛子宫内膜上皮细胞体外培养及鉴定

试验采用酶消化及机械法相结合的方法培养奶牛子宫内膜上皮原代细胞及传代细胞。酶消化法采用0.1%链蛋白酶于4 ℃消化16~20 h,机械法采用手术刀刮取子宫角内膜。获得原代分离的细胞后,采用胰蛋白酶消化法进行细胞的传代培养。试验应用角蛋白抗体对细胞进行免疫细胞化学鉴定,并对第3代奶牛子宫内膜上皮细胞用MTT法绘制增殖曲线。结果显示,细胞在相差显微镜下呈明显的上皮样细胞形态,细胞传代到第8代时,仍能保持与原代细胞相似的细胞形态特征及生长状态。同时细胞角蛋白染色阳性细胞占80％以上。试验结果表明,应用酶消化和机械法可成功分离并培养数量、活力和纯度均较高的子宫内膜细胞,可操作性强,可在具备基本细胞培养条件的实验室应用推广。     

荷斯坦奶牛子宫内膜上皮细胞的体外培养

为建立和完善奶牛子宫内膜上皮细胞的体外培养技术,采用组织培养和胶原酶消化相结合的方法,对中国荷斯坦奶牛的子宫内膜上皮细胞进行了原代分离培养,经胰酶差时消化进行纯化,并以免疫组化法检测角蛋白表达。结果显示,所采用的方法可高效获得呈铺路石样的上皮细胞,角蛋白表达呈阳性,第2~8代的传代细胞在形态特征和增殖状态上保持着与原代细胞相近的生物学特征。     

表皮生长因子对绵羊子宫内膜上皮原代细胞增殖的影响

通过建立和完善绵羊子宫内膜上皮原代细胞体外培养技术,为研究绵羊母体和孕体之间的相互作用机制建立体外着床模型。采用组织块法分离培养子宫内膜上皮细胞,观察其生长情况,并比较不同表皮生长因子（EGF）浓度对子宫内膜上皮细胞增殖的作用效果。结果显示,组织块培养1~2 d后,组织周围迁出子宫内膜上皮细胞,长满60 mm培养皿需9~12 d;经差时消化法纯化后,F1代绵羊子宫内膜上皮细胞纯度可达90%以上,表明所获细胞可用于后续试验;F2代细胞在不同浓度EGF（0、12.5、25、50、75、100 ng/mL）下培养144、168 h,经检测在144 h,12.5、25和100 ng/mL浓度下D_{450 nm}值极显著高于对照组（P<0.01）,50 ng/mL浓度下显著高于对照组（P<0.05）,在168 h,12.5 ng/mL浓度下显著高于对照组（P<0.05）,因此,在12.5 ng/mL EGF作用下效果最佳;F3代细胞在0、12.5 ng/mL浓度下培养不同时间（24、48、72、96、120、144、168、192、216 h）,经检测D_{450 nm}值在144 h差异显著（P<0.05）,168 h后差异极显著（P<0.01）。因此,在培养液中添加12.5 ng/mL EGF可以更加高效、快速得到子宫内膜上皮细胞。     

原代奶牛子宫内膜上皮细胞体外培养方法的改进

目前利用奶牛子宫内膜上皮细胞体外培养技术，建立奶牛子宫内膜炎模型从而减少不可控因素来研究奶牛子宫内膜炎疾病已成为国内外普遍使用的一类方法，因此获得高纯度、同一性的子宫内膜上皮细胞是体外研究的关键环节，国内外所使用方法主要是酶消化法、组织块贴壁法和组织块消化贴壁法。本文旨对先前的方法进行改良，建立一种简便易行、培养周期短、细胞活性及形态良好的原代奶牛子宫内膜上皮细胞体外培养技术。采用健康未孕奶牛子宫为试验材料，取下子宫内膜组织，加入5 mL含2%双抗、40%胰酶的DMEM/F12培养基，4 ℃消化12 h，再将组织移至25 cm²细胞培养瓶中贴壁培养。获得原代细胞后，应用角蛋白-18抗体对细胞进行免疫组化荧光鉴定，并对第3代细胞采用CCK-8法测定不同时间点的D_{450 nm}值，绘制细胞生长曲线。结果显示，培养至第8天，原代细胞基本铺满细胞培养瓶瓶底，有效地缩短了常规组织块贴壁法的周期。通过角蛋白-18免疫组化荧光鉴定，原代上皮细胞的阳性率可达98%以上，相比常规组织块贴壁法来说，纯度明显提高，省去了细胞纯化的操作步骤。细胞增殖状态，符合正常的分裂生长特性。试验结果表明将常规组织块贴壁法进行了优化改良，不仅缩短了培养周期，同时提高纯度，保持细胞活性，为原代奶牛子宫内膜上皮细胞体外培养技术的改良提供一定的参考。     

A passage and storage system for isolated bovine endometrial epithelial and stromal cells

To establish a storage system for isolated endometrial cells, we investigated the basal, oxytocin (OT)- and tumor necrosis factor (TNF) alpha-stimulated production of prostaglandin (PG) F(2alpha) in bovine-passaged and frozen-thawed endometrial cells. Stromal and epithelial cells obtained from cows in the early stage of the estrous cycle (Days 2-5) were frozen at -80 C or further cultured and/or passaged until passage 4 in DMEM/Ham's F-12 supplemented with 10% calf serum. A fresh-unfrozen primary culture and one-time passaged fresh-unfrozen cells were used as the control. When both unfrozen and frozen cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and the cells were stimulated with OT (100 ng/ml) or TNFalpha (1 ng/ml) for 4 h. The passage and freezing of the endometrial cells did not affect their morphology. In primary culture of frozen and unfrozen endometrial cells, OT strongly stimulated PGF(2alpha) production in epithelial cells, and TNFalpha strongly stimulated PGF(2alpha) production in stromal cells (P<0.05). The basal output of PGF(2alpha) in frozen stromal cells was similar to that in unfrozen stromal cells. However, the basal output of PGF(2alpha) in frozen epithelial cells was significantly lower than that unfrozen cells (P<0.05). On the other hand, in passaged cells, the basal level of PGF(2alpha) production was retained until passage 1 in epithelial cells, whereas it was retained until passage 4 in stromal cells. Although epithelial cells responded to OT in PGF(2alpha) production until passage 2 (P<0.05), the stromal cells showed a significant response to TNFalpha until passage 4 (P<0.05). These results suggest that stored cells could be used for studying the physiology of bovine endometrium in vitro until passage 1 in endometrial epithelial cells, and until passage 4 in stromal cells.     

精氨酸对热应激处理的奶牛原代小肠上皮细胞增殖和凋亡的影响

本试验旨在研究精氨酸(L-Arginine,L-Arg)对热应激奶牛原代小肠上皮细胞增殖和凋亡的影响,探究精氨酸对热应激奶牛小肠上皮细胞损伤的修复作用.首先以体外培养的奶牛原代小肠上皮细胞为模型,分为对照组(Con组,37℃)、试验组[42℃培养6h后,更换成无精氨酸培养基(HS组)或者不同浓度(2、4、6、8、10 ...     

EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中血管内皮生长因子基因表达的影响

[目的]观察EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中血管内皮生长因子(vascular endothelial growth factor,VEGF)基因表达的影响,探讨前列腺素类化合物对奶牛子宫内膜组织修复的机制。[方法]分离培养奶牛子宫内膜上皮细胞,采用荧光定量PCR技术检测EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中VEGFmRNA表达的影响。[结果]10-6mol/L的Butaprost作用于奶牛子宫内膜上皮细胞4、8、16、24h可显著(P<0.05)或极显著(P<0.01)地促进VEGFmRNA的表达。[结论]EP2受体激动剂Butaprost能够促进奶牛子宫内膜上皮细胞中VEGF基因的表达。     

奶牛子宫内膜组织体外培养方法的建立及前列腺素E_2和F_（2α）受体分布的研究

[目的]建立体外培养荷斯坦奶牛子宫内膜组织的方法及检测子宫内膜组织中前列腺素E_2和前列腺素F_（2α）受体的分布情况。[方法]挑选新鲜、健康的发情前期奶牛子宫内膜组织,采集双侧子宫角部位内膜组织,体外悬浮培养组织小块并通过组织学形态鉴定培养效果。通过RT-qPCR方法检测PGE_2受体EP1、EP2、EP3（EP3A、EP3B、EP3C、EP3D）、EP4和PGF_（2α）受体FP的表达情况。[结果]HE染色表明,体外培养的奶牛子宫内膜组织结构完整,细胞和腺体形态完好,细胞核完好;发情前期奶牛子宫角部位内膜组织中PGE_2和PGF_（2α）受体均有表达,且表达量存在差异,其中PGE_2受体EP4相对表达量最高,而EP1、EP3B及PGF_（2α）受体FP的相对表达量较低。[结论]成功地建立了奶牛子宫内膜组织的体外培养方法;PGE_2和PGF_（2α）的受体在奶牛子宫内膜组织中均有表达,但不同受体的表达量有所不同。     

脂多糖对体外培养奶牛子宫内膜上皮细胞子宫容受性因子表达的影响

试验旨在通过脂多糖（lipopolysaccharide,LPS）诱导奶牛子宫内膜上皮细胞构建子宫内膜炎体外感染模型,研究子宫内膜炎对奶牛子宫容受性因子大分子转膜黏蛋白-糖蛋白1（MUC-1）、高度保守的分泌型WNT家族亚型糖蛋白（Wnt-7a）、β受体1（IFNAR1）、IFNAR2、Integrin αvβ3 mRNA及蛋白相对表达量的影响,进而阐明子宫内膜炎引起奶牛屡配不孕和繁殖率低下的机制。采用组织块法分离培养奶牛子宫内膜上皮细胞,免疫荧光方法鉴定细胞纯度,不同浓度LPS刺激子宫内膜上皮细胞,在不同时间点用CCK8测定细胞存活率,ELISA检测炎性因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、IL-6、IL-8的分泌变化,实时荧光定量PCR和Western blotting分别检测子宫内膜感染模型中子宫容受性因子MUC-1、Wnt-7a、IFNAR1、IFNAR2、Integrin αvβ3 mRNA及蛋白的表达变化。结果显示,通过组织块法纯化获得的奶牛子宫上皮细胞数量较多,经免疫荧光角蛋白染色证实纯度较高。采用CCK8测定细胞存活率发现,与对照组相比,浓度为0、5、10、50μg/mL的LPS作用6、12、24、48 h后,细胞活力无显著变化（P>0.05）;浓度为100μg/mL的LPS作用24 h时,细胞存活率显著低于对照组（P<0.05）,细胞培养上清中的IL-1β、TNF-α、IL-6、IL-8含量均显著高于对照组及低浓度组（P<0.05）,引起细胞的炎症反应。与对照组相比,模型组MUC-1 mRNA及蛋白的相对表达量均极显著增加（P<0.01）;Wnt-7a mRNA及蛋白的相对表达量均极显著下降（P<0.01）;Integrin αvβ3、IFNAR1和IFNAR2 mRNA相对表达量均极显著下降（P<0.01）,蛋白相对表达量均显著下降（P<0.05）。结果表明,浓度为100μg/mL的LPS作用24 h可成功构建子宫内膜炎体外感染模型,子宫内膜炎对奶牛子宫容受性因子MUC-1、Wnt-7a、IFNAR1、IFNAR2、Integrin αvβ3 mRNA及蛋白表达的影响可能是引起奶牛屡配不孕和繁殖率低下的重要原因。     

孕酮对奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路关键蛋白表达的影响

为研究孕酮对奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路关键蛋白表达的影响,本试验以原代奶牛子宫内膜上皮细胞为材料,添加不同浓度孕酮(0、1、3、5 ng/mL)对其培养,采用CCK-8法检测孕酮对细胞增殖的影响;免疫印迹法检测孕酮对Wnt/β-catenin信号通路关键蛋白表达的影响。结果表明:与对照组相比,添加1、3 ng/mL孕酮组奶牛子宫内膜上皮细胞增殖率提高(P>0.05),5 ng/mL孕酮组细胞增殖率降低(P>0.05);与对照组相比,1 ng/mL和3 ng/mL孕酮组Wnt/β-catenin信号通路关键蛋白β-catenin、cyclin D1和c-Myc的表达水平均升高(P<0.01),而5 ng/mL孕酮则抑制了β-catenin的表达(P<0.05),且cyclin D1和c-Myc蛋白表达较3ng/mL孕酮组有下降趋势(P>0.05)。综上,高浓度孕酮抑制了体外培养的原代奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路的激活和表达,提示该信号通路参与了奶牛产后子宫复旧延迟进程,为进一步研究奶牛子宫内膜的发病机制提供思路。     

大肠杆菌与金黄色葡萄球菌对奶牛子宫内膜组织细胞因子表达及损伤程度的影响

本试验旨在研究体外感染金黄色葡萄球菌与大肠杆菌对奶牛子宫内膜组织中细胞因子白介素-6（IL-6）、IL-1β和IL-8的表达及损伤程度的影响。以体外培养的奶牛子宫内膜组织作为研究对象,采用1×10⁵~1×10⁹ CFU/mL大肠杆菌与金黄色葡萄球菌对奶牛子宫内膜组织进行体外感染,通过实时荧光定量PCR和ELISA方法检测两种细菌刺激后奶牛子宫内膜组织中IL-6、IL-1β及IL-8 mRNA与蛋白的表达量,并用HE染色法观察两种细菌感染后奶牛子宫内膜组织病理学切片。结果显示,1×10⁵~1×10⁹ CFU/mL大肠杆菌体外感染后,奶牛子宫内膜组织中IL-6、IL-1β和IL-8 mRNA表达量均极显著高于空白对照组（P<0.01）;金黄色葡萄球菌感染浓度为1×10⁵、1×10⁶ CFU/mL时,IL-6、IL-1β mRNA表达量极显著高于空白对照组（P<0.01）,感染浓度为1×10⁷ CFU/mL时,IL-6、IL-1β mRNA表达量显著高于空白对照组（P<0.05）,感染浓度为1×10⁶ CFU/mL时,IL-8 mRNA表达量显著高于空白对照组（P<0.05）,其他感染浓度均与空白对照组无显著差异（P>0.05）。奶牛子宫内膜组织感染1×10⁵~1×10⁹ CFU/mL金黄色葡萄球菌和大肠杆菌时,IL-6、IL-1β及IL-8蛋白表达量均极显著高于空白对照组（P<0.01）。相同浓度的大肠杆菌感染奶牛子宫内膜组织后,IL-6、IL-1β及IL-8 mRNA与蛋白表达量均极显著高于金黄色葡萄球菌感染组（P<0.01）。HE切片染色结果显示,大肠杆菌感染后仍有部分上皮细胞保留,而金黄色葡萄球菌感染后上皮细胞全部脱落。本试验结果表明,大肠杆菌与金黄色葡萄球菌感染奶牛子宫内膜组织后,引起的炎症反应不同。大肠杆菌感染后,促炎性细胞因子被显著上调,而金黄色葡萄球菌感染后破坏子宫内膜上皮细胞程度更加严重。     

Crb1在小鼠生后不同发育阶段子宫组织中的表达

应用HE染色方法和免疫荧光组织化学技术对小鼠生后不同发育阶段子宫组织结构的发育以及极性调控蛋白Crb1的定位表达进行了研究,结果显示,随个体发育,子宫管腔及子宫腺腔不断扩大,子宫内膜上皮持续增厚、固有层内子宫腺逐渐发达;4周龄时子宫腔面出现纤毛,随发育进程越来越发达;在各发育阶段的小鼠子宫组织中均有Crb1的表达,随个体发育表达呈现先增强后减弱的趋势,在8周龄时达到最强;Crb1主要定位于子宫内膜上皮细胞和子宫腺上皮细胞的胞膜和胞质,提示Crb1可能与小鼠子宫内膜上皮细胞和腺上皮细胞的极性建立和维持有关。     

奶牛乳腺上皮细胞的二维和三维培养及培养后酪蛋白基因的表达

本试验研究了二维和三维培养对奶牛乳腺上皮细胞(BMEC)形态与β-酪蛋白、κ-酪蛋白、αs1-酪蛋白基因表达的影响.试验采用内蒙古地区3头5岁泌乳期健康荷斯坦奶牛的乳腺组织,将BMEC经3代培养纯化后分别进行二维和三维培养,观察不同培养模式下细胞的生长形态,测定β-酪蛋白、A-酪蛋白、αs1-酪蛋白基因的表达量.结果表明:2种培养模式下的BMEC呈现不同的细胞形态,二维培养的BMEC呈典型铺路石样细胞形态,三维培养的BMEC则形成了类腺泡的聚集物和管腔结构.利用三维培养的BMEC的αs1-酪蛋白基因的表达量显著地高于二维培养(P＜0.05),三维培养的BMEC的β-酪蛋白和κ-酪蛋白基因的表达量极显著地高于二维培养(P＜0.01).结果提示,三维培养的BMEC3种酪蛋白基因的表达量均高于二维培养,三维培养的BMEC在形态上呈现的特点与在体内更加接近.     

仔猪小肠上皮细胞的体外培养及猪流行性腹泻病毒对其感染性研究

本研究旨在建立一种操作简单的仔猪小肠上皮细胞体外培养体系，为猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）的相关研究提供材料。研究以未吃初乳的新生仔猪作为肠道供体，采用肠腔面刮取肠黏膜和机械分离分散的方式进行原代细胞的体外分离。采用0.1%胰蛋白酶差速消化法进行仔猪小肠上皮细胞的纯化；比较新生弱仔猪和正常仔猪作为供体对原代小肠上皮细胞活性的影响；MTT法比较不同代次原代细胞的增殖活性；免疫荧光和实时荧光定量RT-PCR方法检测PEDV毒株CV777在原代小肠上皮细胞感染和增殖情况。结果显示，本研究建立的体外分离培养方法能获得增殖活性良好的原代小肠上皮细胞，具有明显的"S"型细胞增殖曲线。通过胰酶差速消化可得到纯度高、形态单一的小肠上皮细胞，同时细胞连续传代5次仍保持良好的增殖活性。弱仔猪和正常仔猪分离培养的小肠上皮细胞的增殖活性比较显示，两者并没有明显区别，这为降低原代细胞培养的成本提供新的思路。免疫荧光和实时荧光定量RT-PCR结果显示，PEDV可感染本方法分离培养的仔猪原代小肠上皮细胞，并在其中进行复制增殖。本研究建立了一种操作简单、实用性强、成本较低的仔猪原代小肠上皮细胞体外培养方法，该方法培养的原代细胞可作为PEDV分离培养和相关研究的基础材料。     

Culture of Piglet Intestinal Epithelial Cells in vitro and the Infectivity of Porcine Epidemic Diarrhea Virus on the Cells

The study was aimed to establish a simple in vitro culture system for piglet small intestinal epithelial cells,and to provide materials for researches on porcine epidemic diarrhea virus (PEDV).In this study,newborn piglets that did not eat colostrum were used as the initial donors,and the primary cells were separated in vitro by scraping the intestinal mucosa from the intestinal lumen and mechanical separation and dispersion.The piglet intestinal epithelial cells were purified using 0.1% trypsin differential digestion method.Compared the effects of newborn weak piglets and normal piglets as donors on the activity of primary intestinal epithelial cells.MTT method was used to compare the proliferation activity of primary cells of different generations.Immunofluorescence and Real-time RT-PCR were used to detect the infection and proliferation of PEDV strain CV777 in primary intestinal epithelial cells.The results showed that the isolation and culture method in vitro used in this study could obtain primary intestinal epithelial cells with good proliferation activity,with obvious S-type cell proliferation curves.The cells with high purity and single morphology could be obtained by differential digestion,and had good proliferation activity after five consecutive passages.The proliferative activity of intestinal epithelial cells isolated from weak piglets and normal piglets had no obvious difference,and provided new way for reducing the cost of primary cell culture.The results of immunofluorescence and Real-time RT-PCR showed that PEDV could infect the primary intestinal epithelial cells,and replicated and proliferated in them.In this study,we established a simple,practical and low-cost method for in vitro culture of piglet primary small intestinal epithelial cells.The primary cells cultured by this method could be used as basic materials for the isolation and culture of PEDV and related research.     

黄姑鱼肠道上皮细胞体外分离培养及鉴定

本研究旨在建立黄姑鱼(Nibea albiflora)肠道上皮细胞的体外分离培养及鉴定方法,为海水鱼类肠道功能及其发病机制相关研究提供细胞模型.以黄姑鱼肠道组织为研究对象,采用胰蛋白酶(0.25％)、胶原蛋白酶(1 mg/mL)和透明质酸酶(0.16 mg/mL)联合消化法对肠道组织进行不同时间(0、20、40、60和...     

Phyto- and endogenous estrogens differently activate intracellular calcium ion mobilization in bovine endometrial cells

The main purpose of this study was to check whether phyto- and endogenous estrogens influence calcium ion mobilization [Ca(2+)](i) in bovine endometrial cells and whether this action is connected with biological effects i.e. prostaglandin (PG)F(2alpha) production. In our study we used two calcium measurement methods by comparing the microscopic method with widely used quantitative - spectrofluorometric method of [Ca(2+)](i) measurement. We also wanted to confirm whether visualization of calcium ion [Ca(2+)](i) in cells using microscopic method supported by micro image analysis (Micro Image Olympus system) reflects real, qualitative changes in the ion concentration. In both methods a cell-permeable form of fluorescent [Ca(2+)](i) indicator Fura-2 was used. Cultured bovine endometrial epithelial and stromal cells influenced by phorbol-2-myristate-13-acetate (PMA; positive control), estradiol 17-beta (E(2); endogenous estrogen) and active metabolites of phytoestrogens (environmental estrogens) were used as a model to study PGF(2alpha) secretion and [Ca(2+)](i) mobilization in the cells. Equol and para-ethyl-phenol in doses of 10(-8)-10(-6) M increased PGF(2alpha) concentration both in epithelial and stromal cells (P<0.05). In both methods, equol and para-ethyl-phenol did not cause intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P>0.05). Both methods revealed that only E(2) and PMA induced intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P<0.05). The results of both methods were highly correlated (P<0.001; r=0.82 for epithelial cells and r=0.89 for stromal cells). In conclusion, both methods gave approximately the same results and showed that phytoestrogens, in contrast to PMA and E(2), did not cause intracellular [Ca(2+)](i) mobilization in endometrial cells. The obtained results proved that the [Ca(2+)](i) visualization method supported by micro image analysis can produce similar results to the spectrofluorometric method.     

蛋氨酸对奶牛乳腺上皮细胞亚细胞蛋白质组的影响研究

为寻找奶牛乳腺上皮细胞亚细胞结构中与泌乳相关的重要蛋白质、从蛋白质水平揭示乳蛋白合成的调控机制,应用双向凝胶电泳技术分离体外培养的蛋氨酸处理组与正常组奶牛乳腺上皮细胞蛋白质,利用Image Maser 2D软件对图谱进行对比分析,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)和数据库检索鉴定,并采用实时荧光定量PCR技术在mRNA水平上验证2-DE结果。质谱鉴定出8个表达上调的差异蛋白质,大多数差异蛋白质的功能涉及细胞骨架构成、能量代谢等过程。这些差异点的发现为研究奶牛泌乳机理提供了有益的线索。     

siRNA抑制牛子宫内膜上皮细胞Nrf2基因的表达研究

为探讨核因子E2相关因子（nuclear factor E2 related factor，Nrf2）基因沉默及激活后对牛子宫内膜上皮细胞的影响，本试验利用小分子干扰（siRNA）技术及Nrf2的激动剂叔丁基对苯二酚（tBHQ），分别从Nrf2基因的下调和上调表达来研究Nrf2对牛子宫内膜上皮细胞中血红素加氧酶-1（HO-1）和Homebox A10（HOXA10）基因表达的影响。结果显示，经实时荧光定量PCR方法检测，在设计的2条siRNA序列（siRNA-1209和siRNA-1672）中，siRNA-1672在终浓度为75 nmol/L、作用时间24 h时抑制效果较好，抑制效率在80%以上；经Western blotting方法检测，Nrf2蛋白表达水平在转染后96 h极显著下降（P<0.01），而HO-1和HOXA10的mRNA表达量分别下降了60%和70%（P<0.01），蛋白表达量在96 h后极显著或显著下降（P<0.01；P<0.05）。此外，经CCK8方法检测，Nrf2基因表达沉默后，牛子宫内膜上皮细胞增殖能力减弱。而在tBHQ激活Nrf2试验中，经Western blotting方法检测，tBHQ终浓度为30 μmol/L时Nrf2蛋白表达量最高，极显著高于对照组（P<0.01），且HO-1和HOXA10的蛋白表达量与对照组相比也明显上升。结果表明，本试验设计的siRNA-1672能特异性地抑制牛子宫内膜上皮细胞Nrf2的表达，而抑制Nrf2的表达会导致牛子宫内膜上皮细胞增殖能力下降，且Nrf2对牛子宫内膜上皮细胞HO-1和HOXA10基因表达存在调控作用。     

Pseudo-placentational Endometrial Cysts in a Bitch

Cystic alterations of the canine endometrium compromise reproduction and fertility of the bitch and may lead to life-threatening diseases, such as pyometra. Even without clinical evidence, reduction of the uterine lumen by cysts implicates disturbances during migration, nidation and development of the embryo. Several studies point to the high variability of morphology of uterine endometrial cysts but they lack detailed analyses of alterations. In the present study, immunohistochemistry was used to investigate the expression of steroid hormone receptors (oestrogen, progesterone), proliferation activity, inflammation and infection in the cystic affected tissue regions in contrast to the normal endometrium. Oestrogen receptor expression showed a high density of receptors throughout the surface epithelial cells, crypt epithelial cells, glandular epithelial cells and stromal cells of the normal endometrium as well as the cystic affected regions. Proliferation in the cysts was verified in the middle and basal cells of the crypts. Neither in the endometrium nor in the cysts inflammatory processes or evidence of infection could be detected. Furthermore, lectin histochemistry and electron microscopic methods showed that lectin binding patterns and cell morphology of internal epithelial lining and surface epithelium of the cysts can be used to characterize and distinguish different types of cystic alterations. Analogies between epithelial cells of the glandular chambers of the canine placenta and the cystic cellular morphology, steroid hormone receptor distribution as well as lectin binding patterns of the endometrial cysts, as observed in this study, suggest to introduce the term 'pseudo-placentational endometrial cysts'.