前列腺素E_2受体激动剂和雌激素对奶牛输卵管上皮细胞中转化生长因子β_3表达的影响

本试验旨在观察前列腺素E2受体激动剂(布他前列腺素(butaprost))与雌激素对奶牛输卵管上皮细胞中转化生长因子β3(TGFβ3)表达的影响,阐明butaprost和雌激素对奶牛输卵管上皮细胞TGFβ3有无协同调控作用。采用胰酶消化法及机械法分离培养奶牛输卵管上皮细胞,分别将butaprost和雌激素作用于体外培养的奶牛输卵管上皮细胞,采用实时荧光定量PCR技术检测butaprost和雌激素对奶牛输卵管上皮细胞中TGFβ3mRNA表达的影响。结果显示,与0h作用组相比,雌激素作用16、24和48h时对奶牛输卵管上皮细胞TGFβ3的表达量均极显著升高(P0.01),4h的表达量极显著降低(P0.01);且受体激动剂butaprost和雌激素有协同调控TGFβ3的效应;加入吲哚美辛后能有效抑制内源性前列腺素对TGFβ3表达的作用。结果表明,butaprost和雌激素可调控奶牛输卵管上皮细胞TGFβ3mRNA的表达。     

雌激素与前列腺素受体对奶牛输卵管上皮细胞输卵管蛋白合成和分泌的影响

旨在研究雌激素与前列腺素受体对奶牛输卵管上皮细胞输卵管蛋白合成与分泌的调控作用。用荧光定量PCR检测了奶牛输卵管上皮细胞输卵管蛋白mRNA的表达;采用Western blot检测了奶牛输卵管上皮细胞输卵管蛋白的表达。同时,加入雌激素(10~(-10)mol/L)和butaprost(PE受体激动剂,10~(-6)mol/L),雌激素(10~(-10)mol/L)和fluprostenol(PF受体激动剂,10-6mol/L),分别观察对输卵管蛋白表达的影响。结果表明:雌激素与PGE_2受体对奶牛输卵管上皮细胞输卵管蛋白的分泌调控具有协同作用;雌激素与PGF_(2α)受体对奶牛输卵管上皮细胞输卵管蛋白的分泌调控具有间接拮抗作用。     

前列腺素F_(2α)受体激动剂fluprostenol对奶牛输卵管上皮细胞合成分泌细胞因子表达的影响

为验证前列腺素类化合物对输卵管分泌细胞因子有调控作用,以前列腺素F2α受体激动剂fluprostenol对奶牛输卵管上皮细胞合成分泌细胞因子TGF-α、TGF-β3、FGF-2、LIF和GM-CSF的调控机制为研究对象,采用实时荧光定量PCR技术检测吲哚美辛(10-5 mol/L)作用不同时间(2,4,8,16,24,48h)对奶牛输卵管上皮细胞TGF-α、TGF-β3、FGF-2、LIF和GM-CSF mRNA表达的影响。结果显示,fluprostenol对奶牛输卵管上皮细胞中TGF-α和FGF-2mRNA有正调控的作用,均在4h时mRNA的表达量达到峰值;fluprostenol对奶牛输卵管上皮细胞中LIF和GMCSF mRNA有双重调控作用,均是先短暂显著促进(P<0.05)其mRNA的表达,之后表现为极显著抑制(P<0.01),并均在2h时达到表达量峰值;fluprostenol对奶牛输卵管上皮细胞中TGF-β3mRNA起到正调控作用,作用48h时与空白对照组相比极显著升高(P<0.01),达到峰值。     

EP2受体和FP受体对奶牛输卵管上皮细胞输卵管蛋白的影响

采用免疫荧光法,使用角蛋白鉴定奶牛输卵管上皮细胞;采用荧光定量PCR法检测奶牛输卵管上皮细胞输卵管蛋白mRNA的表达;采用Wsetern blot法检测了奶牛输卵管上皮细胞输卵管蛋白的表达。结果显示:加入butaprost(EP2受体激动剂,10~(-6) mol/L)能促进输卵管蛋白的表达,加入fluprostenol(FP受体激动剂,10~(-6) mol/L)能抑制输卵管蛋白的表达。结果表明:PGE2在奶牛输卵管上通过激活EP2受体上调输卵管蛋白的表达,PGF2α通过激活FP受体下调输卵管蛋白的表达。     

雌激素对奶牛输卵管上皮细胞前列腺素E2和F2α合成酶mRNA表达的影响

为了揭示雌激素(estrogen,E₂)对奶牛输卵管上皮细胞中前列腺素E₂合成酶(PGES)和前列腺素F_2α合成酶(PGFS)mRNA表达的影响,探讨E₂对奶牛输卵管生殖生理的调节作用,本试验采用了体外培养荷斯坦奶牛输卵管上皮细胞技术,分不同时间(0、2、4、8、16、24和48 h)和不同浓度(10^-12、10^-11、10^-10、10^-9 mol/L)添加雌激素E₂(以不加雌激素作空白对照),采用荧光定量PCR 技术检测PGES和PGFS mRNA表达。不同浓度的E₂或同一浓度不同刺激时间的E₂均能增加PGES的表达,但4 h浓度为10^-10 mol/L时与空白对照相比差异极显著(P<0.01),而PGFS在24 h添加浓度为10^-12 mol/L E₂时与空白对照相比差异极显著(P<0.01)。试验结果表明,E₂对培养的奶牛输卵管上皮细胞PGES和PGFS mRNA的表达有促进作用,说明雌激素E₂对前列腺素酶PGES和PGFS mRNA的表达具有调控作用。     

雌激素对奶牛输卵管组织COX-1和COX-2表达的影响

试验旨在阐明雌激素(E₂)对体外培养的奶牛输卵管组织中环氧合酶-1(cyclooxygenase-1,COX-1)与环氧合酶-2(cyclooxygenase-2,COX-2)基因表达的影响。分离培养奶牛输卵管组织,用不同浓度的E₂作用于奶牛输卵管组织,采用实时荧光定量PCR与Western blotting检测COX-1与COX-2 mRNA和蛋白的表达量。结果表明,E₂作用浓度为10^-11 mol/L时奶牛输卵管组织中的COX-1与COX-2 mRNA相对表达量达到高峰;E₂作用时间为8 h时COX-1 mRNA的相对表达量达到高峰,E₂作用时间为2 h时COX-2 mRNA的相对表达量达到高峰;浓度为10^-11 mol/L的E₂作用不同时间后,COX-1蛋白的相对表达量在8~48 h显著升高;没有检测到COX-2蛋白的表达。     

雌激素对奶牛输卵管上皮细胞中mPGES-1表达的影响

观察雌激素(β-雌二醇)对奶牛输卵管上皮细胞膜结合型前列腺素E2合酶-1(mPGES-1)表达的影响,并探讨β-雌二醇对奶牛输卵管上皮细胞合成分泌PGs调控的作用机制。采用胰酶消化法及机械法分离培养奶牛输卵管上皮细胞,应用荧光定量RT-PCR技术检测β-雌二醇对奶牛输卵管上皮细胞mPGES-1 mRNA表达的影响,应用Incell Western技术检测β-雌二醇对奶牛输卵管上皮细胞mPGES-1蛋白表达的影响。结果显示,与空白对照相比,β-雌二醇能够显著和极显著地促进奶牛输卵管上皮细胞中mPGES-1基因和蛋白的表达。本试验结果表明,β-雌二醇能够对奶牛输卵管上皮细胞中mPGES-1产生调节作用。     

肝脏X受体(LXRα)对奶牛子宫内膜炎调节机制的研究

为了探索肝脏X受体(LXRα)对脂多糖(LPS)诱导的奶牛子宫内膜上皮细胞炎症反应的调节机制，研究采用小鼠体内试验及奶牛子宫内膜上皮细胞体外试验，用LXRα激动剂T0901317激活LXRα,子宫灌注0.2 mg/mL LPS 50μL,检测奶牛子宫内膜上皮细胞活性、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、亚硝酸盐、前列腺素E₂(PGE₂)等炎性细胞因子的表达情况，对核因子κB(NF-κB)、糖原合成酶激酶3β(GSK3β)-重组与合成蛋白(Nrf2)信号通路的影响。结果表明：10,20,40μmol/L的T0901317对奶牛子宫内膜上皮细胞没有毒性作用，对TNF-α、IL-1β、亚硝酸盐、PGE₂的表达具有极显著的抑制作用(P<0.01),可降低磷酸化蛋白65(p-p65)、磷酸化蛋白ⅠκB(p-ⅠκB)的磷酰化水平，提高磷酸化葡萄糖合成激酶3β(p-GSK3β)、Nrf2及Nrf2调控的抗氧化因子血红素氧化酶-1(HO-1)的表达。说明LXRα可以通过调节GSK3β-Nrf2信号通路抑...     

催乳激素对山羊脂肪酸合酶基因转录活性的影响

为探讨催乳激素对体外培养的山羊乳腺上皮细胞脂肪酸合酶（fatty acid synthase，FASN）基因转录活性的调控作用，试验分别用不同浓度的胰岛素（insulin，INS）、雌激素（estradiol，E₂）、催乳素（prolactin，PRL）及不同激素组合（INS+PRL、E₂+INS+PRL）处理山羊乳腺上皮细胞24 h，提取细胞总RNA，采用实时荧光定量PCR检测催乳激素对FASN基因mRNA表达水平的影响。细胞转染山羊FASN基因启动子报告基因载体，同样用不同浓度的胰岛素、雌激素、催乳素及激素组合处理24 h，利用双荧光素酶报告基因系统检测催乳激素对FASN基因启动子活性的影响。结果发现，用雌激素、催乳素处理山羊乳腺上皮细胞后，FASN基因启动子活性及mRNA水平极显著或显著上调（P<0.01；P<0.05），雌激素浓度为10、100 μmol/L和催乳素浓度为0.1和1 μg/mL时效果最为明显，而胰岛素对FASN基因的启动子活性及mRNA水平没有显著影响（P>0.05）。用不同激素组合（INS+PRL、E₂+INS+PRL）处理细胞均能显著上调FASN基因启动子活性及mRNA表达水平（P<0.05）。结果表明，雌激素和催乳素能够调控FASN基因的转录活性，为进一步研究泌乳过程中FASN基因的分子调控机制提供理论依据。     

1,25(OH)2D3及VDR敲除对山羊附睾头上皮细胞β防御素家族表达的影响

旨在研究1,25(OH)₂D₃是否通过VDR途径调节山羊附睾头上皮细胞β防御素基因表达。本试验选取3只6月龄太行黑山羊，分别采集附睾头组织。采用差速贴壁法分离山羊附睾头上皮细胞，用细胞免疫荧光鉴定上皮细胞纯度。添加100 nmol·L^-1 1,25(OH)₂D₃处理附睾头上皮细胞以及筛选出敲除效率最高的pCas9/gRNA1质粒载体进行细胞转染，同时设置阴性对照组和空白对照组，每组3个重复孔。附睾头上皮细胞经1,25(OH)₂D₃处理以及VDR基因敲除后，分别用qRT-PCR检测VDR和17种β防御素基因的表达，用Western blot检测VDR蛋白和3种β防御素蛋白的表达。结果表明，1,25(OH)₂D₃能极显著提高VDR、gBD124、gBD126和gBD104a的mRNA和蛋白表达(P<0.01),同时极显著提高gBD104、gBD109tr1、gBD109tr2、gBD113...     

前列腺素E2和F2α对LPS刺激后奶牛子宫内膜上皮细胞COX-1和COX-2表达的影响

试验旨在阐明前列腺素E₂（prostaglandin E₂,PGE₂）和F_2α（prostaglandin F_2α,PGF_2α）对体外培养的奶牛子宫内膜上皮细胞中环氧合酶-1（cyclooxygenase-1,COX-1））与环氧合酶-2（cyclooxygenase-2,COX-2）表达的影响。培养奶牛子宫内膜上皮原代细胞和传代细胞,第4代细胞以1×10⁶个/孔接种于6孔板,以10^-7mol/L PGE₂和PGF_2α分别预处理细胞24 h,以100 ng/mL细菌脂多糖（lipopolysaccharides,LPS）刺激细胞4、8和12 h后分别提取RNA和总蛋白质,采用实时荧光定量PCR与Western blotting等技术检测COX-1与COX-2 mRNA和蛋白质的表达量。结果表明,与对照组相比,COX-1 mRNA表达量在PGE₂单独作用4、8和12 h后显著上调（P<0.05）;COX-2 mRNA表达量在PGE₂单独作用4和12 h后显著上调（P<0.05）,PGE₂单独处理使COX-1、COX-2蛋白表达量均显著上调（P<0.05）。与对照组相比,LPS刺激8和12 h时COX-1 mRNA表达量显著下调（P<0.05）,LPS刺激后COX-1蛋白表达量无显著变化（P>0.05）;LPS刺激后4、8和12 h时COX-2 mRNA表达量显著上调（P<0.05）,LPS刺激后COX-2蛋白表达量显著上调（P<0.05）。与LPS单独处理组相比,LPS+PGE₂处理组在8和12 h时COX-1和COX-2 mRNA表达量均显著上调（P<0.05）,同时COX-1和COX-2蛋白表达量也显著上调（P<0.05）。PGF_2α在LPS未刺激和刺激后对COX-1和COX-2 mRNA的表达无显著影响（P>0.05）,仅在PGF_2α单独处理8和12 h后COX-1 mRNA表达量上调（P<0.05）。两种激素联合处理与各自单独处理及LPS单独刺激相比,对COX-1和COX-2 mRNA表达具有一定的协同诱导作用。     

1,25(OH)2D3以双向方式影响猪前体脂肪细胞增殖分化的研究

1,25（OH）₂D₃是维生素D的主要活性形式,影响人和动物脂肪形成,为探究其在猪脂肪细胞增殖分化中的作用,试验从3~5日龄仔猪皮下脂肪组织分离培养前体脂肪细胞,并以浓度为0、0.1、1、10、100和1 000 nmol/L的1,25（OH）₂D₃分别处理,在培养的0、1、2、4、6、8和10 d采用MTT比色法检测细胞增殖活性;在诱导分化后0、1、2、4、6、8和10 d,以油红O染色提取法和实时荧光定量PCR检测细胞成脂分化及分化标志基因过氧化物酶体增殖物激活受体γ（PPARγ）和脂肪酸合成酶（FAS）表达。结果显示,0.1和1 nmol/L 1,25（OH）₂D₃显著促进猪前体脂肪细胞增殖（P<0.05）,而浓度为10~100 nmol/L时则抑制细胞增殖（P<0.05）;0.1和1 nmol/L 1,25（OH）₂D₃显著抑制猪前体脂肪细胞分化（P<0.05）,降低PPARγ和FAS mRNA表达水平（P<0.05）,但在浓度为10和100 nmol/L时,显著促进猪前体脂肪细胞分化（P<0.05）,上调PPARγ和FAS mRNA表达（P<0.05）;浓度达1 000 nmol/L时,可能对细胞有毒性作用。综合以上结果,低浓度1,25（OH）₂D₃促进猪前体脂肪细胞增殖,而通过下调PPARγ表达抑制分化;高浓度1,25（OH）₂D₃抑制猪前体脂肪细胞增殖,通过上调PPARγ表达促进分化。1,25（OH）₂D₃对猪前体脂肪细胞增殖和分化具有双向作用。     

Effect of GDF9 on Sheep Cumulus Cell Proliferation

The purpose of this study was to investigate the effect of growth differentiation factor 9 (GDF9) on the gene expression of cumulus cells expansion and hormone receptors as well as hormone secretion,in order to provide evidence for the role of GDF9 in the development of sheep cumulus cells.Sheep cumulus cells were used as the research object in this study,and were cultured for 48 h by adding different concentrations (0,50,100,200,400 ng/mL) GDF9 to low serum cell culture medium.Total RNA were extracted from the cells,using β-actin as the reference gene,Real-time quantitative PCR technology were used to detect the cumulus cells expansion related genes hyaluronic acid synthase gene 2 (HAS2),prostaglandin lead oxide synthase 2 (PTGS2),pentraxin 3 (PTX3) and hormone receptor genes follicle-stimulating hormone receptor (FSHR),luteinizing hormone receptor (LHR) and estrogen receptors (E₂R).Using the enzyme-linked immunosorbent assay (ELISA) method to test the content of E₂ and P₄.The results showed that HAS2,PTX3,FSHR,E₂R and LHR mRNA relative expression of 200 ng/mL GDF9 group was extremely significantly higher than the control group and other GDF9 groups (P<0.01),PTGS2 mRNA relative expression was extremely significantly higher than the control group and 50,400 ng/mL GDF9 groups (P<0.01),and significantly higher than 100 ng/mL GDF9 group (P<0.05).When added 400 ng/mL GDF9,the relative mRNA expression of all the mentioned-above genes were all extremely significantly lower than that of the 200 ng/mL GDF9 group.Moreover,the E₂ secretion level was extremely significantly higher than that of the control group and 50 ng/mL GDF9 group (P<0.01),significantly higher than that of the 100 ng/mL GDF9 group(P<0.05),while had no significant difference from the 200 ng/mL GDF9 group (P>0.05).When added 100,200 and 400 ng/mL GDF9,the concentration of P₄ was significantly higher than the control group (P<0.05),and there was no significant difference from the 50 ng/mL group (P>0.05),and there was no significant difference between 100,200 and 400 ng/mL GDF9 groups (P>0.05).To sum up,GDF9 could promote the expansion of sheep cumulus cells and participated in the regulation of hormone secretion of sheep cumulus cells.     

GDF9对绵羊卵丘细胞增殖的影响

本试验旨在探究生长分化因子9（GDF9）对卵丘细胞扩展相关基因和激素受体基因表达量及激素分泌的影响,为GDF9在绵羊卵泡发育中的作用提供依据。以绵羊卵丘细胞为研究对象,通过在低血清细胞培养液中添加不同浓度（0、50、100、200、400 ng/mL）的GDF9,培养绵羊卵丘细胞48 h后,提取细胞总RNA,利用实时荧光定量PCR技术,以β-actin为内参基因,检测卵丘细胞扩展相关基因透明质酸合酶2（HAS2）、前列腺素内过氧化物合酶2（PTGS2）、穿透素3（PTX3）及激素受体相关基因卵泡刺激素受体（FSHR）、促黄体生成素受体（LHR）和雌激素受体（E₂R）的mRNA相对表达量;利用酶联免疫吸附法（ELISA）测定培养液中卵丘细胞分泌的雌二醇（E₂）和孕酮（P₄）含量。结果显示：在细胞培养液中添加200 ng/mL GDF9时,HAS2、PTX3、FSHR、E₂R和LHR的mRNA相对表达量极显著高于对照组与其他处理组（P<0.01）;PTGS2 mRNA相对表达量极显著高于对照组、50和400 ng/mL GDF9组（P<0.01）,显著高于100 ng/mL GDF9组（P<0.05）。当添加400 ng/mL GDF9时,各基因mRNA相对表达量均极显著低于200 ng/mL GDF9组（P<0.01）;E₂分泌量极显著高于对照组与50 ng/mL GDF9组（P<0.01）,显著高于100 ng/mL GDF9组,与200 ng/mL GDF9组差异不显著（P>0.05）。100、200和400 ng/mL GDF9组P₄分泌量显著高于对照组（P<0.05）,与50 ng/mL GDF9组没有显著差异（P>0.05）,且3组之间差异不显著（P>0.05）。综上所述,GDF9能够促进绵羊卵丘细胞扩展,并参与绵羊卵丘细胞激素分泌的调控。     

Effects of Estrogen on COX-1 and COX-2 Expression in Bovine Oviduct Smooth Muscle

The aim of this study was to clarify the effects of estrogen (E₂) on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) gene expression in the cow oviduct tissue in vitro.Quantitative Real-time PCR and Western blotting were used to analyze the mRNA and protein expression of COX-1 and COX-2 at different time points after different concentrations of E₂ treatment.The results indicated that the relative expression of COX-1 and COX-2 mRNA reached the highest levels with 10^-11 mol/L E₂ treatment,the relative expression of COX-1 mRNA reached the highest level at 8 h after treatment;The relative expression of COX-2 mRNA reached the highest level at 2 h after treatment.The relative expression of COX-1 protein increased significantly at the range from 8 to 48 h after E₂ treatment.However,we did not detect COX-2 protein expression.     

Effects of Estrogen and Progesterone on Canine Endometrial Stromal Cells Proliferation and Progesterone Receptor Expression

In this study different digestion and isolation methods were applied to obtain canine endometrial stromal cells (ESCs), different concentration levels were set for estrogen (E₂) and progesterone (P₄), then MTT method was used to measure their effect on cell proliferation in vitro, also cell immunohistochemistry was used for cell identification and measurement of effect on progesterone receptors (PR) expression.The results indicated that E₂ (15, 30 and 100 pg/mL) showed no significant regulations on both cell proliferation and PR expression, P₄ (15 and 30 ng/mL) had significant promoting effect on proliferation of ESCs (P<0.05), P₄ (3, 15 and 30 ng/mL) showed significant inhibitory effect on PR expression (P<0.05), the regulation level was related to concentration and acting time.     

Construction of Specifically Targeted Chimeric Canine Parvovirus DNA Vaccine and Its Efficacy Assessment

To design chimeric DNA vaccine targeted to antigen-presenting cells with enhanced efficacy to induce immunization.The plasmid containing the gene encoding the extracelluar domain of canine cytotoxic T-lymphocyte antigen 4(CTLA-4₁₂₅),was directly fused to the gene fragment for major antigenic epitopes of VP2(VP2₂₂₈) by molecular engineering technology.The plasmid containing VP2₂₂₈ alone was also constructed to serve as control and transfection of COS-7 cells with the two resultant plasimds was performed respectively,followed by assay of Western blotting.The mice were immunized with the constructed two plasimds,respectively.After immunization,the antibodies anginst CPV in the immunized mice at different times were measured by HI.The spleen lymphocyte proliferation response was determined by lymphocyte proliferation assay,and the interferon-γ (IFN-γ) expression level of the mouse lymphocytes were measured by ELISA.The eukaryotic expression plasimds of CTLA-4₁₂₅-VP2₂₂₈ fusion protein or VP2₂₂₈ alone were cloned.Western blotting showed that the two recombinant proteins could be expressed.Immunization results showed that the antibody levels in serum of CTLA-4₁₂₅-VP2₂₂₈-immunized mice were significantly higher than that of VP2₂₂₈-immunized mice (P<0.05).The lymphocyte stimulation indexes and secreted IFN-γ levels of the CTLA-4₁₂₅-VP2₂₂₈-immunized mice were significantly higher than that of VP2₂₂₈-immunized mice (P<0.05 and P<0.01),respectively.CTLA-4₁₂₅-VP2₂₂₈ chimeric DNA vaccine stimulates strong immune response in mice,making it possible for further exploration into chimeric DNA that target the antigen to APCs.