苜蓿皂苷对小白鼠体内溶血的研究

为了探究摄入苜蓿皂苷(alfalfa saponin, AS)是否会引起血管内溶血,选取180只健康小白鼠,按体重和性别随机分为6个处理,每个处理3个重复,每个重复10只。将1mL 0.0125、0.025、0.05、0.1、0.2 g·mL^-1皂苷分别灌胃Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组小鼠,对照组灌胃等量的生理盐水(1 mL),试验期为30 d。检测红细胞数目(red blood cell, RBC)、形态、压积(hematocrit, HCT)、平均红细胞体积(erythrocyte mean corpuscular volume, MCV)、平均血红蛋白浓度(mean corpuscular hemoglobin, MCH)、血浆游离血红蛋白(free hemoglobin, FHb)、网织红细胞计数(reticulocyte count, RET)及红细胞渗透脆性最大、最小抵抗值(maximum/minimum osmotic fragility of red blood cells, OF_max/OF_min),采血和骨髓制作观察血涂片及骨髓涂片,制作实质性器官心、肝的切片。结果表明: 1)灌胃苜蓿皂苷后,小白鼠血常规中红细胞数目和血细胞比容均降低且Ⅴ组显著低于对照组(P<0.05),平均血红蛋白浓度均高于对照组且Ⅳ、Ⅴ组与对照组相比差异显著(P<0.05)。2)Ⅳ、Ⅴ组血浆游离血红蛋白含量极显著高于对照组(P<0.01);Ⅳ组网织红细胞计数极显著高于对照组(P<0.01)。3)随着灌胃剂量的加大,红细胞逐渐出现轻度大小不等,着色较深,形态异常的现象。4)红细胞渗透脆性最大抵抗值显著升高且差异显著(P<0.05),最小抵抗值有逐渐升高的趋势。5)灌胃后粒系比有不同程度的降低,Ⅴ组与对照组相比差异显著(P<0.05);红系比则有不同程度的升高,Ⅴ组与对照组相比差异显著(P<0.05);粒红比不但降低而且变化明显,Ⅳ、Ⅴ组显著低于对照组(P<0.05)。 6)灌胃对实质性器官没有影响但是组织更易碎。综上,高剂量的苜蓿皂苷能够引起血管内溶血。     

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.     

甲状腺素对猪小肠上皮细胞miR-let-7a基因表达及细胞增殖的影响

miR-let-7a在动物细胞的分化、增殖与凋亡等方面发挥越来越重要的作用。甲状腺激素(TH)作用非常广泛,机体的每个细胞几乎都是TH作用的靶细胞,其可以促进组织分化、生长和成熟。本实验用甲状腺素(T4)浓度分别为(0、0.02、0.03、0.05、0.075、0.1、0.2μmol/L)在体外培养猪的小肠上皮细胞。结果表明:T4处理组的细胞体积形态相对于空白对照组没有明显变化;当T4添加浓度为0.03μmol/L时,细胞的增殖率显著低于其他组(P0.05);当T4浓度为0~0.03μmol/L时,let-7a的表达随着添加剂量的增加而升高,浓度从0.03~0.2μmol/L变化时,let-7a的表达呈现降低趋势,浓度为0.03μmol/L时表达量极显著高于其他组(P0.01)。let-7a的表达量与细胞增殖呈负相关。     

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

草鱼mst2在免疫应答中的作用机制

为了初步阐明草鱼哺乳动物STE20样蛋白激酶2基因(mammalian sterile 20-like kinase 2, mst2)在机体免疫中的作用机制,实验采用RNA-Seq技术对干扰mst2后经脂多糖(lipopolysaccharide,LPS)应激的草鱼肾细胞系(Ctenopharyngodon idella kidney cell lines,CIK)进行了转录组测序与验证分析。测序原始数据经De novo拼接与组装后共获得22 374个独立功能基因(unigenes),其中已知功能基因为21 199个,预测的新基因为1 175个。干扰mst2后经LPS应激的unigenes表达差异分析表明,对照组与实验组之间共存在38个差异基因(differentially expressed genes,DEGs),其中上调基因16个,下调基因22个。利用实时荧光定量PCR技术(quantitative real-time PCR, qRT-PCR)对38个DEGs的RNASeq结果进行验证,结果显示,qRT-PCR和RNA-Seq分析一致,说明RNA-Seq分析结果可靠。采用RNA干扰技术干扰mst2后经LPS处理,CIK细胞转录组中DEGs参与免疫代谢的途径主要有MAPK信号通路、内吞作用途径、自噬途径和细胞因子受体相互作用途径。凋亡相关基因检测结果显示,干扰mst2并经LPS处理后,促凋亡基因(fas、bad1、bad2、caspase-3、caspase-8和caspase-9)转录水平上调,抗凋亡基因(bcl2)转录水平下调,证明干扰mst2后经LPS处理会诱发细胞发生凋亡。综上所述,mst2可通过调控凋亡相关过程参与机体免疫反应。本研究结果初步阐明了草鱼mst2参与机体免疫应答的分子机理,可为草鱼细菌性疾病防控提供一定的基础理论参考。     

Effects of component of some Chinese herbs on proliferation of human umbilical vein endothelial cells in vitro

AIM: To observe the effects of some component of Chinese herbs for external use on proliferation of human umbilical vein endothelial cells (HUVEC) and investigate the mechanism of promoting tissue repair. METHODS: The method of MTT was used to examine the effects of Rg1, Rh1, perlolyrine, cinnamyl aldehyde, muscone, astragaluspolysaccharin (APS), velver antler polypeptide (VAP) and soluble extract of boswellia carterii birdw (BCB) on proliferation of HUVEC. RESULTS: APS did not promote proliferation of HUVEC at 9.75 mg/L-2.5 g/L; Rh1 promoted proliferation of HUVEC at 1.94 mg/L-0.5 g/L (P<0.05 or P＜0.01), and Rg1 inhibited proliferation of HUVEC at 31 mg/L (P<0.05); VAP promoted proliferation of HUVEC at 1 mg/L-0.5 g/L with optimal dose of 10 mg/L (P<0.01), Cinnamyl aldehyde promoted proliferation of HUVEC at 2 g/L(P＜0. 05); Muscone and soluble extract of BCB inhibited proliferation of HUVEC at 1 g/L, 0.5-2.5 kg/L(P＜0. 01), respectively; Perlolyrine inhibited proliferation of HUVEC at 0.125 g/L-0.5 g/L(P＜0. 01). CONCLUSION: The external herbs for supplementing Qi and warming Yang can promote HUVEC proliferation and improve angiogenesis during tissue repair. The external herbs for promoting blood circulation and accelerating capillary movement may have influence upon other stages of tissue repair.     

猪流行性腹泻病毒SHpd/2012株侵染Vero细胞特性分析

为了探究PEDV(猪流行性腹泻病毒)流行毒株SHpd/2012侵染细胞的特性,研究采用IFA(间接免疫荧光)及绘制病毒一步生长曲线等方法,对感染PEDVSHpd/2012株后的Vero细胞(非洲绿猴肾细胞)病变过程进行研究。结果发现,CPE(细胞病变)主要表现为细胞肿胀、变圆、皱缩、聚集成合胞体甚至从培养瓶壁大量脱落。IFA结果显示,接毒后12h病毒开始增殖,在接毒后36h时感染细胞数量最多,其后病毒增殖速度逐步下降,60h时趋于平稳。病毒一步生长曲线结果显示,SHpd/2012株感染细胞后24~60h的毒价一直处于较高水平,60h后开始下降。此外,本研究成功克隆并真核表达了PEDV S蛋白,为研究PEDV与受体蛋白的互作及病毒的致病机制奠定基础。     

不同中药对中性粒细胞迁移微血管内皮细胞后溶菌酶释放量的影响

试验旨在探讨脂多糖（LPS）刺激大鼠肠黏膜微血管内皮细胞（RIMVECs）对跨内皮迁移中性粒细胞（PMNs）溶菌酶释放量的影响及白头翁、黄连、黄柏、秦皮、马齿苋的干预作用。选取0、0.1、1、10、100 μg/mL LPS刺激RIMVECs,采用ELISA法检测12和24 h细胞培养上清液中IL-6水平以确定后续试验所需LPS浓度。建立PMNs跨RIMVECs黏附模型和迁移的Transwell模型,采用细胞计数法、染色法、ELISA方法评价5种中药对LPS刺激RIMVECs后PMNs的黏附情况、迁移率及其溶菌酶释放量的影响。结果显示,与对照组相比,10 μg/mL LPS作用RIMVECs后24 h时的IL-6水平极显著升高（P<0.001）;与LPS组相比,黄连、白头翁组的PMNs黏附率显著降低（P<0.05）,溶菌酶释放量极显著升高（P<0.001）,秦皮、白头翁组迁移率显著降低（P<0.05）。提示LPS刺激内皮细胞显著抑制了PMNs杀菌酶的释放,中药黄连、白头翁可以显著减轻这种抑制作用,PMNs杀菌酶的释放可能与内皮细胞的功能完整性有关,本试验为进一步筛选有效中药水溶性成分提供了理论依据。     

Lipopolysaccharide and cytokines modulate leukotriene (LT)B4 and LTC4 production by porcine endometrial endothelial cells

Uterine inflammatory response is mediated by inflammatory mediators including eicosanoids and cytokines produced by immune and endometrial cells. Interactions between lipopolysaccharide (LPS) and cytokines, and leukotrienes (LTs) in endothelium, important for the host defence during the inflammation, are unknown. We studied the effect of LPS, tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐4 and IL‐10 on 5‐lipooxygenase (5‐LO), LTA₄ hydrolase (LTAH) and LTC₄ synthase (LTCS) mRNA and protein expression, LTB₄ and LTC₄ release from porcine endometrial endothelial cells, and cell viability. For 24 hr, cells were exposed to LPS (10 or 100 ng/ml of medium) and cytokines (each 1 or 10 ng/ml). 5‐LO mRNA/protein expression augmented after incubation with larger doses of LPS, TNF‐α, IL‐4 and IL‐10 and smaller dose of IL‐1β. Larger dose of TNF‐α, smaller doses of LPS and IL‐1β and both doses of IL‐10 increased LTAH mRNA/protein expression. LTAH protein content was up‐regulated by larger dose of LPS, but it was reduced in response to both doses of IL‐4. LTCS mRNA expression was elevated by larger doses of LPS, IL‐4 and IL‐10 or both doses of TNF‐α and IL‐1β. LTCS protein level increased after treatment with both doses of IL‐1β, IL‐4 and IL‐10, smaller dose of LPS and larger dose of TNF‐α. Both doses of LPS and larger doses of TNF‐α and IL‐10 increased LTB₄ release. LPS, IL‐1β and IL‐10 at smaller doses, or TNF‐α and IL‐4 at larger doses stimulated LTC₄ release. Smaller doses of TNF‐α and IL‐1β or both doses of IL‐4 enhanced the cell viability. This work provides new insight on the participation of LPS, TNF‐α, IL‐1β, IL‐4 and IL‐10 in LTB₄ and LTC₄ production/release from porcine endometrial endothelial cells, and the effect of above factors on these cells viability. The used cellular model gives the possibility to further establish the interactions between inflammatory mediators.