不同日龄犊牛睾丸中细胞周期调控基因CyclinB1和p34~(cdc2)表达的差异

旨在探究不同日龄犊牛睾丸中细胞周期调控基因CyclinB1和p34cdc2的表达情况。本试验选取发育正常、体重相似的5~6d荷斯坦哺乳公犊18头,随机分为3组,每组6头,饲喂相同的基础日粮,分别在30(断奶)、60和90d时从各组分别随机抽取2头,采集其睾丸备用;通过qRT-PCR技术检测不同日龄犊牛睾丸中细胞周期调控基因CyclinB1和p34cdc2 mRNA表达规律,免疫组化技术对睾丸CyclinB1和p34cdc2进行定位分析。结果表明:90d时CyclinB1和p34cdc2 mRNA表达量显著高于30与60d(P0.05),但30与60d两种基因mRNA表达量均差异不显著(P0.05);p34cdc2蛋白在不同日龄犊牛表达差异显著(P0.05);CyclinB1蛋白在60、90d的表达量显著高于30d(P0.05),但60和90d差异不显著(P0.05)。综上表明,CyclinB1和p34cdc2 mRNA和蛋白在睾丸的表达量随着犊牛日龄的增加而增加,且不同的日龄CyclinB1和p34cdc2 mRNA和蛋白表达量存在一定的差异。     

CCND2在雌性牦牛生殖轴系中的表达分布

细胞周期蛋白CCND2(Cyclin-D2)是细胞增殖和细胞周期中G1/S期转变的关键调控因子,在癌症、营养代谢以及卵泡发育中参与调控.为研究CCND2在雌性牦牛(Bos grunniens)生殖轴系(HPOA轴)中的表达分布及其对季节性繁殖的作用机制,选取健康发情期雌性牦牛的下丘脑、垂体、卵巢组织,采用实时荧光定量P...     

沉默信息调节因子2相关酶1对乳脂、乳蛋白合成和乳腺炎的调节及其可能机制

沉默信息调节因子2相关酶1(SIRT1)是Sirtuins家族成员，可以对染色质和非组蛋白进行去乙酰化，在乳脂、乳蛋白合成以及乳腺炎的调控中发挥重要作用。本文主要综述了SIRT1通过AMP依赖的蛋白激酶（AMPK）、蛋白酪氨酸激酶-2(JAK2)、哺乳动物雷帕霉素靶蛋白（mTOR）和核转录因子-κB(NF-κB)等信号通路调控奶牛乳脂、乳蛋白合成以及乳腺炎的分子机制，以期为奶牛在乳脂、乳蛋白合成以及乳腺炎的靶向治疗提供理论支撑。     

胰岛素样生长因子-1对细胞周期的影响

胰岛素样生长因子-1(IGF-1)是一类具有类胰岛素功能活性的多肽,对多种细胞的增殖和分化起到重要的调控作用。血清中的IGF-1主要由肝脏分泌,随血液运输到靶组织后,与靶组织细胞膜上的1型IGF受体(IGF-1R)结合后,激活相应的受体后信号转导途径,提高细胞周期蛋白表达,加速细胞周期运转,促进细胞增殖。本文就IGF-1促进细胞增殖的作用机理进行阐述。     

siRNA干扰Wnt11对鸭成肌细胞增殖的影响

为了探究Wnt11(无翅型MMTV整合位点家族成员11)对鸭骨骼肌成肌细胞增殖的影响以及可能与Wnt11相关的信号通路,采用体外分离培养鸭骨骼肌成肌细胞,脂质体介导siRNA(小干扰RNA)转染干扰基因的表达,qRT-PCR(实时荧光定量PCR)检测基因表达变化,Ed U(5-乙炔基-2'-脱氧尿苷)染色分析细胞增殖情况,流式细胞术检测细胞周期变化及信号通路抑制剂处理。结果显示:干扰Wnt11的表达后,增殖相关基因CCND1(细胞周期蛋白D1)、CDK6(周期蛋白依赖性激酶6)和PCNA(增殖细胞核抗原)的表达量极显著降低(P0.01),Ed U阳性细胞数极显著降低(P0.01),处于S期的细胞数显著降低(P0.05);当用Akt(蛋白激酶B)抑制剂处理鸭成肌细胞后,Wnt11表达量显著降低(P0.05),而干扰Wnt11表达后,PI3K(磷脂酰肌醇-3-羟激酶)和Akt的表达量显著升高(P0.05)。提示:Wnt11在鸭成肌细胞增殖过程中有重要调控作用,Wnt11发挥调控作用需要PI3K/Akt信号通路参与,且二者间存在反馈作用。     

家蚕细胞周期素E基因在5龄幼虫组织表达的剪切变体和表达谱特征

细胞周期素E(cyclinE)是调控细胞周期从G1期进入S期的关键因子。利用家蚕基因组数据库信息设计引物,克隆家蚕cyclinE在5龄幼虫不同组织表达的cDNA序列,获得该基因的6种不同剪切变体,这6种剪切变体前4个外显子和最后一个外显子都相同,变化主要集中在第5~7外显子处。同源性比对显示,不同物种来源的cyclinE周期蛋白盒区域具有较高的保守性,有35个氨基酸的共同保守序列,该区域可能也是家蚕cyclinE的特异识别并结合细胞周期素依赖蛋白激酶的功能区域。利用实时定量PCR分析显示,cyclinE在家蚕5龄第3天幼虫的中肠、脂肪体、丝腺、脑、马氏管、精巢、卵巢、血液等8种组织、器官中均有表达,并且在脂肪体中的表达相对较高,推测cyclinE基因在家蚕脂肪体细胞分裂过程中发挥重要作用。     

哺乳动物雷帕霉素靶蛋白信号通路调控猪肠上皮细胞精氨酸转运的机制

本研究旨在基于哺乳动物雷帕霉素靶蛋白(mTOR)信号通路探讨猪肠上皮细胞增殖和精氨酸(Arg)转运的调控机制。在含100或350μmol/L Arg的培养基中添加(10 nmol/L)或不添加(0 nmol/L)雷帕霉素(Rap),培养猪肠上皮细胞(IPEC-J2细胞)3 d后,对细胞活力、细胞周期以及Arg转运、增殖和凋亡相关通路基因和蛋白表达进行检测。结果表明:1)添加Rap极显著降低了G2期和S期细胞数量(P0.01),而提高Arg浓度有效缓解了Rap对细胞增殖的抑制作用;Rap通过激活磷脂酰肌醇-3-羟激酶(PI3K)-蛋白激酶(Akt)-B细胞淋巴瘤2(Bcl2)信号通路抑制细胞凋亡。2)添加Rap抑制mTOR信号通路后极显著提高了Arg摄取率(P0.01),极显著提高了100μmol/L Arg培养下细胞中阳离子氨基酸转运载体2(CAT2)的mRNA和蛋白表达量(P0.01);进一步试验证明蛋白激酶Cα(PKCα)-细胞外信号调节激酶(Erk)/cFos-CAT2信号通路可能是Rap促进CAT2表达,进而提高Arg摄取的重要通路。综上可知,Rap应激下猪肠上皮细胞增殖被抑制,提高Arg浓度能有效缓解Rap对细胞增殖的抑制作用;Rap通过调控PKCα-Erk/cFos-CAT2信号通路促进猪肠上皮细胞对Arg的摄取,且提高Arg浓度可促进细胞对Arg的摄取。结果提示,mTOR信号通路在调控猪肠上皮细胞Arg利用过程中发挥重要作用。     

Wnt/β-连环蛋白信号通路在哺乳动物子宫发育中的调控作用及其机制

机体内生理活动程序化的有序运行均依赖于不同信号传导通路之间的相互协调,其中Wnt(名称来源于果蝇无翅基因Wingless和小鼠致癌基因int-1)信号通路受到学者们的广泛关注,已经成为分子生物学和细胞生物学领域的研究热点。本文研究了Wnt/β-连环蛋白(β-catenin)信号通路在哺乳动物子宫发育中的调控作用及其机制,综述了糖原合酶激酶-3β(GSK-3β)、结肠腺瘤样息肉基因(APC)、支架蛋白(Axin)和成骨细胞抑制因子(Dkk)对Wnt/β-catenin信号通路的调控机制及Wnt/β-catenin信号通路的核内激活,旨在进一步揭示子宫内调节机制,并为子宫疾病的治疗提供借鉴。     

敲低SQLE基因对奶牛乳腺上皮细胞增殖及凋亡的影响

为了探讨角鲨烯环氧酶(squalene epoxidase,SQLE)基因对体外培养奶牛乳腺细胞凋亡及增殖的影响,本研究用RNAi技术敲低奶牛乳腺上皮细胞中SQLE基因;用实时荧光定量PCR方法检测奶牛乳腺上皮细胞中SQLE基因及凋亡和周期相关基因的表达;用CCK-8法检测其对细胞增殖的影响;用周期和凋亡检测试剂盒筛选细胞,用流式细胞术准确计数处于不同周期的细胞数和凋亡细胞数。结果显示,奶牛乳腺上皮细胞转染siRNA后,SQLE基因相对表达量显著下降(P0.05),细胞增殖受到极显著抑制(P0.01);G1期细胞数量显著下降(P0.05),G2期细胞数量没有显著性改变,S期细胞数量显著上升(P0.05);肿瘤坏死因子超家族成员6(又称Fas或CD5)的相对表达量显著上升(P0.05),细胞周期蛋白依赖性激酶抑制剂1B(cyclin dependent kinase inhibitor 1B,P27)相对表达量显著上升(P0.05),而细胞周期素D1(Cyclin D1)、B淋巴细胞瘤因子2(B-cell lymphoma-2,Bcl-2)、细胞周期蛋白依赖性激酶抑制剂1A(cyclin dependent kinase inhibitor 1A,P21)、Bcl-2相关X蛋白(Bcl-2 associated X,apoptosis regulator,Bax)显著下降(P0.05)。综上所述,敲低SQLE基因通过调节相关基因的表达抑制乳腺上皮细胞的增殖。     

苜蓿源miR168b通过靶向肉碱棕榈酰转移酶1A对奶牛乳腺上皮细胞增殖、凋亡和脂滴积累的调控研究北大核心CSCD

本试验旨在探究苜蓿源miRNAs(mtr-miRNAs)对奶牛乳腺上皮细胞(BMECs)增殖和凋亡的影响。试验首先通过CCK8、5-乙炔基-2′脱氧尿嘧啶核苷(EdU)、流式细胞术、基因和蛋白定量等方法检测苜蓿源-miR168b(mtr-miR168b)对BMECs增殖、凋亡和周期的影响。然后,敲低mtr-miR168b靶基因肉碱棕榈酰转移酶1A(CPT1A)表达后,检测了BMECs的活力和凋亡。通过诱导转染后细胞,用氟硼二吡咯(Bodipy)染色和油红O染色检测mtr-miR168b和CPT1A基因干扰对BMECs脂滴合成的影响。结果表明:1)mtr-miR168b高表达极显著降低了BMECs活力(P<0.01)。2)mtr-miR168b高表达极显著抑制了BMECs增殖(P<0.01),延长了细胞G1~S期进程,极显著促进了细胞凋亡(P<0.01)。3)mtr-miR168b在BMECs中高表达极显著抑制了细胞增殖基因周期蛋白依赖性激酶4(CDK4)、增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)和细胞周期蛋白D2(Cyclin D2)的基因的表达水平(P<0.01),极显著增加了凋亡标志基因Bcl2关联X蛋白(BAX)基因的表达水平(P<0.01),极显著抑制了PCNA蛋白表达水平(P<0.01),极显著增加了BAX蛋白表达水平(P<0.01),并且mtr-miR168b显著抑制了蛋白激酶B(AKT)和哺乳动物雷帕霉素靶蛋白(mTOR)基因的表达水平(P<0.05)。4)mtr-miR168b高表达抑制了BMECs中脂滴的积累。5)干扰mtr-miR168b的靶基因CPT1A,对BMECs脂滴积累也有抑制作用。综上所述,mtr-miR168b可通过抑制BMECs增殖,促进细胞凋亡,并通过靶向CPT1A基因抑制BMECs中脂滴的生成。试验结果可为mtr-miRNAs调控牛奶乳脂合成提供分子层面的技术支撑。     

CDKs(细胞周期依赖性蛋白激酶)调控细胞周期中的作用

细胞周期是细胞生命活动的基本过程,由细胞周期蛋白(Cyclin)依次融活相应的细胞周期依赖性蛋白激酶(CDKs)所推动的。本文综述了CDKs在细胞周期各个不同阶段的调控机制。     

Butyrate-induced apoptosis and cell cycle arrest in bovine kidney epithelial cells: involvement of caspase and proteasome pathways

Beyond their nutritional effect, short-chain fatty acids, especially butyrate, modulate cell differentiation, proliferation, motility, and in particular, they induce cell cycle arrest and apoptosis. A bovine kidney epithelial cell line (Madin-Darby bovine kidney; MDBK) was used to investigate the cell cycle regulatory and apoptotic effects of butyrate. Butyrate not only induced apoptosis but also induced cell cycle arrest at the G1/S boundary and M/G2 in MDBK cells (P < 0.01). The cell responses were concentration-dependent (r(2) = 0.9482, P <0.001). In examining possible mechanisms for the apoptosis and cell cycle arrest induced by butyrate, the results showed that butyrate treatment activates caspase-3 activities and induces accumulation of acetylated histone. At least two proteins, cdc6 and cdk1, become targeted for destruction on butyrate treatment. These two proteins are downregulated (P < 0.01 and P < 0.05, respectively) by proteolytic pathways. Moreover, the proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) reverses the cell cycle arrest induced by butyrate, indicating a multiprotein crosstalk wherein the ubiquitination/ proteasome pathway interacted with the caspase-signaling pathway. Because the proteasome inhibitor MG-132 blocked activation of caspase-3, these results functionally locate the proteasome pathway upstream of the caspase pathway. All these results indicate that butyrate functions as both a nutrient and signaling molecule regulating cell growth and proliferation.     

Effect of EGF on expression and localization of maturation‐promoting factor,mitogen‐activated protein kinase,p34cdc2 and cyclin B during different culture periods on in vitro maturation of canine oocytes

This study aimed to investigate the localization of MPF, MAPK, p34^cdc2 and cyclin B1 proteins, before and after treatment with EGF during different moments of oocyte maturation. The ovaries obtained from 350 domestic dogs were aseptically isolated, immersed in physiological solution and transported at 4°C. In the laboratory, the ovaries were sectioned for the release of cumulus–oocyte complexes. Cumulus–oocyte complexes were selected and divided into treatment groups with and without EGF and cultured for 24, 48 and 72 hr. Immunofluorescence was used for the detection and the localization of MAPK, MPF, p34^cdc2 and cyclin B1 proteins. We observed that the expression and localization of MPF, MAPK, p34^cdc2 and cyclin B1 proteins are associated with meiosis resumption and cell cycle progression, and that EGF influences cell signalling pathways by promoting alterations in the localization of these proteins, improving the acquisition of oocyte competence. This is the first report of the localization of crucial proteins for meiosis progression in domestic dogs and identification of the expression and localization of proteins for cell cycle progression performed in this study represented a step of great importance to elucidate the mechanisms involved in the meiosis block in domestic dogs, allowing the advance in this research area.     

Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates

ABSTRACT: The present study examined the immunological response of antigen presenting cells (APC) to genotype-I isolates of porcine reproductive and respiratory syndrome virus (PRRSV) infection by analysing the cytokine profile induced and evaluating the changes taking place upon infection on immunologically relevant cell markers (MHCI, MHCII, CD80/86, CD14, CD16, CD163, CD172a, SWC9). Several types of APC were infected with 39 PRRSV isolates. The results show that different isolates were able to induce different patterns of IL-10 and TNF-α. The four possible phenotypes based on the ability to induce IL-10 and/or TNF-α were observed, although different cell types seemed to have different capabilities. In addition, isolates inducing different cytokine-release profiles on APC could induce different expression of cell markers.     

Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini-pig (Sus Scrofa)

To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.     

Targeting to porcine sialoadhesin receptor improves antigen presentation to T cells

Antibody-mediated targeting of antigen to specific antigen presenting cells (APC) receptors is an attractive strategy to enhance T cell immune responses to weak immunogenic antigens. Here, we describe the characterization of two monoclonal antibodies (mAb) against different epitopes of porcine sialoadhesin (Sn) and evaluate in vitro the potential of targeting this receptor for delivery of antigens to APC for T cell stimulation. The specificity of these mAb was determined by amino acid sequence analysis of peptides derived from the affinity purified antigen. Porcine Sn is expressed by macrophages present in the border between white and red pulp of the spleen and in the subcapsular sinus of lymph nodes, an appropriate location for trapping blood and lymph-borne antigens. It is also expressed by alveolar macrophages and monocyte-derived dendritic cells (MoDC). Blood monocytes are negative for this molecule, but its expression can be induced by treatment with IFN-a. MAb bound to Sn is rapidly endocytosed. MAb to sialoadhesin induced in vitro T cell proliferation at concentrations 100-fold lower than the non-targeting control mAb when using T lymphocytes from pigs immunized with mouse immunoglobulins as responder cells and IFN-a treated monocytes or MoDC as APC, suggesting a role of sialoadhesin in antigen uptake and/or delivery into the presentation pathway in APC.     

17β-雌二醇对仔猪支持细胞周期的影响

为了研究17β-雌二醇对仔猪睾丸支持细胞周期的影响,实验采用流式细胞术测定了细胞周期中DNA含量,分析了细胞周期细胞增殖指数(PI)和S期细胞指数(SPF)的变化。结果表明:低浓度的17β-雌二醇(10-10～10-7mol/L)能够促进支持细胞周期的转化,其中17β-雌二醇浓度为10-9mol/L时的作用最强;而高浓度的17β-雌二醇(10-6mol/L)则抑制支持细胞周期的转化。17β-雌二醇(10-9mol/L)能以时间依赖性促进支持细胞周期转化,其中24h的作用最明显。ERK1/2抑制剂U0126能减少17β-雌二醇诱导的细胞周期的转化。cAMP的激动剂8-Br-cAMP促进了细胞周期的转化,而抑制剂Rp-cAMP阻止了17β-雌二醇诱导的细胞周期的转化。表明17β-雌二醇对细胞周期的调节呈现一定的时间-剂量依赖性,cAMP和ERK1/2级联参与了这一过程。     

Cyclin D1 protein expression in human thyroid gland and thyroid cancer

Cell cycle progression is facilitated by cyclin dependent kinases (CDKs) that are activated by cyclins, including Cyclin D1 and inhibited by CDK inhibitors. Evidence of the involvement of cyclin gene alterations and over expression of various cyclins in human cancer is growing. The role of Cyclin D1 in malignant progression of papillary carcinomas of the thyroid has yet to be established. We therefore studied the expression of Cyclin D1 protein in thyroid carcinomas of young Kuwaiti patients (36 cases of conventional papillary thyroid carcinoma, 12 cases of its follicular variant, one case of tall cell thyroid carcinoma and one case of medullary carcinoma) using immunohistochemistry. In 23 patients (46%) circumscribed areas of cells were detected that showed a distinct to strong nuclear staining for immunoreactive Cyclin D1 whereas the remaining bulk of the carcinoma cells were negative or only showed a slight cytoplasmic staining. None of the tested clinical or path histological parameters showed a statistically significant correlation with the focal immunostaining. This does not rule out that the detected foci with positive nuclear Cyclin D1 immunostaining are areas where a progressive transformation to a more malignant phenotype occurs which eventually leading to lymph node and distant metastases.     

流产型布鲁氏菌Omp22基因酵母双杂交诱饵载体的构建及鉴定

通过PCR获得流产型布鲁氏菌Omp22基因的编码区,将其克隆到pSOS载体中,构建酵母双杂交系统的诱饵载体pSOS-Omp22。测序正确后,将重组质粒导入cdc25H检测其表达产物对酵母细胞有无毒性和自激活作用,且在酵母细胞中定位是否正确。结果表明,获得了正确的流产型布鲁氏菌Omp22基因编码区,并成功克隆到pSOS诱饵载体中,且转化有诱饵载体的cdc25H在SD/Glucose(-L)营养缺陷平板上生长良好,说明表达产物对酵母细胞无毒性,对报告基因也无自激活作用且其定位正确。表明pSOS-Omp22可应用在酵母双杂交系统中,用于寻找巨噬细胞cDNA文库中与Omp22相互作用的蛋白质。     

A blinded randomized controlled trial evaluating the usefulness of a novel diet (aminoprotect care) in dogs with spontaneous food allergy

Aminoprotect Care (APC) is a novel diet composed of aminoacids, potato proteins and corn starch. The objectives of this study were to determine whether Maltese-Beagle atopic (MBA) dogs hypersensitive to corn exhibited clinical signs and changes in immunological markers after being fed APC. The study was designed as a blinded randomized controlled crossover experiment. Ten MBA dogs with signs of allergy within five days of ingesting corn were selected. Dogs were randomized to be fed either their maintenance diet with corn or APC for five days. After a washout of two weeks, diets were switched. Before and daily during each intervention, skin lesions were graded by an investigator while pruritus was assessed by another. Before and at the end of each intervention, the percentage of circulating CD4+CCR4+, corn-activated CD4+ T-lymphocytes and serum corn-specific IgE levels were measured and ratios of post:pre values calculated. During this trial, pruritus and skin lesions increased significantly in MBA dogs when ingesting corn while no such increase occurred when fed APC. Total, median and maximal pruritus values were significantly higher in MBA dogs ingesting corn compared to APC. There were no significant differences between interventions in the immunological parameters assessed. In summary, even though APC contains corn starch to which corn-sensitive MBA dogs often react, the ingestion of APC did not lead to significant increases in skin lesions or pruritus. Aminoprotect Care might prove valuable for management of food allergies. These experimental observations must be validated in large field studies.