中草药抗球散超微粉对柔嫩艾美耳球虫感染鸡免疫机能的影响

为探讨中草药抗球散超微粉防治鸡柔嫩艾美耳球虫(E.tenella)的作用机制,将150只20日龄供试鸡随机均分为Ⅰ组(感染给中草药组)、Ⅱ组(感染不给药组)和Ⅲ组(不感染不给药组)。Ⅰ组、Ⅱ组人工感染柔嫩艾美耳球虫,Ⅰ组于感染前1 d在饲料中添加1%的中草药抗球散超微粉,分别测定供试鸡血清免疫球蛋白IgG、IgM、IgA含量和外周血T淋巴细胞百分率,试验结束时测定脾脏和法氏囊指数。结果显示:与Ⅱ组相比,Ⅰ组的IgG、IgM、IgA抗体水平整体上呈上升趋势,IgG抗体水平于感染后13、17、21 d分别显著提高10.06%、14.78%、10.63%,IgM抗体水平于感染后13 d显著提高18.18%,IgA抗体水平于感染后17 d显著提高17.95%;Ⅰ组的外周血T淋巴细胞百分率于感染后14、21 d分别显著提高7.20、6.20个百分点;试验结束时,Ⅰ组的脾脏指数和法氏囊指数分别极显著提高46.72%和43.82%。以上结果表明,中草药抗球散超微粉能够促进柔嫩艾美耳球虫感染鸡免疫器官发育、增强机体免疫功能。     

蛋氨酸三肽对奶牛乳腺上皮细胞酪蛋白和小肽转运载体基因表达量的影响

本试验旨在研究蛋氨酸三肽(Met-Met-Met)对奶牛乳腺上皮细胞(BMECs)酪蛋白和小肽转运载体基因表达量的影响。采用酶消化法培养的第3代奶牛乳腺上皮细胞为模型,各处理在培养基中分别添加0(对照)、40、50、60、70和80μg/mL的蛋氨酸三肽,每个处理5个重复,每个重复1个培养孔,分别培养细胞24、48和72h,检测奶牛乳腺上皮细胞的相对增殖率,整体试验重复2次,确定最佳培养时间;各处理在培养基中分别添加0(对照)、40、50、60、70和80μg/mL的蛋氨酸三肽,每个处理3个重复,每个重复1个培养孔,以最佳培养时间培养,用实时定量PCR法检测酪蛋白基因的表达量,确定适宜蛋氨酸三肽浓度,整体试验重复3次;以最佳培养时间和适宜蛋氨酸三肽浓度培养细胞,以未添加蛋氨酸三肽的培养基为对照,每个处理3个重复,每个重复1个培养孔,测定小肽转运载体基因的表达量,整体试验重复3次。结果表明:在培养基中添加蛋氨酸三肽培养奶牛乳腺上皮细胞24h时,相对增殖率最高;培养基中加入60μg/mL的蛋氨酸三肽培养细胞24h,αs1-酪蛋白和β-酪蛋白的基因表达量最高,同时发现奶牛乳腺上皮细胞中小肽转运载体1和小肽转运载体2基因表达量显著高于对照处理(P0.05)。综上所述,培养基中添加60μg/mL的蛋氨酸三肽能够提高奶牛乳腺上皮细胞酪蛋白和肽转运载体基因的表达量。     

表皮生长因子调控猪肠上皮细胞中钠依赖Ⅱb型磷转运蛋白表达的细胞信号通路研究

本研究旨在探讨表皮生长因子(EGF)调控猪小肠上皮细胞IPEC-J2中钠依赖Ⅱb型磷转运蛋白(NaPi-Ⅱb)表达的分子机制。试验分别用EGF受体酪氨酸激酶抑制剂(tyrphostin AG1478)、蛋白激酶A(PKA)抑制剂(H89)、蛋白激酶C(PKC)抑制剂(k4393)、p38抑制剂(SB203580)、细胞外信号调节激酶(ERK)抑制剂(PD98059)、c-Jun氨基末端激酶(JNK)抑制剂(anisomycin)与EGF共同处理IPEC-J2细胞,利用Western blot检测相关通路蛋白及目的蛋白(NaPi-Ⅱb)的表达水平。结果显示:相较于对照组,EGF处理后NaPi-Ⅱb表达水平显著降低(P0.05);相较于无抑制剂组,EGF受体、PKA、PKC、丝裂原活化蛋白激酶(MAPK)/p38、MAPK/ERK1/2、MAPK/JNK的特异性抑制剂处理IPEC-J2后,NaPi-Ⅱb表达水平显著提高(P0.05),其中添加MAPK/ERK1/2特异性抑制剂显著降低了MAPK/ERK1/2在Tyr204位点的磷酸化水平(P0.05),添加MAPK/JNK的特异性抑制剂显著降低了MAPK/JNK1/2/3在Thr183和Tyr185位点的磷酸化水平(P0.05),说明该2组抑制剂对该通路的抑制作用是通过降低上述位点的磷酸化水平实现的。本研究结果表明EGF受体、PKA、PKC、p38、ERK和JNK均介导了EGF调控IPEC-J2细胞中NaPi-Ⅱb的表达。     

Conversion of Goat Fibroblasts into Lineage‐Specific Cells Using a Direct Reprogramming Strategy

Direct reprogramming is an efficient strategy to convert one cell type to another. In this study, due to the failure of maintaining the undifferentiated state of goat embryotic stem‐ and induced pluripotent stem‐like cells in vitro, we explored an alternative way to directly convert goat fibroblasts to lineage‐specific cells. The ‘Yamanaka factors’ was ectopically expressed in fibroblasts for a short term to situate cells in a metastable state. By culturing with lineage‐specific media for 1–2 weeks, the cardiomyocyte‐like cells and neurocyte‐like cells were generated and confirmed by the quantitative RT‐PCR and immunocytochemical staining. The metastable‐state cells could also be converted into oocyte‐like cells (OLCs) after culturing in media with retinoic acid (RA) and bovine follicular fluid (bFF) for 2–3 weeks. The generated OLCs were surrounded by cumulus granulosa cell‐like cells and formed a structure resembling goat cumulus‐oocyte complex from ovaries. This primary follicular structure could be developed further in oocyte mature medium and expressed germ cell‐specific markers. In addition, we found that the induction efficiency was higher and OLC cell size was bigger in bFF than in RA treatment. Altogether, the direct reprogramming of goat fibroblasts into lineage‐specific cells can facilitate stem cell research in domestic animals.     

Improved porcine model for Shiga toxin‐producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 10¹¹ colony‐forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum‐deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.     

Effects of melatonin on maturation,histone acetylation,autophagy of porcine oocytes and subsequent embryonic development

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1 nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1 nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle‐derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.     

Immortalization and characterization of a new canine mammary tumour cell line FR37‐CMT

Here we describe the establishment of a new canine mammary tumour (CMT) cell line, FR37‐CMT that does not show dependence on female hormonal signaling to induce tumour xenografts in NOD‐SCID mice. FR37‐CMT cell line has a stellate or fusiform shape, displays the ability to reorganize the collagen matrix, expresses vimentin, CD44 and shows the loss of E‐cadherin which is considered a fundamental event in epithelial to mesenchymal transition (EMT). The up‐regulation of ZEB1, the detection of phosphorylated ERK1/2 and the downregulation of DICER1 and miR‐200c are also in accordance with the mesenchymal characteristics of FR37‐CMT cell line. FR37‐CMT shows a higher resistance to cisplatin (IC₅₀>50 µM) and to doxorubicin (IC₅₀>5.3 µM) compared with other CMT cell lines. These results support the use of FR37‐CMT as a new CMT model that may assist the understanding of the molecular mechanisms underlying EMT, CMT drug resistance, fostering the development of novel therapies targeting CMT.     

Establishment and Application of Loop-mediated Isothermal Amplification for Rapid Detection of Porcine Epidemic Diarrhea Virus

This study was aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of porcine epidemic diarrhea virus (PEDV), and provide a simple, sensitive, accurate and reliable tool for diagnosis of PEDV.The conservative PEDV N gene (GenBank accession number: KT799997) of PEDV was selected as a target to design six specific primers.The reaction system and temperature of LAMP were optimized, and the LAMP method for specific amplification of PEDV was established. Results showed that the PEDV LAMP detection method was established successfully, and it could detect PEDV specifically at 60℃ for 60 min,and the detection limit was 91 copies/μL, which was one hundred-fold higher than conventional RT-PCR method.75 clinical samples were detected by LAMP and PCR, respectively, the coincidence of LAMP and PCR was about 97.3%. All the data suggested that the LAMP assay had strong specificity, high sensitivity, simple operation, low equipment requirement, and was suitable for rapid detection of PEDV clinical samples.