Construction and Identification of Mammary Gland-specific Expression Vector of Bovine Tracheal Antimicrobial Peptide (TAP)

[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and KpnI, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein （ EGFP）, and transfected into bovine mammary epithelial cells （bMEC）, COS-7 cells and lactating rabbit mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue was detected by semi-quantity RT-PCR. [ Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition, semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfeeted with pBLG-EGFP-TAP. [ Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.

Expression of Human GCSF in Mammary Gland of Mice by Injection of Plasmid DNA

The vectors carrying the genes coding for the proteins of interest are of unpredictable efficiency in transgenic animals. The expression vector of mammary gland (pINGG) containing GCSF genomic DNA was injected into mouse mammary gland, and expression was detected in the milk of mice. The result showed that mammary gland injection method could provide a convenient transient system to confirm vector validity.

THE EXPRESSION OF APOPTOSIS RELATED GENES IN THE PROCESS OF CANCERATION OF ATYPICAL HYPERPLASIA OF MAMMARY DUCT

Objective： To investigate the expression of apoptosis related genes p53 and bcl-2 in atypical hyperplasia of mammary duct and the relationship between the gene expression and oncogenesis of breast. Methods： mRNA of apoptosis related gene p53 and bcl-2 were detected by in situ hybridization in 44 cases of atypical ductal hyperplasia. p53 protein expression was detected by immunohistochemistry. The data were compared with those of 6 cases of benign hyperplasia and 26 cases of breast carcinoma. Results： The expression of p53 mRNA was 66.7% in benign hyperplasia, 40% in atypical ductal hyperplasia （55.6% in mild, 41.7% in medium, 26.1% in severe） and 19.2% in carcinoma （of which 21.4% were intraductal carcinoma and 16.7% were invasive）. The expression of p53 protein was negative in benign hyperplasia, 24% in atypical hyperplasia （mild 11.1%, medium 25%, severe 34.8%）, 38.5% in carcinoma （intraductal carcinoma 35.7%, invasive ductal carcinoma 41.7%）. The expression of bcl-2 was negative in benign hyperplasia, 78.6% in intraductal carcinoma, 83.3% in invasive ductal carcinoma. Conclusion： Loss and mutation of p53 gene and excessive expression bcl-2 mRNA were detected in severe atypical ductal hyperplasia.

Stable Expression of Antibacterial Peptide CecropinB in Dairy Goat Mammary Gland Epithelial Cells

The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.

Effect of exogenous somatotropin and staged ovariectomy on mRNA expression of select ECM-related proteins in mammary tissue of prepubertal Holstein calves

Growth hormone (GH) and estrogen are essential stimulators of mammary cell proliferation and mammary development as mammals near puberty. Mammary ductal growth requires modifications of the extracellular matrix (ECM) for this tissue expansion to occur. Our purpose was to evaluate the effects of exogenous GH and ovariectomy (known to impact estrogen production) on gene expression of selected ECM proteins in the mammary parenchyma (PAR) and mammary fat pad (MFP) of prepubertal calves. Our hypothesis was that both GH and ovariectomy would alter the mRNA expression of multiple mammary ECM proteins. However, treatment with GH significantly reduced the expression of only fibronectin in PAR. However, the mRNA expression of all of the ECM proteins tested was numerically lower in PAR from GH treated calves. In contrast, staged ovariectomy decreased expression of fibronectin and heat shock protein 90 but increased expression of epimorphin in mammary PAR. In the MFP expression of Rac-1 and fascin were increased. These findings suggest that effects of exogenous GH on mammary gland composition are only marginally dependent on alterations in ECM proteins but the more pronounced effects of ovariectomy (reduced PAR mass and altered myoepithelial ontogeny) are more likely linked to changes in expression of ECM proteins.

Induced in vitro Expression of Human Lactoferrin in Goat Mammary Gland Epithelial Cell

[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign protein at the cell level.[Method]Goat mammary gland epithelial cell transfected by human lactoferrin gene was inducted by culturing in DMEM/F12 medium supplemented with 5 mg/L insulin,5 mg/L prolactin and 1 mg/L hydrocortisone.Supernatant was collected per 6 hours and concentrated.Expression situation of foreign protein were detected by SDS-PAGE and Western blotting.[Result]There was target protein expression in the induced culture medium,which molecular weight was about 42 kD.[Conclusion]The method used in this study can induce goat mammary gland epithelial cell to express foreign gene,it lays a foundation for researching heterologous expression of foreign gene and producing mammary gland bioreactor.

Identification of Differentially Expressed MicroRNAs During the Development of Chinese Murine Mammary Gland

MicroRNAs （miRNAs） are endogenous -22 nucleofide-long noncoding RNAs. In this study, to investigate miRNA expression profiles and their functions in mammary gland development, we have used microarray as well as qRT-PCR, to analyze the miRNA expression changes along the murine mammary cycle during pregnancy, particularly on transition from pregnancy to lactation. It shows that every developmental stage of the mammary gland has its own mjRNA expression pattern. Compared with virgin and involution, some miRNAs such as miR-138 and miR-431 are downregulated, whereas, some miRNAs such as miR-133 and miR-133a-133b are upregulated during pregnancy and lactation. These results indicate that miRNAs are functionally involved in mammary gland development.

柯乐猪异常乳腺组织的TMT蛋白组学分析

【目的】基于串联体量标记(TMT)定量蛋白组学技术探讨柯乐猪母猪乳腺发育的调控机制,为选育工作提供依据。【方法】选取乳腺发育正常和异常的柯乐猪母猪各3头,采集乳腺组织,利用TMT定量蛋白组学技术探究正常和异常乳腺组织的差异表达蛋白,筛选与乳腺发育相关的差异关键蛋白,并对差异表达蛋白进行生物信息学分析。【结果】从柯乐猪乳腺组织中共筛选出474个差异表达蛋白,包括245个上调蛋白、229个下调蛋白;基因本体(GO)功能注释和富集分析显示,差异表达蛋白主要富集在上皮细胞分化、皮肤形成和肾小管上皮细胞分化等生物学过程中,主要分布在胞外区和细胞外空隙中,参与调节肽酶抑制剂活性和肽链内切酶活性;京都基因与基因组百科全书(KEGG)富集分析显示,差异表达蛋白主要富集在补体系统、过氧化物酶体增殖物激活受体和金黄色葡萄球菌感染等信号通路上;结合差异表达蛋白互作网络分析,筛选出10个核心差异表达蛋白[上调蛋白有P14287(骨桥蛋白)、F1RXF9(角蛋白25)、I3LDS3(角蛋白10)、Q75QW1(上皮细胞黏附分子)、A0A5S8KLN1(丛生蛋白)、F1RW75(桥粒素),下调蛋白有A0A286ZT13(白蛋白)、P06867(血纤维蛋白溶酶原)、F1SCC9(含丝氨酸蛋白酶抑制剂结构域的蛋白)、P50447(α-1-抗胰蛋白酶)]。【结论】采用TMT定量蛋白组学技术能有效筛选出母猪正常和异常乳腺组织中的差异表达蛋白。

采食高淀粉高油日粮奶牛的泌乳性能、乳成分变化及乳腺差异表达基因筛选

【目的】研究高淀粉高油日粮对奶牛泌乳性能和乳腺组织转录组的影响,探索乳脂下降综合征(Milk fat depression,MFD)的分子机制。【方法】选用8头荷斯坦泌乳奶牛,随机分为对照组和处理组(4头/组),采用2×2反转试验设计,分为2期,每期23 d,期间2组对调。每期的前16 d,对照组奶牛饲喂低淀粉低油的基础饲粮,对照组日粮的泌乳净能为6.78 MJ/kg;处理组奶牛在基础饲粮基础上添加266 g/kg(DM基础)细粉碎玉米和46 g/kg(DM基础)大豆油;后7 d 2组奶牛均喂基础饲粮,处理组日粮的泌乳净能为7.66 MJ/kg。分别于每期的第1、4、7、10、13、16、19和22天测定产奶量和乳成分,第16天采集乳腺组织用于检测转录本的变化。【结果】与对照组相比,处理组第13、16天奶牛的干物质采食量和产奶量显著降低(P<0.05);饲喂高淀粉高油饲粮7 d后乳脂率开始降低,其中,第10、13、16和19天的乳脂率和乳脂产量显著降低(P<0.05);第13、16和19天的乳蛋白、乳糖和非脂固形物含量显著提高(P<0.05)。在奶牛乳腺对照组和处理组中共检测到235个显著差异表达的基因,其中,64个上调、171个下调。GO分析表明,差异表达基因主要与炎症反应、α−氨基酸代谢、有机氮化合物代谢、细胞发育、脂质生物合成和脂肪细胞分化等相关;KEGG分析表明,差异表达基因主要与轴突导向、NF-κB信号通路、钙信号通路等相关。结合差异表达基因与泌乳性能的相关性分析,本研究筛选出了部分与脂质调控相关的基因作为研究奶牛MFD状态下脂质代谢的候选基因,包括在乳腺中下调表达的FGFR4、VDR、HTR2B、CCL21和TYRP1。【结论】高淀粉高油饲粮饲喂奶牛降低了干物质采食量、产奶量和乳脂率,提高了乳蛋白、乳糖和非脂固形物含量,下调了奶牛乳腺中脂肪酸合成相关基因的表达。本研究为科学配制日粮和研究MFD的分子机制研究提供了数据参考。

脂多糖与钙离子共处理的牛乳腺上皮细胞miRNA表达谱的比较分析

【目的】探索钙离子对牛乳腺上皮细胞炎症反应的调控作用,比较分析脂多糖与钙离子共处理的牛乳腺上皮细胞的miRNA表达谱。【方法】使用脂多糖处理牛乳腺上皮细胞构建炎症反应模型,检测不同浓度(0.5~6.6 mmol/L)钙离子和脂多糖(50μg/mL)共处理的牛乳腺上皮细胞的炎症因子转录水平。提取正常、脂多糖处理、脂多糖与钙离子共处理牛乳腺上皮细胞样本中总RNA,每组各3个样本。对9个RNA样本进行质量评估,并对其开展miRNA测序和生物信息学分析,最后对3组共有差异表达miRNA进行实时荧光定量PCR验证。【结果】在牛乳腺上皮细胞炎症模型中,高浓度钙离子可抑制炎症因子的转录。9个样本的clean reads在raw reads中占比为93.86%~98.18%,序列分布长度均集中在21~24 nt之间。3组样本的组内相似性高,而组间差异度高。3组样本共鉴定出47个差异表达miRNA,共有差异表达miRNA分别为bta-miR-11980、bta-miR-1246和bta-miR-677。GO功能和KEGG通路富集分析显示,差异表达miRNA的靶基因显著富集于细胞通讯调节、信号调节、内膜系统、系统发育、细胞发育过程、细胞分化等GO条目,显著富集于HIF-1信号通路、ErbB信号通路、TNF信号通路、突触囊泡周期、肌醇磷酸代谢途径等通路。实时荧光定量PCR验证结果显示,3组样本共有差异表达miRNA(bta-miR-677和bta-miR-1246)表达趋势与转录组测序结果一致。【结论】钙离子通过调控牛乳腺上皮细胞内bta-miR-11980、bta-miR-1246和bta-miR-677等差异表达miRNA的表达模式,间接影响牛乳腺上皮细胞炎症反应进程。

荷斯坦奶牛DPP6和PRKN基因的生物信息学及表达规律分析

【目的】旨在探究前期GWAS筛选出的与荷斯坦奶牛产奶相关的二肽基肽酶样6(Dipeptidyl Peptidase⁃like 6,DPP6)和E3泛素连接酶基因(Parkin gene,PRKN)的生物学性质及表达特性,为后续验证DPP6和PRKN基因对于荷斯坦奶牛相关调控机制研究提供基础。【方法】利用ProtParam、Phyre2、NetPhos 3.1等软件分析DPP6和PRKN的理化性质与蛋白结构,并采用实时荧光定量PCR(Real⁃Time quantitative PCR,RT⁃qPCR)检测DPP6和PRKN基因在荷斯坦奶牛的心脏、脾脏、肝脏、肺脏、肾脏、乳腺、子宫、卵巢、瘤胃、黄体10个组织中的表达规律;通过脂多糖(Lipopolysaccharide,LPS)诱导乳腺上皮细胞(bMECs),初步检测DPP6和PRKN基因在炎症反应中的表达模式。【结果】DPP6蛋白编码863个氨基酸,与山羊的亲缘关系最近;PRKN蛋白编码488个氨基酸,其中甘氨酸含量最多,其序列与马鹿的相似性最高,保守型较强。本研究推测2个物种的DPP6和PRKN蛋白可能具有相似的生物学功能,并且2个蛋白均属于不稳定亲水性蛋白。DPP6和PRKN基因在组织中普遍表达。其中,DPP6基因在乳腺、肺脏、脾脏以及肾脏组织中的表达量显著高于其他组织,而PRKN基因在乳腺、肾脏、瘤胃组织中显著高表达。此外,相比于空白对照组,DPP6与PRKN基因均能够在LPS诱导的bMECs中极显著表达(DPP6基因极显著下调,PRKN基因极显著上调)。【结论】DPP6与PRKN基因可能在调控荷斯坦奶牛乳房炎症过程中发挥潜在作用,为后期荷斯坦奶牛DPP6与PRKN两个基因在奶牛bMECs炎症反应相关调控机制研究提供理论依据。

Bzw2 Promotes Proliferation and Lactation of Mammary Epithelial Cell in Dairy Goat

Mitosis of mammary epithelial cell is foundation of mammal lactation. We developed a strategy of combined application of generation of longer cDNA fragments from the serial analysis of gene expression （SAGE） tags for gene identification （GLGI） to screen and identify genes influencing lactating ability of mammary epithelial cell in dairy goat. GLGI as a new tag identification technique was brought about with SAGE. Bzw2 was found as a candidate gene related to lactation by screening Long-SAGE library of mammary gland in dairy goat. Bzw2 cDNA was synthesized by switching mechanism at 5＂-end of RNA transcript （SMART） technology. The mRNA level of Bzw2 was relatively higher in early lactation than in other development stages of mammary gland. The proliferation of mammary epithelial cell was inhibited by transfecting specific shRNA of Bzw2. The mRNA levels of Stat5, Csn2 and Prlr were also down-regulated, suggesting the lactating ability of mammary epithelial cell was attenuated after Bzw2 RNAi. The reduction of mammary epithelial cell growth and lactation by Bzw2 RNAi was rescued through over-expression of Bzw2. These results revealed that Bzw2 might play an important role in lactation though the molecular mechanism was still unclear.

β-谷甾醇对小鼠乳腺上皮细胞乳蛋白和乳脂肪合成的影响

为探讨β-谷甾醇对泌乳动物乳腺乳蛋白和乳脂肪合成的影响,研究首先利用MTT法筛选出对小鼠乳腺上皮细胞生长增殖不受抑制的β-谷甾醇剂量;采用qRT-PCR技术检测β-谷甾醇处理乳腺上皮细胞后乳蛋白合成相关基因和乳脂肪合成相关基因的mRNA表达水平。结果表明:较高剂量(40~120μmol/L)的β-谷甾醇明显抑制小鼠乳腺上皮细胞的活力(P<0.05),故后续试验添加5、10、20μmol/L的β-谷甾醇处理乳腺上皮细胞。β-谷甾醇明显提高酪蛋白基因CSN1S1、CSN2、CSN3及乳蛋白合成通路相关基因JAK2、STAT5、mTOR、S6K1的mRNA表达水平(P<0.05),但5、10μmol/L的β-谷甾醇对4EBP1基因的mRNA表达水平无显著影响(P>0.05)。β-谷甾醇显著上调乳脂肪合成关键调控因子SREBF1、PPARG、PPARGC1A和脂肪酸合成相关基因ACC、FAS、SCD的mRNA表达(P<0.05)。由此可见,β-谷甾醇能通过调节小鼠乳腺上皮细胞乳蛋白和乳脂肪合成相关基因的表达,进而促进乳腺细胞乳蛋白和乳脂肪的合成。

穿刺冲洗术治疗哺乳期乳腺脓肿182例疗效观察及治疗后哺乳结局分析

目的 探讨哺乳期乳腺脓肿经超声引导下穿刺冲洗术治疗效果,并分析影响治疗后病人哺乳结局的相关因素。方法收集2017年1月至2020年12月郑州大学第三附属医院收治经超声引导下穿刺冲洗术治疗的182例哺乳期乳腺脓肿病人的临床资料,哺乳结局根据治疗后是否回奶,分为回奶组27例和继续母乳喂养组155例。从产次、产后时间(产后初次发病时间)、脓肿位置、脓肿长径、致病菌、穿刺次数等方面,采用χ^(2)检验进行单因素分析,采用logistic回归模型进行哺乳结局的多因素分析。结果 本研究纳入182例病人,其中165例经过超声引导下穿刺冲洗术联合抗生素治疗后达到治愈标准,治愈率90.7%,治疗后回奶病人27例,回奶率14.8%(27/182)。单因素分析结果显示,哺乳结局与时间、脓肿位置、穿刺次数有相关性(均P<0.05);多因素分析结果显示,中央区脓肿[OR=11.61,95%CI:(2.70,49.85),P=0.001]、脓腔≥5 cm[OR=3.60,95%CI:(1.12,11.51),P=0.031]和穿刺次数[OR=10.08,95%CI:(2.04,49.60),P=0.004]是影响病人治疗后回奶的独立危险因素,产后时间长[OR=0.08,95%CI:(0.02,0.34),P=0.001]是病人治疗后回奶的保护性因素。结论 超声引导下穿刺冲洗术治疗哺乳期乳腺脓肿效果良好,产后时间≤42 d、中央区脓肿、脓腔≥5 cm、增加穿刺次数更容易导致病人回奶。

奶牛乳房炎相关IRF5基因CDS区克隆及表达分析

【目的】研究奶牛干扰素调节因子5(Interferon regulatory factor 5,IRF5)基因在奶牛乳腺组织及乳腺上皮细胞炎症中的表达模式。【方法】利用RT-PCR和测序技术克隆得到IRF5基因的编码区(Coding sequence,CDS)序列,并采用生物信息学方法分析IRF5基因及其特性;利用qPCR技术检测IRF5基因及相关干扰素调节因子3(Interferon regulatory factor 3,IRF3)和MyD88基因的表达量。【结果】IRF5基因的CDS长度为1500 bp,编码499个氨基酸,存在47个潜在磷酸化位点,氨基酸组成中脯氨酸(Pro)和亮氨酸(Leu)所占比例较高。IRF5蛋白分子式为C_(2524)H_(3901)N_(671)O_(728)S_(20),主要存在于细胞核和线粒体,理论等电点为5.38,是碱性亲水且不稳定的非分泌蛋白。蛋白互作分析结果显示,IRF5蛋白可能与骨髓分化初级反应蛋白(MyD88)、白细胞介素-1受体相关激酶1(IRAK1)、C-C基序趋化因子4(CCL4)、组蛋白乙酰转移酶p300(EP300)和核受体共激活因子3异构体X1(NCOA3)等蛋白相互作用,与机体或细胞的炎症反应有关。qPCR结果表明,与健康奶牛相比,IRF5基因在乳房炎奶牛的乳腺组织中的表达量极显著上调(P<0.01)。在乳腺上皮细胞中,与对照组(0 h)相比,IRF5及其相关的Myd88和IRF3基因在LPS诱导乳腺上皮细胞炎症反应的3、6和12 h的表达量均极显著上调(P<0.01)。【结论】IRF5基因在奶牛乳房炎过程中表达量上调,可能通过上述基因参与促进奶牛乳房炎症反应。

水牛ACSL基因家族全基因组及表达分析

为鉴定水牛长链脂酞辅酶A合成酶(long-chain fatty Acyl-CoA synthetases,ACSLs)的家族成员并探究该基因家族在泌乳方面的功能,通过生物信息学技术对水牛ACSL基因家族进行了motif、基因结构分析、共线性分析、不同物种系统进化分析,运用qPCR技术检测ACSLs在不同组织的mRNA表达量。结果表明:水牛ACSL基因家族共有5个家族成员并分别命名为bbu.ACSL1、bbu.ACSL3、bbu.ACSL4、bbu.ACSL5和bbu.ACSL6。染色体分布情况结果显示:bbu.ACSL1位于1号染色体,bbu.ACSL3位于2号染色体,bbu.ACSL4位于25号染色体,bbu.ACSL5位于23号染色体,bbu.ACSL6位于9号染色体。这5个ACSL氨基酸数在699~722 aa,所有蛋白的等电点除ACSL6外均大于7.00。Motif分析发现,除ACSL3和ACSL4缺少motif8外,其余均含有预测出的Motif 1~10,且排序相同。物种内共线性分析结果显示,水牛和普通牛中均不存在串联重复和片段重复,种间共线性分析结果显示,水牛ACSL与普通牛ACSL存在4对片段重复基因。不同物种ACSL系统发育分析显示,ACSL基因家族具有较高的保守性,其中水牛与普通牛和牦牛的聚类更为相近。不同组织表达量分析发现ACSL1、ACSL3在乳腺组织的mRNA表达量高于其他组织,ACSL6在大脑的mRNA表达量高于其他组织,ACSL4在肺部的mRNA表达量高于其他组织,ACSL5在肝的mRNA表达量高于其他组织。根据RNA-seq测序结果分析不同泌乳时期表达量结果显示,ACSL1在整个泌乳期均有高表达量,尤其是在泌乳第50、140和280天。ACSL3和ACSL5在泌乳第7天和第50天高表达,但随着泌乳天数增加,表达量降低。ACSL4和ACSL6在泌乳第7天表达量最高,但在整个泌乳期的表达量均远低于该家族的其他3个成员。

重组克柔念珠菌14-3-3蛋白诱导奶牛乳腺上皮细胞炎症反应的分子机制

旨在研究重组克柔念珠菌(Candida krusei)14-3-3蛋白(rCK14-3-3)对奶牛乳腺上皮细胞(MAC-T)炎性反应的分子机制,为系统研究克柔念珠菌损伤奶牛乳腺上皮细胞的机制提供理论依据。采用原核表达的方法构建克柔念珠菌BMH 1基因重组质粒并转化入大肠杆菌BL21(DE3)中,经诱导表达纯化后进行Western blot鉴定;CCK-8法筛选rCK14-3-3蛋白对MAC-T的最适作用浓度和时间;Western blot检测rCK14-3-3蛋白对MAC-T的Toll样受体、MAPK信号通路、NF-κB信号通路主要蛋白相对表达量的影响;ELISA检测rCK14-3-3蛋白对MAC-T中相关炎性因子IL-1β、IFN-γ、IL-18、TNF-α、IL-6、IL-12相对表达量的影响。结果显示:成功构建了pET-21a-BMH1重组表达载体,经诱导表达纯化后获得有生物学活性的rCK14-3-3蛋白,纯度达90%以上,浓度为0.2 mg·mL^(-1)。与空白对照组相比,采用50μg·mL^(-1)的rCK14-3-3作用MAC-T,TLR2、TLR4、MyD88在1、3 h时表达量极显著升高(P<0.01),6 h时,TLR4表达量显著降低(P<0.05),TLR2、MyD88表达量无显著变化;p-JNK在1 h时的表达量无显著变化,3、6 h时极显著降低(P<0.01);p-ERK在1、3、6 h时的表达量显著或极显著升高(P<0.05,P<0.01);p-p38在1 h时的表达水平极显著降低(P<0.01),3、6 h时极显著升高(P<0.01);p-IκB-α、p-p65在1、3、6 h时的表达量均极显著升高(P<0.01)。IL-1β、IFN-γ、IL-18、TNF-α在作用1、3、6 h后均呈现出显著或极显著的上调趋势(P<0.05,P<0.01);IL-6在作用1、3 h后极显著上升(P<0.01),6 h后显著降低(P<0.05);IL-12在作用1 h后极显著上升(P<0.01),3、6 h后极显著降低(P<0.01)。本研究成功表达具有生物学活性的rCK14-3-3蛋白,该蛋白可通过TLR2/4-MAPK/NF-κB信号通路诱导MAC-T发生炎性反应,rCK14-3-3蛋白可作为引起MAC-T炎症的重要因子之一。

大肠杆菌型奶牛乳房炎对产奶性状相关基因表达的影响

旨为从基因水平上探究大肠杆菌(Escherichia coli)型乳房炎奶牛的产奶量和乳品质下降的原因。本实验利用脂多糖(LPS)诱导乳腺上皮细胞(bMECs)建立细胞炎症模型,并采用实时荧光定量PCR技术检测了奶牛产奶量与乳蛋白相关基因(CSN1S1、CSN2和LALBA)、乳脂调控相关基因(FABP3和LPL)以及炎症与产奶量相关基因MFGE8在奶牛乳腺组织和bMECs炎症反应中的表达量。结果表明,与对照组(0 h)相比,上述6个基因在E.coli型乳房炎奶牛的乳腺组织中表达量均极显著下调(P<0.01)。此外,上述6个基因在LPS诱导bMECs产生炎症反应的3、6、12和24 h的bMECs中表达量也均极显著下调(P<0.01)。在E.coli型奶牛乳房炎发生或bMECs产生炎症后,上述6个基因的表达水平均受到抑制,解释了E.coli型乳房炎导致产奶量减少、乳蛋白率和乳脂率下降的原因。

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.

Microarray analysis of gene expression profiles in the bovine mammary gland during lactation

Mammary glands undergo functional and metabolic changes during virgin,lactation and dry periods.A total of 122 genes were identified as differentially expressed,including 79 up-regulated and 43 down-regulated genes during lactation compared with virgin and dry periods.Gene ontology analysis showed the functional classification of the up-regulated genes in lactation,including transport,biosynthetic process,signal transduction,catalytic activity,immune system process,cell death,and positive regulation of the developmental process.Microarray data clarified molecular events in bovine mammary gland lactation.