利用亮甲酚蓝染色法预测牛卵母细胞发育潜能的研究

利用亮甲酚蓝(Brilliant cresyl blue,BCB)对牛未成熟卵母细胞进行染色,根据染色结果的不同对牛未成熟卵母细胞进行分组培养,观察不同组间卵母细胞发育潜能与细胞凋亡的差异,探讨以BCB着色判定牛卵质量的可行性.本试验在牛卵母细胞成熟培养前用26μmol·L-1BCB染色90 min作为处理组(BCB+和BCB-),以未染色卵母细胞作为对照组;以卵内谷胱甘肽的浓度作为胞质成熟的判定指标,以卵母细胞体外受精后囊胚率作为卵子发育能力的判定指标,同时参照卵母细胞的凋亡检测,综合评定卵子的质量.结果表明:(1)着色组(BCB+)卵母细胞成熟率与对照组和无色组(BCB-)间差异显著(P＜0.05);(2)成熟卵母细胞中谷胱甘肽的浓度较培养前差异显著(P＜0.05);(3)经BCB染色,卵母细胞在成熟后,BCB+组的成熟率、凋亡率较对照组、BCB-组差异显著(P＜0.05);(4)体外受精后,BCB+组的囊胚率和囊胚细胞数较对照组和BCB-组差异显著(P＜0.05).由此可见,牛卵母细胞经BCB的染色选择可以作为判断卵母细胞未来发育潜能的一个标记.     

肠道氨基酸和小肽转运载体的基因表达、影响因素与分子调控机制

饲粮蛋白质在瘤胃或肠道被降解成多肽,并进一步被分解成氨基酸和小肽,然后被吸收、利用。肠道氨基酸的转运受多种氨基酸转运载体,如中性、酸性和碱性氨基酸转运载体等的调节,小肽的转运则由小肽转运载体1介导。目前,对氨基酸及小肽转运载体基因的表达和功能调节相关的分子机制还不清楚,有待于进一步研究。本文综述了动物小肠肽与氨基酸转运载体等,重点介绍了其基因表达调节的分子机制、影响因素以及营养调控方面的研究进展。     

Efficient Transgene Expression in IVF and Parthenogenetic Bovine Embryos by Intracytoplasmic Injection of DNA–Liposome Complexes

Transgenic animals constitute an important tool with many biotechnological applications. Although there have been advances in this field, we propose a novel method that may greatly increase the efficiency of transgenic animal production and thereby its application. This new technique consists of intracytoplasmic injection of liposomes, in bovine oocytes and zygotes, to introduce exogenous DNA. In the first experiment, we evaluated embryo development and EGFP expression in In Vitro Fertilization (IVF) embryos injected with different concentrations of exogenous DNA–liposome complexes (0.5, 5, 50, 500 ng pCX‐EGFP/μl). The highest EGFP‐embryos rates were obtained using 500 ng pCX‐EGFP/μl. In the second experiment, we evaluated embryo development and EGFP expression following the injection of DNA–liposome complexes into pre‐fertilized oocytes and presumptive zygotes, 16 and 24 h post‐fertilization. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp–liposome was injected 16 h post‐fertilization. The percentages of positive embryos for the 24‐h post‐fertilization and pre‐fertilization groups were 30.1 and 6.3, respectively. Blastocysts that developed from injected zygotes were analysed by PCR, confirming the presence of transgene in all embryos. Finally, we examined the embryo development and EGFP expression of parthenogenetic embryos that resulted from the injection of egfp–liposome complexes into pre‐activated oocytes, and 3 and 11 h post‐activated oocytes. The group with the highest expression rate (48.4%) was the one injected 3 h post‐activation. In summary, this study reports the efficient, reproducible and fast production of IVF and parthenogenetic embryos expressing EGFP, by the intracytoplasmic injection of liposomes to introduce the foreign DNA.     

牛乳铁蛋白肽基因LfcinB的合成及真核表达载体的构建

根据APD抗菌肽数据库报道的牛乳铁蛋白肽LfcinB氨基酸序列,合成编码LfcinB基因的2条互补的寡核苷酸链,退火后在5′端和3′端分别形成含有BamH I和Xho I位点粘性末端的双链DNA.真核表达载体pcDNA3.1(+)经Bam H I和Xho I限制性内切酶双酶切处理后与合成的LfcinB基因进行连接,连接产物转化E.coli DH5α感受态细胞,提取质粒DNA进行测序.测序结果显示,牛乳铁蛋白肽LfcinB基因成功克隆到pcDNA3.1(+)真核表达载体中,为进一步表达和抗菌活性研究奠定物质基础.     

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。     

营养物质对基因表达的调控作用

本文阐述了营养物质对基因表达调控的作用机理，营养物质对基因表达的调控途径以及其在动物生产中的应用前景。     

Antioxidative Effects of Astaxanthin against Nitric Oxide‐Induced Oxidative Stress on Cell Viability and Gene Expression in Bovine Oviduct Epithelial Cell and the Developmental Competence of Bovine IVM/IVF Embryos

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co‐culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide‐induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl‐2, Caspase‐3 and Bax) by RT‐PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co‐cultured with BOEC pre‐treated with astaxanthin (500 μm ) in the presence or absence of sodium nitroprusside (SNP, 1000 μm ) for 24 h. Cell viability in BOEC treated with SNP (50–2000 μm ) lowered, while astaxanthin addition (50–500 μm ) increased it in a dose‐dependent manner. Cell viability in astaxanthin plus SNP (1000 μm ) gradually recovered according to the increase in astaxanthin additions (100–500 mm ). The LPO in astaxanthin group (50–500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT‐PCR. Bcl‐2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase‐3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6–7 days under co‐culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μm astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.     

Expression of Cyclins and Cyclin‐Dependent Kinase Inhibitors in Granulosa Cells from Bovine Ovary

The regulation of granulosa cell proliferation is complex, and it is essential for normal follicular development in mammals. The aim of this study was to examine the expression of cyclins and their inhibitors in the granulosa cells of follicles at different developmental stages. Follicles were classified into three groups: oestrogen‐inactive dominant follicles (EIDs), oestrogen‐active dominant follicles (EADs) and pre‐ovulatory follicles (POs). The expression of CCND2 (cyclin D2) mRNA was significantly higher in granulosa cells from EADs and POs than in those from EIDs. The expression of CCND3 (cyclin D3) mRNA was significantly higher in granulosa cells from EADs than in those from other follicles. CCND1 (cyclin D1), CCNE1 (cyclin E1) and CCNE2 (cyclin E2) mRNA expression did not differ among the different follicular stages. The expression of CDKN1A (p21^cip1) and CDKN1B (p27^kip1) mRNA was significantly higher in granulosa cells from EIDs and POs, respectively, than in those from other follicles. Expression of CDKN2D (p19^INK4d) mRNA did not differ among the different follicular stages. Taken together, our study suggested that cyclins and their inhibitors are associated with granulosa cell proliferation at specific follicular developmental stages.     

秦川牛CAP2基因CDS区克隆及表达谱分析

[目的]克隆秦川牛CAP2基因的编码区序列（CDS）,分析该基因在不同组织及原代脂肪细胞分化过程中的表达特征。[方法]采用RT-PCR方法扩增秦川牛CAP2基因的CDS区,运用ProtPram、TMpred、ProtFun 2.1 Server等在线网站进行CAP2蛋白的生物信息学分析;通过油红O染色和qPCR检测成脂标志基因PPARγ和FABP4基因的表达水平以构建牛原代脂肪细胞诱导分化体系;利用qPCR检测分析CAP2基因在秦川牛7种组织（心、肝、脾、肺、肾、肌肉和背脂）和原代脂肪细胞分化过程（0~10 d）中的表达。[结果]秦川牛CAP2基因CDS区长1 461 bp,编码486 个氨基酸,氨基酸序列主要由无规卷曲和α-螺旋构成。CAP2基因在脂肪组织中高表达,极显著（P<0.01）高于其他组织。牛原代脂肪细胞诱导分化过程中,PPARγ和FABP4基因的表达量逐渐上升,与第0天相比第10 天时表达量达到最大（P<0.01）;与对照组相比,诱导分化组脂滴积累明显增加;CAP2基因表达量也随时间推移逐渐上升,以0 d为对照,第10天表达量最高（P<0.001）。[结论]成功克隆了秦川牛CAP2基因全长1 461 bp的编码区;CAP2基因在牛脂肪组织中以较高水平表达,且CAP2基因可能参与脂肪生成与分化过程,预测CAP2基因可能是促进成脂分化的转录因子,对维持牛脂肪细胞状态发挥关键作用,可能作为秦川牛肉质性状的候选基因,该研究结果为进一步揭示牛CAP2基因的功能提供基础资料。     

牛流行热病毒糖蛋白基因的原核表达与抗原性鉴定

为探索可用来开发牛流行热病毒(bovine ephemeral fever virus,BEFV)疫苗和诊断试剂的候选基因,本研究针对BEFV糖蛋白(G)基因设计了2对特异性引物,用PCR方法扩增基因片段,PCR产物经Xho Ⅰ和Nde Ⅰ双酶切后亚克隆到表达载体pET-30上,将鉴定正确的重组质粒(480-pET-30、1107-pET-30)转化大肠杆菌BL21(DE3)感受态细胞,培养阳性菌株D_{600 nm}值为0.6~1.0,37 ℃下用1.0 mmol/L IPTG诱导表达目的蛋白,将其纯化之后进行SDS-PAGE和Western blotting免疫原性分析。同时,应用间接ELISA、动物免疫试验及交叉反应试验对目的蛋白进行分析。结果表明,首次发现的1 107 bp基因片段是分段表达的,具有很好的生物活性和特异性,更适合作为开发疫苗和诊断试剂的候选基因,为今后建立BEFV的血清学诊断方法及疫苗研发提供了理论基础。     

Effects of Lysine Dipeptides on the Expression of Genes Related to Milk Protein Synthesis and Amino Acid Transporters in Bovine Mammary Tissue

The study was carried out to investigate the effects of various lysine (Lys) dipeptides on gene expression related to milk protein and amino acid transporters in bovine mammary tissue.Mammary tissues were obtained from midlactating Holstein dairy cows and cultured under lactogenic hormones (prolactin, hydrocortisone and insulin) with different ratios of various Lys-peptide (Lys-Lys (KK), Lys-His (KH), Lys-Phe (KF), Lys-Leu (KL)and Lys-Thr (KT)).They isometrically substituted 0, 5%, 10%, 15% and 20% of free Lys (total concentration of Lys was 210 μg/mL), respectively.After 48 h treatment, mammary tissues were harvested for gene expression analysis by Real-time PCR.The results showed that:Compared with the free Lys group with no Lys substitution, when the KH replaced Lys at ratio of 15%, the KF replacement ratio was 10%, KK, KL and KT replacement ratio was 5%, the CSN1S1 mRNA abundance were the highest (P<0.05);When the KK replaced Lys at ratios of 5%, 10%, 15% and 20%, KH replacement ratios were 10% and 15%, KF replacement ratio was 15%, KL replacement ratios were 5% and 20% and KT replacement ratios were 5%, 10% and 15%, the SLC15A2 gene expression was significantly enhanced (P<0.05); when the KH replaced lysine at ratio of 15%, KK and KL replacement ratio was 10%, KF and KT replacement ratio was 5%, the abundance of SLC6A14⁺ mRNA was the highest (P<0.05);When the KH replaced Lys at ratio of 15% and 20%, KK replacement ratios were 5%, 10% and 15%, KL replacement ratio was 5% and 10%, the mRNA abundance of mTOR was significantly increased (P<0.05).The results indicated that the Lys dipeptides might be able to promote amino acid uptake and milk protein related gene expression in bovine mammary gland tissues.     

Insulin‐Like Growth Factor‐1 Regulates the Expression of Luteinizing Hormone Receptor and Steroid Production in Bovine Granulosa Cells

Luteinizing hormone LH plays important roles in follicular maturation and ovulation. The effects of LH are mediated by LH receptor (LHR) in the ovary. However, the factors that regulate the expression of LHR in bovine granulosa cells (GCs) are not well known. Insulin‐like growth factor‐1 (IGF‐1) is known to play a key role in the acquisition and maintenance of functional dominance. To better understand the roles of LHR expression and IGF‐1, we conducted three experiments to determine (i) mRNA expression of LHR in the GCs of developing follicles, (ii) the effects of IGF‐1 on LHR mRNA expression in cultured GCs and (iii) the effects of IGF‐1 on estradiol (E2), progesterone (P4) and androstenedione (A4) production by non‐luteinized GCs. In experiment 1, small follicles (<6 mm Ø) expressed lower levels of LHR than mid‐sized follicles (6–8 mm Ø) and large follicles (≥9 mm Ø) expressed the highest levels of LHR mRNA (p < 0.05). In experiment 2, IGF‐1 (1 and 100 ng/ml) increased (p < 0.05) the expression of LHR mRNA in GCs from small and large follicles. In experiment 3, IGF‐1 (0.1–100 ng/ml) increased A4 and E2 in GCs from both small and large follicles but increased P4 only in large follicles. IGF‐1 in combination with LH (0.1 and 1 ng/ml) increased P4 and A4 in large follicles, and increased E2 and A4 in GCs of small follicles. These findings strongly support the concept that IGF‐1 upregulates LHR mRNA expression as well as A4 and E2 production in GCs and that IGF‐1 is required for determining which follicle becomes dominant and acquires ovulatory capacity.     

牛乳头瘤病毒2型L1基因的原核表达及多克隆抗体的制备

试验旨在表达牛乳头瘤病毒（BPV）的L1衣壳蛋白,并制备其多克隆抗体。对疑似牛乳头状瘤的病料进行病理组织学检查,采用L1基因简并引物FAP59/FAP64进行扩增,然后设计特异性引物,将衣壳蛋白L1基因亚克隆至pET-30a （+）表达载体中,构建重组原核表达载体pET30a-BPV2-L1,在E.coli Rosetta^TM（DE3） pLysS宿主菌中表达重组蛋白rBPV2-L1,并以纯化的rBPV2-L1蛋白为免疫原制备兔抗BPV2-L1蛋白多克隆抗体,随后间接ELISA检测其效价,并进行间接免疫荧光试验分析。病理组织切片观察结果显示,病变部分表皮细胞增生、角质过度并出现细胞挖空等BPV感染的组织病变情况;序列分析确定BPV分离株的基因型为2型,L1基因编码区长1 494 bp,可编码一个含有497个氨基酸的衣壳蛋白,蛋白分子质量为55.5 ku;与各型BPV的L1氨基酸序列系统进化树分析结果显示,其与BPV2-SW01（GenBank登录号：KC878306.1）、BPV2（GenBank登录号：KX113620.1）处于同一遗传进化分支,且属于Delta属成员;诱导表达的rBPV2-L1蛋白主要以包涵体形式存在于沉淀中;间接ELISA法检测多克隆抗体效价高达1：163 840,间接免疫荧光分析表明,兔抗BPV2-L1多克隆抗体能与瞬时表达的BPV2-L1蛋白发生特异性反应。以上结果证实,本研究成功克隆、表达了BPV2 L1基因,且重组蛋白rBPV2-L1具有较好的免疫原性,所制备的BPV2-L1蛋白多克隆抗体具有良好的免疫活性,为进一步研究BPV2亚单位疫苗奠定基础。     

Pre‐ and Post‐Partum Mild Underfeeding Influences Gene Expression in the Reproductive Tract of Cyclic Dairy Cows

Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post‐partum. This study investigated the effects of pre‐ and post‐partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post‐partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post‐partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi‐ and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post‐partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post‐partum patterns of IGF‐1, insulin, β‐hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid‐related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post‐partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post‐partum dairy cows.     

Goat cathelicidin‐2 is secreted by blood leukocytes regardless of lipopolysaccharide stimulation

It has been reported that goat cathelicidin‐2, an antimicrobial peptide, localizes in leukocytes and is present in milk. Here, we examined whether cathelicidin‐2 is secreted by leukocytes. Different concentrations (10⁵–10⁸ cells/mL) of blood leukocytes were cultured for 0–48 h with or without lipopolysaccharide (LPS). After culture, the concentrations of cathelicidin‐2 in the conditioned media were measured. Blood was collected from male goats 0–24 h after the intravenous injection of Escherichia coli O111:B4 LPS. The plasma cathelicidin‐2 concentrations were determined and the blood leukocytes immunostained with anti‐cathelicidin‐2 antibody to calculate the proportion of cathelicidin‐2‐positive cells in the total leukocytes. When higher concentrations of leukocytes were cultured, the cathelicidin‐2 concentrations in the media increased significantly, whereas the addition of LPS to the media caused no further increase. The plasma cathelicidin‐2 concentrations did not increase with time after LPS infusion. The proportion of cathelicidin‐2‐positive cells in the total leukocytes was significantly reduced 1 h after LPS injection compared with that at 0 h, but increased again at 6 h and thereafter. These results suggest that cathlicidin‐2 is secreted by leukocytes even without LPS stimulation, whereas LPS may be required for cathelicidin‐2‐containing leukocytes to be recruited from the blood to tissues showing inflammation.