Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

CnAβ基因对大鼠RBL-2H3细胞脱颗粒的影响

过敏是人和养殖动物常见的疾病，而且发生的频率呈现逐渐增加的趋势。为了深入了解过敏反应的机制，本研究利用CRISPR/Cas9基因编辑技术，构建钙调磷酸酶A beta （calcineurin A beta，CnAβ）基因敲除的大鼠RBL-2H3细胞株，并探讨CnAβ基因对RBL-2H3细胞生长及脱颗粒的影响。选取大鼠CnAβ基因第一外显子为敲除靶点，设计并合成3个单导向RNA （single guide RNA，sgRNA），构建pX459-CnAβ-sgRNA质粒，并用脂质体3000将构建好的质粒转染到RBL-2H3细胞内；利用嘌呤霉素对转染细胞进行筛选，通过DNA测序验证获得CnAβ基因敲除的RBL-2H3细胞株；并检测CnAβ基因缺失对细胞增殖和脱颗粒的影响。结果表明，成功构建了CnAβ单基因敲除的大鼠RBL-2H3细胞株；CnAβ基因缺失对RBL-2H3细胞增殖、细胞的颗粒形成以及颗粒含量无显著影响，但显著抑制由细胞表面受体介导的RBL-2H3细胞脱颗粒作用。该研究结果有助于深入了解动物过敏性疾病的发生机制，为动物过敏性疾病的预防提供了理论基础。     

黄曲霉毒素B1、玉米赤霉烯酮和呕吐毒素的累加细胞毒性研究

由于饲料中多种霉菌毒素并存的几率比较高，本研究以仔猪肠上皮细胞（IPEC-J2）为模型，研究黄曲霉毒素B1（AFB1）、玉米赤霉烯酮（ZEA）和呕吐毒素（DON）的叠加细胞毒性。细胞毒性试验选用AFB1、ZEA和DON三种毒素作为响应面Box-Behnke设计的三个因素，以AFB1：10、20、30 μg/L，ZEA：150、300、450 μg/L，DON：500、1000、1500 μg/L作为Box-Behnke设计的三个编码水平。利用响应面设计构建得到17组复合霉菌毒素组合，以其对IPEC-J2细胞活力的影响作为参考指标，得到对细胞损伤程度最高和最低的霉菌毒素添加比例。结果表明：经方程预测后，得到细胞活力最低（霉菌毒素毒性最高）的AFB1、ZEA和DON组合为30、150 μg/L和1500 μg/L，经测定细胞活力为32.32%|得到细胞活力最高（霉菌毒素毒性最低）的AFB1、ZEA和DON组合为10、150 μg/L和600 μg/L，经测定细胞活力为53.01%。该结果为多种霉菌毒素叠加毒性的研究提供了依据。 [关键词] IPEC-J2细胞|黄曲霉毒素B1|玉米赤霉烯酮|呕吐毒素|细胞毒性     

褪黑激素对长毛兔毛囊细胞凋亡的影响

为研究褪黑激素对皖系长毛兔毛囊细胞凋亡的影响及相关机制,选择60只皖系长毛兔,随机均分成4组,1组不埋植褪黑激素(对照),另3组每只兔按25 mg(低剂量组)、40 mg(中剂量组)和55 mg(高剂量组)分别埋植褪黑激素,饲养70 d后,进行组织病理学观察和RNA的转录组高通量测序及q PCR表达分析。结果表明:通过组织切片观察,证实55 mg褪黑激素处理能有效抑制毛囊细胞凋亡,维持细胞正常形态;通过对高剂量组和对照组测序,得到25个差异表达基因,其中有14个基因上调,11个基因下调,涉及通路包括嗜T细胞病毒感染、坏死性凋亡、甲状腺癌、铁死亡、I型糖尿病和胰岛素信号通路,其中坏死性凋亡、铁死亡通路与长毛兔毛囊凋亡相关;选取4个与凋亡相关的差异表达基因进行荧光定量验证,得到新基因MSTRG.3561表达显著上调,SLC25A5表达下调,但差异无统计学意义。     

Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

Effect of miR-29b-mediated TGF-β/Smad signaling pathway on progression of hepatic fibrosis in rats

AIM: To investigate the role of microRNA-29b (miR-29b)-mediated TGF-β/Smad signaling pathway in the activation of hepatic stellate cells (HSC) and its effect on the progression of hepatic fibrosis in rats.METHODS: Hepatic liver fibrosis rat model was established, and its HSC were isolated. Normal rat HSC were also obtained and identified in vitro. RT-qPCR and Western blot were used to detect the alterations of miR-29b, TGF-β/Smad signaling pathway-related proteins and liver fibrosis marker proteins in the acquired cells. Finally, the direct targeting binding of miR-29b to TGF-β1 was identified by dual-luciferase reporter assay system.RESULTS: With the activation of HSC, the expression of miR-29b gradually decreased (P<0.01), while the expression of collagen type I and α-smooth muscle actin gradually increased (P<0.01). At the same time, the expression of Smad2/3/4 was significantly increased, and the expression of Smad7 was significantly decreased (P<0.01). Dual-luciferase reporter assay showed that miR-29b bound directly to "UCUCUCCGU" in the 3'UTR of TGF-β1, indicating that TGF-β1 was a downstream target gene of miR-29b.CONCLUSION: miR-29b may be involved in the inhibition of HSC activation and migration, thereby inhibiting the process of liver fibrosis. The biological function of miR-29b may be through the direct targeting of TGF-β1, thus regulating and inhibiting the TGF-β/Smad signaling pathway.     

Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

Determination of Festuca filiformis seedbank characteristics,seedling emergence and herbicide susceptibility to aid management in lowbush blueberry (Vaccinium angustifolium)

Festuca filiformis is a common perennial grass in lowbush blueberry fields, but little is known about the general biology, seedbank characteristics, seedling recruitment or susceptibility of seedlings to currently registered herbicides. The objectives of this research were to determine (i) the presence of F. filiformis seedbanks in lowbush blueberry fields, (ii) whether F. filiformis seedbanks accumulate near the soil surface in lowbush blueberry fields, (iii) the dormancy status of fresh F. filiformis seeds, (iv) the temporal patterns of seedling recruitment in established F. filiformis populations, (v) whether F. filiformis has a vernalisation requirement for flowering in lowbush blueberry and (vi) susceptibility of F. filiformis seedlings to various herbicides currently registered in lowbush blueberry. Festuca filiformis formed a seedbank in lowbush blueberry fields, with an average of 1660 ± 272–5680 ± 1409 seedlings m^?2 emerging from soil cores collected from two infested fields. Most seeds were located at the soil surface, providing opportunities for seedbank management through predation or burning. Fresh seeds lacked dormancy and readily germinated, although germination was reduced by dark conditions. New seedlings emerged in spring and autumn and required vernalisation to flower. Seedlings were susceptible to several currently registered herbicides in lowbush blueberry, although mortality rates were highest in plants treated with glufosinate, flumioxazin, glufosinate + flumioxazin and terbacil. Growers should avoid movement of seeds on machinery, and additional research should be conducted to determine the effects of registered herbicides on F. filiformis seedling recruitment under field conditions in lowbush blueberry.     

Effect of CHOP expression knock-down on apoptosis of renal tubular epithelial cells

AIM:To study the effect of C/EBP homologous protein (CHOP) on the apoptosis of renal tubular epithelial HK2 cells. METHODS:The serum mRNA levels of CHOP in the patients with acute kidney injury and healthy controls were detected by qPCR. In vitro, renal tubular epithelial HK2 cells were divided into control group, negative group (transfected with negative control siRNA), si-CHOP group (transfected with CHOP siRNA), and induced by transforming growth factor-β1 (TGF-β1). The viability of the cells was measured by MTT assay, and the apoptotic rate was analyzed by flow cytometry. The protein levels of nuclear antigen Ki-67, proliferating cell nuclear antigen (PCNA), caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with the healthy controls, the serum mRNA levels of CHOP in the patients with acute kidney injury were increased significantly (P<0.05). Transfection with CHOP siRNA significantly decreased the expression of CHOP in the renal tubular epithelial HK2 cells (P<0.05). Knock-down of CHOP expression by siRNA significantly increased the viability of renal tubular epithelial HK2 cells (P<0.05), decreased the apoptotic rate (P<0.05), increased the expression of Ki-67 and PCNA (P<0.05), and down-regulated the protein level of cleaved caspase-3 (P<0.05). CONCLUSION:The serum mRNA levels of CHOP were increased in the patients with acute kidney injury. Knock-down of CHOP expression inhibits the apoptosis of renal tubular epithelial cells by regulating the expression of proliferation-and apoptosis-related proteins.     

新型鸭呼肠孤病毒在DF-1细胞系中的增殖特性

为了研究新型鸭呼肠孤病毒(new-type duck reovirus,NDRV)在鸡胚成纤维细胞DF-1中的增殖特性,将NDRV JDM10毒株接种到DF-1细胞,连续传代后,通过观察病毒对细胞的致病变效应,测定半数组织感染量(TCID₅₀)、RT-PCR检测、间接免疫荧光(IFA)及Western-blot免疫学检测,探索NDRV对DF-1细胞的作用效果。结果表明,NDRV JDM10毒株在DF-1细胞中能有效增殖,产生明显的致病变效应;RT-PCR检测成功扩增出1条大小为1 001 bp的条带;病毒蛋白在细胞中获得了良好的表达,并与抗NDRV σC单克隆抗体发生特异性反应;NDRV JDM10毒株在感染DF-1细胞后会经历潜伏期、快速增长期、稳定期3个时期,并在72 h病毒效价达到峰值,TCID₅₀为1×10^-7.90·(0.1mL)^-1。本研究为进一步研究NDRV的致病机理和研制细胞疫苗奠定了基础。