肿瘤坏死因子(TNFα)与妊娠

TNFα对妊娠既有正调节作用又有负调节作用 ,这些作用随着妊娠各时期靶细胞的类型不同甚至同一类型在不同动物而显示出明显的差异。妊娠中 ,适量的 TNFα可以促进分解代谢 ,满足胎儿能量需要 ,也可能参与胎儿和胎盘组织的生长分化调节 ,在保护胎儿作为同种异体自身宿主的复杂免疫反应中 ,与胎儿胎盘分泌的其它多肽性激素一起发挥作用。过量的 TNFα则严重影响胎儿的正常生长 ,或造成胚胎丢失及流产。本文从子宫局部 TNFα的来源 ,TNFα合成的调节 ,TNFα对妊娠的影响等方面综述了该领域的研究进展。     

用ELISA检测伊氏锥虫血清中TNF的研究

本文分别用鼠抗TNF单克隆抗体T_5和E_6,通过ELISA对伊氏锥虫阳性血清中TNF进行了检测。在受检的164份血清中,T_5检测出现28份阳性血清。E_6检测出现22份阳性血清。两种单抗检测后均为阳性反应的有21份血清,其阳性检出率为12.80%。阳性血清中TNF的含量平均为180ng/ml,范围在0.36ng/ml—1600ng/ml之间。结果首次证实伊氏锥虫感染时TNF的存在,对进一步研究伊氏锥虫的免疫及免疫病理具有重要意义。     

pCMV－TNFα的构建及其在动物细胞中的表达

以ＸｂａⅠ和ＥｃｏＲＶ双酶消化ｐＢＴ１获得肿瘤坏死因子α（ＴＮＦα）ｃＤＮＡ基因片段，以ＸｂａⅠ和ＳｍａⅠ酶切点定向克隆入质粒载体ｐＣＭＶ４，构建了人ＴＮＦα重组质粒ｐＣＭＶ－ＴＮＦα。采用ＤＮＡ—磷酸钙共沉淀法转染ＣＯＳ７和ＮＩＨ３Ｔ３细胞，收集转染后不同时间的细胞培养上清，检测ＴＮＦα的表达水平和生物学活性。ＥＬＩＳＡ检测表明，两种细胞转染后，其培养上清中均有ＴＮＦα抗原存在，而载体等对照则测不出ＴＮＦα。以Ｌ９２９细胞检测其细胞毒活性，发现ＣＯＳ７细胞于转染后４８ｈ表达水平最高，活性高达１６Ｕ／ｍＬ以上，但７ｄ时测不出ＴＮＦα活性；ＮＩＨ３Ｔ３细胞于转染后２４ｈ活性最高（８Ｕ／ｍＬ），９ｄ时尚有表达，但不足１Ｕ／ｍＬ。     

pCMV—TNFα的构建及其在动物细胞中的表达

以Ｘｂａ Ｉ和Ｅｃｏ ＲＶ双酶消化ｐＢＴ１获得肿瘤坏死因子α（ＴＮＦα）ｃＤＮＡ基因片段，以Ｘｂａ Ｉ和Ｓｍａ Ｉ酶切点定向克隆入质粒载体ｐＣＭＶ４，构建了人ＴＮＦα重组质粒ｐＣＭＶ－ＴＮＦα。采用ＤＮＡ－磷酸钙共沉淀法转染ＣＯＳ７和ＮＩＨ３Ｔ３细胞，收集转染后不同时间的细胞培养上清，检测ＴＮＦα的表达水平和生物学活性。ＥＬＩＳＡ检测表明，两种细胞转染后，其培养上清中均有ＴＮＦα抗原存在，而载体等     

棘腹蛙TNFα基因的克隆与序列分析

为了解棘腹蛙TNFα基因的相关信息,对棘腹蛙的TNFα基因进行了克隆和序列分析。克隆了全长为936 bp、ORF长度为675 bp的TNFα-Pb基因;对该基因的蛋白质结构分析显示,TNFα-Pb的信号肽由44个氨基酸组成,随后由2个α螺旋,和10个β折叠形成小分子蛋白;系统进化分析发现,TNFα-Pb与两栖类聚为一枝,与高等哺乳动物存在功能分化的可能。本研究为了解棘腹蛙的生物反应调节机制奠定了一定基础。     

TNF—a的生物活性及其在肝脏损伤中的作用

早期发现ＴＮＦ－ａ具有杀死肿瘤或抑制肿瘤增殖的作用。后来发现ＴＮＦ－ａ具有广泛的生物活性，既可参与机体的免疫功能的调节，又在感染、组织损伤、炎症、休克等方面超重要介导作用；近年来还发现ＴＮＦ－ａ在中枢神经系统以及许多生物因子诱导的肝损伤过程中也起重要作用。     

Transfer of tumour necrosis factor-α via colostrum to foals

This study aimed to determine whether TNF-α is transferred to equine neonates via colostrum and the relationship between TNF-α and IgG concentrations in the equine neonate. Colostrum, presuckle and postsuckle foal serum samples were collected from healthy mares and their foals. Equine TNF-α ELISA and IgG SRID kits were used to determine the concentrations of TNF-α and IgG, respectively. Statistical analysis was performed using the Spearman rank correlation. TNF-α concentrations in all presuckle foal serum were below the limit of detection in 15/16 foals and increased in postsuckle foal serum to a mean concentration of 7.7 x 10(4) pg/ml. TNF-α concentrations in postsuckle foal serum and colostrum showed significant correlation (rho=0.668; P=0.005). However, TNF-α and IgG concentrations in colostrum or postsuckle foal serum did not correlate (rho<-0.016; P>0.05). Ratios of TNF-α/IgG in colostrum or postsuckle foal serum showed significant correlation (rho=0.750; P=0.0008). These results indicate that TNF-α is transferred to the foal via colostrum absorption and may play a role in early immunity.     

Antiviral activity of rChIFN-α against vesicular stomatitis virus and Newcastle disease virus: a novel recombinant chicken interferon-α showed high antiviral activity

The sequences of 27 chicken interferon-alpha (ChIFN-α) genes were obtained from GenBank. The gene sequences were compared and homology between them was determined by using a bio-software. On the basis of these results, a new rChIFN-α peptide sequence with 194 amino acids was assembled. Thereafter, on the basis of the new amino acid sequences and by using the most frequently occurring codes of Pichia pastoris, and a 582 bp gene sequence was formed. In order to amplify this non-templated gene, 16 primers were designed, and their gene sequences were synthesized, and amplified. This amplified gene sequence was cloned into the expression vector pPICZα-A to construct a recombination plasmid named pPICZ-rChIFN-α. Then, the recombination plasmid was induced to express the rChIFN-α protein. The results demonstrated that the recombinant plasmid pPICZ-rChIFN-α was successfully expressed in P. pastoris. Furthermore, rChIFN-α had a considerable antiviral activity against both Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV). Therefore, this method of gene engineering could give direction to research on the key amino acids in the interferon or analogous proteins and enable the construction of proteins with high antiviral activity, which can be used both for research and industrial purposes.     

番鸭IFN-α mRNA实时荧光定量RT-PCR检测方法的建立

根据GenBank中鸭IFN-α基因(GenBank登录号:JF894229)序列设计并合成引物,并以鸭β-actin基因(GenBank登录号:GU564232)为内参,采用SYBR Green I染料法,建立检测鸭IFN-αmRNA的实时荧光定量Real-time PCR方法(RT-PCR)。将IFN-α和β-actin基因分别克隆至pMD18-T载体上,鉴定出阳性重组质粒为标准品(p-IFN-α和p-β-actin),分别建立番鸭IFN-α和β-actin实时荧光定量RT-PCR方法。结果表明,Ct值在13.27~27.54与11.86~25.32范围内呈现良好的线性关系,相关系数均大于0.99(r>0.99),扩增产物的熔解曲线分析均各自只出现1个特异性单峰,无引物二聚体,Tm值分别为(93.6±0.26)℃和(85.3±0.15)℃,最低检测限分别为9.37×102copies/μL和3.71×101copies/μL,检测周期从样品的处理到荧光定量PCR结束仅需4 h左右。本研究建立的鸭IFN-α基因实时荧光定量PCR灵敏度高、特异性强、检测周期短,为番鸭IFN-αmRNA的定量分析奠定了基础。     

小鼠输卵管、子宫和卵巢组织中的TGF-α mRNA活性

采用ＲＴ－ＰＣＲ技术分别检测了排卵期和怀孕１￣５ｄ小鼠输卵管、子宫及卵巢组织中的转化生长因子－α（ＴＧＦ－α）ｍＲＮＡ活性。结果发现,这一期间内小鼠输卵管、子宫及卵巢中均有较强的ＴＧＦ－αｍＲＮＡ表达,其中输卵管和卵巢中的表达量变化不大,而子宫中的表达量在怀孕５ｄ时明显减少,这进一步证外源（旁分泌）ＴＧＦ－α在早期胚胎发育中可能起重要作用。     

Prostaglandin F2α metabolite levels in normal and uterine-infected postpartum cows

The stable metabolite of prostaglandin F₂, 15 keto-13, 14-dihydroprostaglandin F₂ (PGFM), was measured from peripheral blood samples collected at specified intervals postpartum from 7 normal dairy cows and 4 cows with apparent endometritis. Plasma PFGM levels were significantly (P<.05) elevated for the first 5 days postpartum in the cows with endometritis (ranging from 4.0 to 5.0 ng/ml) compared to the controls (approximately 1.0 ng/ml). Beyond 5 days postpartum, plasma PGFM levels were not significantly different and decreased to approximately 0.4 ng/ml by day 13 in both groups. Time to uterine involution was not different between groups (less than 30 days). Therefore, uterine infections in cows during the puerperium was associated with elevated circulating PGFM levels. These findings and the observation that PGF₂ is not uterotonic in the puerperal cow do not suggest a therapeutic use of PGF₂ in order to evacuate the uterus.     

以雏鸡肝细胞为靶细胞检测鸡TNFα细胞毒活性方法的建立

鸡源性肿瘤坏死因子α(TNFα)样品检测,通常使用微量细胞病变法,操作繁琐费时,尽管国外有利用生物学方法测定的报道,但因需要特定的细胞系作为靶细胞[1],使鸡TNFα检测难以开展。然而,TNFα活性检测对鸡免疫、抗感染和抗肿瘤等方面的机制研究具有重要的意义,为克服靶细胞...     

Pro-inflammatory and pro-apoptotic responses of TNF-α stimulated bovine mammary endothelial cells

Coliform mastitis may be severe in periparturient cows due to enhanced expression of pro-inflammatory cytokines that contribute to disease pathogenesis. Tumor necrosis factor (TNF)-α is implicated with the severity of coliform mastitis by provoking inflammatory responses in affected tissues. The endothelium is an integral organ in regulating inflammatory responses and loss of endothelial integrity may be fatal. Studies in humans suggest that endothelial cell apoptosis may be a consequence of TNF-α exposure and contributes to the development of sepsis, however, its impact on bovine mammary endothelial cells (BMEC) is unknown. We sought to determine the inflammatory and apoptotic responses of primary BMEC exposed to TNF-α in vitro. Stimulation of endothelial monolayers with TNF-α resulted in significant increase of toll-like receptor 4, interleukin-6 and -8, and intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 gene expression in a time-dependent manner. Caspase-8 and caspase-3 mRNA expression, as well as caspase enzyme activity, also increased significantly following TNF-α stimulation. Cell viability assessed by ATP activity and BMEC apoptosis determined by flow cytometry revealed no significant changes across time with TNF-α stimulation. Results suggest that TNF-α stimulation, at the dose used in this study, can elicit a pro-inflammatory response in BMEC, but not induce apoptosis. The impact of TNF-α on mammary vascular function and the subsequent impact on the pathophysiology of severe coliform mastitis warrant further investigation.     

Production of prostaglandin F2α and estrogen by embryonal membranes and endometrium and metabolism of prostaglandin F2α by embryonal membranes,endometrium and lung from gilts

A 3 hr incubation of endometrium and embryonal membranes was used to assess potential contributions of these tissues to the prostaglandin F₂α (PGF₂α) and unconjugated estrogen (UE) present in the uteri of pregnant gilts. Metabolism of [³H]PGF₂α was determined during a 6 hr incubation of endometrium, lung and embryonal membranes to assess the contribution of these tissues in conversion of PGF₂α to less active forms during the estrous cycle and early pregnancy. Tissue was collected from 12 cyclic gilts on days 13, 16 or 19 and from 17 pregnant gilts on days 13, 16, 19 and 25 after the onset of estrus (day 0). Concentration of PGF₂α (ng/g of tissue) in incubation medium after incubation of endometrium at 37 C was 4- to 6-fold greater (P<.001) on days 16 and 19 for cyclic gilts than for pregnant gilts. Concentration of PGF₂α in medium after incubation of embryonal membranes recovered on days 13 and 16 was similar to that found after incubation of endometrium from cyclic gilts on days 16 and 19. Percentage of [³H]PGF₂α converted by endometrium and lung tissue to other metabolites (61.8 and 79.5%, respectively) did not differ significantly among days of the cycle or pregnancy. The percentage of [³H]PGF₂α metabolites recovered as [³H]13,14-dihydro-15-keto-PGF₂α (PGFM) for endometrium (50.3%) and for lung (64.6%) was not affected significantly by pregnancy status.Embryonal membranes recovered on days 13 and 16 converted more (P<.05) [³H]PGF₂α to other metabolites (%/μg of DNA) than embryonal membranes recovered on days 19 and 25, lung or endometrium. The % of [³H]PGF₂α metabolites recovered as [³H]PGFM for embryonal membranes increased (P<.05) from 37.4 on day 13 to 68.6 on day 25. Concentration of UE (ng/g of tissue) in medium after incubation of embryonal membranes from day 13 was about 100-fold greater than for endometrium. Concentration of UE and estrone sulfate (E₁SO₄) (ng/g of tissue) in medium after incubation was greater (P<.05) for endometrium from pregnant gilts on day 25 than that for all days of the cycle or pregnancy. Concentration of UE in medium after incubation of endometrium or embryonal membranes was not significantly affected by incubation treatment, but more E₁SO₄ accumulated in the presence of indomethacin (P<.01). These results indicate that endometrium from pregnant gilts produces less PGF₂α than that of cyclic gilts in vivo and this may contribute to the maintenance of corpora lutea. The high concentrations of PGF₂α and estradiol in uteri of pregnant gilts may originate from embryonal membranes and be converted to biologically less active forms before leaving the uterus.     

Inhibin-α immunohistochemical expression in mature and immature canine Sertoli and Leydig cells

Formalin-fixed, paraffin-embedded sections from 32 canine pairs of testes were immunohistochemically examined for Inhibin-α (INHα). Samples were subdivided into two groups (group 1, neonates; group 2, puppies and adults) and results statistically compared. Inhibin-α was significantly expressed only in Sertoli cells of neonatal testes, while in Leydig cells it was expressed without significant difference between groups. These results suggest that, in dogs, INHα expression switches from Sertoli to Leydig cells during testicular maturation and that, in adult, Leydig cells represent the main source of INHα.     

PPARα siRNA干扰对骡鸭原代肝细胞脂质代谢基因表达影响研究

为揭示PPARα基因对骡鸭肝细胞PPAR信号通路中脂质代谢基因的调控作用,试验以16日胚龄的骡鸭为材料,用Ⅳ型胶原酶法分离原代肝细胞,分离的原代肝细胞经形态学观察、糖原染色和白蛋白(ALB)检测鉴定后用于后续试验;构建4对干扰PPARα基因的siRNA,分别转染骡鸭原代肝细胞,转染12 h后用荧光显微镜检测转染效率,转染48 h后用油红O染色检测肝细胞脂质分泌情况,筛选干扰效率最高的siRNA,qRT-PCR检测该siRNA干扰PPARα基因后PPAR信号通路上脂质代谢相关基因的表达情况。结果显示:Ⅳ型胶原酶法分离的骡鸭原代肝细胞活性较好,ALB的分泌量维持在较高水平,糖原染色发现大量细胞存在双核和多核状态,干扰效率最高的PPARα-1344组与对照组相比脂质分泌量减少,原代肝细胞中LPL、FABP、FAS和ACBP基因表达量极显著下调(P0.01),SCD基因表达量显著下调(P0.05),APO-A1、CYP7A1和CPT1基因表达量差异不显著(P0.05)。研究表明:分离的骡鸭原代肝细胞纯度好、活性高,PPARα基因可能促进脂肪在肝脏中沉积,与骡鸭的肥肝形成密切相关。