Analysis of PPP1R11 expression in granulosa cells during developmental follicles of yak and its effects on cell function

The aims of this study were to analyse the protein phosphatase 1 regulatory subunit 11 (PPP1R11) expression and cellular localization in yak follicles and investigate its effects on cell proliferation, apoptosis and oestrogen secretion in granulosa cells (GCs). Ten healthy and non-pregnant female yaks (4-year-old) were used as experimental animals. The mRNA relative expression level of PPP1R11 in GCs from small (<3.0 mm), medium (3.0–5.9 mm) and large (6.0–9.0 mm) follicles was detected by RT-qPCR, and the cellular localization of PPP1R11 protein was detected by immunohistochemistry staining (IHC). After isolation, culture and identification of yak GCs in vitro, si-PPP1R11 and si-NC (negative control) were transfected into GCs. RT-qPCR and immunofluorescence staining were used to evaluate the interference efficiency, and ELISA was performed to detect oestrogen concentration. Then, EdU staining and TUNEL staining were conducted to analyse cell proliferation and apoptosis. In addition, the oestrogen synthesis, proliferation- and apoptosis-related genes were detected by RT-qPCR after knockdown PPP1R11. The results showed that PPP1R11 is mainly located in ovarian GCs, and the expression levels of PPP1R11 in GCs from large follicles were significantly higher than that from medium and small follicles. Transfection of si-PPP1R11 into GCs could significantly inhibit the expression of PPP1R11. Interestingly, the oestrogen secretion ability and the expression level of oestrogen pathway-related genes (STAR, CYP11A1, CYP19A1 and HSD17B1) were also significantly downregulated. Moreover, the proportion of positive cells was decreased, and cellular proliferation-related genes (PCNA, CCNB1 and CDC25A) were significantly downregulated after knockdown PPP1R11. However, the proportion of apoptotic cells was increased, and apoptosis-related genes (BAX, CASP3 and P53) were significantly upregulated. Taken together, this study was the first revealed the expression and cellular localization of PPP1R11 in yak follicles. Interference PPP1R11 could reduce oestrogen secretion, inhibit proliferation and promote apoptosis in GCs, which provided a basis for further studies on the regulatory mechanism of PPP1R11 in follicle development.     

前列腺素E2和F2α对LPS刺激后奶牛子宫内膜上皮细胞COX-1和COX-2表达的影响

试验旨在阐明前列腺素E₂（prostaglandin E₂,PGE₂）和F_2α（prostaglandin F_2α,PGF_2α）对体外培养的奶牛子宫内膜上皮细胞中环氧合酶-1（cyclooxygenase-1,COX-1））与环氧合酶-2（cyclooxygenase-2,COX-2）表达的影响。培养奶牛子宫内膜上皮原代细胞和传代细胞,第4代细胞以1×10⁶个/孔接种于6孔板,以10^-7mol/L PGE₂和PGF_2α分别预处理细胞24 h,以100 ng/mL细菌脂多糖（lipopolysaccharides,LPS）刺激细胞4、8和12 h后分别提取RNA和总蛋白质,采用实时荧光定量PCR与Western blotting等技术检测COX-1与COX-2 mRNA和蛋白质的表达量。结果表明,与对照组相比,COX-1 mRNA表达量在PGE₂单独作用4、8和12 h后显著上调（P<0.05）;COX-2 mRNA表达量在PGE₂单独作用4和12 h后显著上调（P<0.05）,PGE₂单独处理使COX-1、COX-2蛋白表达量均显著上调（P<0.05）。与对照组相比,LPS刺激8和12 h时COX-1 mRNA表达量显著下调（P<0.05）,LPS刺激后COX-1蛋白表达量无显著变化（P>0.05）;LPS刺激后4、8和12 h时COX-2 mRNA表达量显著上调（P<0.05）,LPS刺激后COX-2蛋白表达量显著上调（P<0.05）。与LPS单独处理组相比,LPS+PGE₂处理组在8和12 h时COX-1和COX-2 mRNA表达量均显著上调（P<0.05）,同时COX-1和COX-2蛋白表达量也显著上调（P<0.05）。PGF_2α在LPS未刺激和刺激后对COX-1和COX-2 mRNA的表达无显著影响（P>0.05）,仅在PGF_2α单独处理8和12 h后COX-1 mRNA表达量上调（P<0.05）。两种激素联合处理与各自单独处理及LPS单独刺激相比,对COX-1和COX-2 mRNA表达具有一定的协同诱导作用。     

ZEA、DON及其联合染毒对CTLL-2细胞因子分泌的影响

为研究镰刀菌毒素玉米赤霉烯酮（ZEA）、脱氧雪腐镰刀菌烯醇（DON）及其联合作用对动物免疫功能的影响,试验以CTLL-2细胞（细胞毒性T淋巴细胞株）为材料,用不同浓度的ZEA （0、5、10、20 μg/mL）、DON （0、0.5、1、2 μg/mL）及联合（空白组、5 μg/mL ZEA、0.5 μg/mL DON、5 μg/mL ZEA＋0.5 μg/mL DON）处理CTLL-2细胞48 h,采用ELISA法检测了细胞内及培养上清液中颗粒酶B （GZMB）、穿孔素（PFP）、干扰素γ（IFN-γ）和肿瘤坏死因子α（TNF-α）等细胞因子的含量。结果显示,ZEA、DON能够降低CTLL-2细胞胞内及培养上清液中PFP、GZMB、IFN-γ的浓度,增加TNF-α浓度,染毒组与对照组相比均有显著或极显著差异（P＜0.05;P＜0.01）,且均呈剂量效应关系;ZEA、DON联合染毒表现为加性效应。结果表明,ZEA、DON及其联合作用可通过影响免疫细胞因子的分泌,降低免疫细胞杀伤活力,间接影响机体体液免疫和细胞免疫的负调节,从而导致动物机体免疫机能下降。     

Effects of ZEA,DON and Their Combined Effects on the Secretion of Cytokines in CTLL-2 Cells

To analyze the effects of ZEA or/and DON on the immunologic function,CTLL-2 cell was used as experimental materials. After exposing the CTLL-2 cell to different concentrations of ZEA (0,5,10,20 μg/mL),DON (0,0.5,1,2 μg/mL) and ZEA+DON (blank group,5 μg/mL ZEA,0.5 μg/mL DON,5 μg/mL ZEA+0.5 μg/mL DON) for 48 h,the concentration of GZMB,PFP,IFN-γ and TNF-α in CTLL-2 cell and supernatant fluid were detected by ELISA method. The result showed that DON and ZEA could reduce the concentration of PFP,GZMB and IFN-γ in the CTLL-2 cells and the culture supernatant fluids,and increase the concentration of TNF-α. The intoxicated group had significant or extremely significant difference compared to control group (P <0.05;P <0.01),which showed the dose effect. The group exposed to the combined of ZEA and DON showed an additive effect. The result indicated that ZEA,DON and the two combined could reduce the killing activity of immunological cell by affecting the secretion of immunological cytokines, and indirectly affect the negative feedback regulation of humoral immunity and cellular immunity,which led to the decline of immune function in animals.     

Cytotoxic Effects of Serum from Equine Grass Sickness Cases on Neuro-2a and PC12 Tet-Off Cell Lines: Implication for Using In Vitro Methods as Antemortem Diagnostic Tools

A wide variety of clinical and paraclinical methods have been used for diagnosis of equine grass sickness (EGS), but none of them could absolutely confirm the diagnosis, and postmortem pathologic examination is still considered the final step in precise diagnosis of EGS. Use of in vitro cell toxicity caused by EGS serum on neuronal cell lines was investigated. Three well-known cytotoxic methods were used to investigate the cytotoxicity of EGS serum on neuro-2a and genetically engineered PC12 Tet-Off P53 cells. The results of alamar blue reduction as an index for mitochondrial activities and intracellular adenosine triphosphate content assays, but not neutral red uptake, indicate that the EGS serum may affect the mitochondrial function and cellular metabolism at up to 60% of the cases. The results of present study might be used for diagnosis of EGS cases. Further studies with high sample size may lead us to uncover the pathogenesis of EGS and to increase the sensitivity and applicability of in vitro techniques as diagnostic tools.     

80%赖氨酸硫酸盐与98%赖氨酸盐酸盐对生长中期草鱼生长性能、消化吸收能力和消化器官生长发育影响的比较研究

本试验通过比较80%赖氨酸硫酸盐[80%L-Lys·H2SO4,简称80赖氨酸(80-Lys)]与98%赖氨酸盐酸盐[98%L-Lys·HCl,简称98赖氨酸(98-Lys)]对生长中期草鱼生长性能、消化吸收能力和消化器官生长发育的影响,探讨80-Lys和98-Lys在草鱼上的生物效价并确定以80-Lys为赖氨酸(Lys)添加形式时饲料中Lys的适宜含量。选择初始体重为275.80 g左右的健康草鱼540尾,随机分成6组(每组3个重复,每个重复30尾鱼),分别饲喂Lys含量为0.8%(基础饲料)、1.0%、1.2%、1.4%和1.6%的添加80-Lys的饲料及Lys含量为1.2%的添加98-Lys的饲料60 d。结果表明:与基础饲料相比,饲料中添加适宜水平的80-Lys使饲料Lys含量达到1.2%时可显著提高生长中期草鱼的增重率(W GR),特定生长率(SGR),采食量(FI),全肠脂肪酶、淀粉酶活力,肝胰脏谷草转氨酶(GOT)和谷丙转氨酶(GPT)活力,前、中、后肠碱性磷酸酶(AKP)、肌酸激酶(CK)活力,肝体指数与肠体指数以及前、后肠皱襞高度(P0.05),显著降低血清GOT和GPT活力(P0.05),且80-Lys对上述指标的作用效果显著优于98-Lys(P0.05);此外,还可显著提高生长中期草鱼的饲料效率(FE),全肠胰蛋白酶活力,前、中、后肠Na+,K+-ATP酶(Na+,K+-ATPase)和γ-谷胺酰转肽酶(γ-GT)活力,肠长与肠长指数以及中肠皱襞高度(P0.05),但80-Lys对上述指标的作用效果与98-Lys差异不显著(P0.05)。由此得出,与98-Lys相比,80-Lys能更有效地提高生长中期草鱼的消化吸收能力,进而促进其生长。以80-Lys为Lys添加形式,以SGR和FE为标识,生长中期草鱼(276~667 g)饲料中Lys的最适含量分别为1.31%(占饲料蛋白质的4.68%)和1.27%(占饲料蛋白质的4.54%)。