Incidence and causes of pregnancy loss after Day 70 of gestation in Thoroughbreds     

Jessica M. Roach  Alastair K. Foote  Ken C. Smith  Kristien L. Verheyen  Amanda M. de Mestre 《Equine veterinary journal》2021,53(5):996-1003

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Interleukin-1β inhibits AKT to down-regulate eNOS Ser1177 phosphorylation in human umbilical vein endothelial cells     

LUO Ming-hao  HU Shu-peng  WANG Rui-yu  Li Chang  Lü Ding-yi  YANG Xi-yang  LUO Su-xin 《园艺学报》2000,36(10):1739-1744

AIM To investigate whether interleukin-1β （IL-1β） regulates endothelial nitric oxide synthase （eNOS） phosphorylation at Ser1177 site in human umbilical vein endothelial cells （HUVECs）, and to explore its possible mechanism. METHODS The HUVECs were randomly divided into normal control group, tumor necrosis factor-α （TNF-α） group, IL-1β group, IL-6 group, SC79 ［protein kinase B （PKB/AKT） specific agonist］ group and SC79+IL-1β group. Western blot was used to determine the protein levels of eNOS, p-eNOS-Ser1177, AKT and p-AKT-Ser473 in the HUVECs. Chemical colorimetry was used to detect the nitric oxide （NO） content in the culture medium of HUVECs. RESULTS No statistically significant difference of p-eNOS-Ser1177 level in HUVECs treated with TNF-α and IL-6 was observed as compared with normal control group （P>0.05）, while the protein level of p-eNOS-Ser1177 in the HUVECs and the content of NO in the culture medium of HUVECs decreased significantly in IL-1β group （P<0.05）, and the protein level of p-AKT-Ser473 in the HUVECs was decreased as compared with normal control group （P<0.05）. The AKT agonist SC79 blocked the down-regulation effect of IL-1β on p-eNOS-Ser1177 level in the HUVECs and NO content in the culture medium of HUVECs （P<0.05）. CONCLUSION IL-1β down-regulates the protein level of p-eNOS-Ser1177 in HUVECs and affects the activity of eNOS, which may be involved in AKT/eNOS signaling pathway.  相似文献   

Effect of xeroderma pigmentosum group D gene on proliferation of human umbilical arterial smooth muscle cells induced by Ox-LDL     

XIA Zi-rong  LI Qing  XIA Zhen  LI Ju-xiang  HONG Kui  WU Yan-qing  WU Qin-hua  CHENG Xiao-shu 《园艺学报》2017,33(12):2238-2244

AIM: To investigate the effects of xeroderma pigmentosum group D (XPD) gene on the proliferation of human umbilical arterial smooth muscle cells (HUASMCs) induced by oxidized low-density lipoprotein (Ox-LDL). METHODS: The recombinant plasmid pEGFP-N2/XPD was transfected into HUASMCs by liposome. The cells were divided into blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, Ox-LDL group, Ox-LDL+pEGFP-N2 group and Ox-LDL+pEGFP-N2/XPD group. The proliferation rate of the cells was detected by MTT and EdU assays. The apoptotic rate and cell cycle distribution were analyzed by flow cytometry. The protein levels of XPD, caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with blank control group, the expression of XPD was increased in pEGFP-N2/XPD group (P<0.05). According to the results of MTT and EdU assays, the cell proliferation in pEGFP-N2/XPD group was reduced compared with blank control group (P<0.05). Compared with Ox-LDL group, the cell proliferation in Ox-LDL+pEGFP-N2/XPD group was significantly inhibited (P<0.05). According to the results of flow cytometry, the cell proportion of S phase decreased and the G₀/G₁-phase cell proportion increased significantly in pEGFP-N2/XPD group and Ox-LDL+pEGFP-N2/XPD group compared with blank control group and Ox-LDL group, repectively (P<0.05). Compared with blank control group and Ox-LDL group, the protein level of Bcl-2 decreased and the protein levels of Bax and cleaved caspase-3 increased in pEGFP-N2/XPD group and Ox-LDL+pEGFP-N2/XPD group, respectively (P<0.05). CONCLUSION: XPD inhibits the proliferation of HUASMCs and promotes their apoptosis, and reduces the promoting effect of Ox-LDL on the proliferation of HUVSMCs. XPD may be the target for treatment of atherosclerosis.  相似文献   

Role of endoplasmic reticulum stress in trimethylamine N-oxide-induced oxidative stress in human umbilical vein endothelial cells     

WEN Ren-hui  KE Shi-ye  SHI Guang-yao  LIU Ding-hui  YU Xian-guan  LING Ye-sheng  ZHU Jie-ming  QIAN Xiao-xian 《园艺学报》2020,36(2):193-199

AIM: To investigate whether endoplasmic reticulum stress is involved in trimethylamine N-oxide (TMAO)-mediated oxidative stress in human umbilical vein endothelial cells (HUVECs). METHODS: The cell viability was examined by CCK-8 assay. The cells were stained by DCFH-DA, and the intracellular level of reactive oxygen species (ROS) was observed by phase-contrast microscopy and detected by flow cytometric analysis. The protein levels of phospho-IRE-1α, IRE-1α and GRP78/BiP were detected by Western blot. RESULTS: TMAO exerted no significant effect on the viability of HUVECs. For a long period (>24 h), even a low concentration (10 μmol/L) of TMAO increased the oxidative stress level in the HUVECs (P<0.05). TMAO increased the phosphorylation level of IRE-1α and significantly up-regulated the protein level of GRP78/BiP in HUVECs (P<0.01). Pretreatment with STF-083010, an inhibitor of IRE1α, for 1 h reduced TMAO-induced oxidative stress in HUVECs (P<0.05). CONCLUSION: Endoplasmic reticulum stress is involved in TMAO-induced oxidative stress in HUVECs.  相似文献   

CRISPR/Cas9介导的绵羊示踪脐带间充质干细胞系的建立     

李松美  仇雨歌  陈胜男  王晓萌  王春生 《中国农业科学》2021,54(2):400-411

【目的】利用CRISPR/Cas9技术建立绵羊示踪脐带间充质干细胞系,为间充质干细胞的临床治疗与分化机制研究奠定基础。【方法】根据绵羊ROSA26的基因组序列,利用在线工具ZiFiT Targeter Version 4.2设计合成3对引物,利用点突变法,以px330质粒为模板分别进行PCR,DpnⅠ去除质粒DNA后,PCR产物自身环化,酶切测序鉴定,构建以绵羊Rosa26为靶标基因的sgRNA/Cas9载体,构建的质粒含Cas9和向导RNA(single-guide RNA,sgRNA) 表达盒,由U6启动子驱动表达。将上述载体分别利用脂质体转染绵羊脐带间充质干细胞(sUMSCs),提取其基因组PCR后进行T7E1酶切,琼脂糖电泳分析条带灰度以检测载体编辑活性。根据sgRNA序列在绵羊ROSA26靶位点的上下游设计并合成左、右同源臂扩增引物,提取绵羊全基因组为模板分别进行PCR扩增得到左右同源臂,回收纯化后分别与pMD19-Simple连接,酶切测序鉴定获得左、右同源臂重组质粒。根据PCR引入的酶切位点,将左同源臂质粒和Donor表达载体DC-DON-SH02 ROSA26进行酶切连接,鉴定获得左臂重组打靶载体,使用相同方法将右同源臂质粒连接到左臂打靶载体上,鉴定获得Donor打靶载体,载体携带嘌呤霉素抗性基因和绿色荧光蛋白（GFP）报告基因。在生长良好的sUMSCs中加入不同浓度的嘌呤霉素,观察细胞存活时间,确定最佳抗性筛选浓度和时间。利用脂质体共转sgRNA/Cas9载体和Donor载体到绵羊间充质干细胞,在其ROSA26位点切割DNA双链,在DNA断裂处通过同源重组方式引入报告基因,转染48 h后进行嘌呤霉素抗性筛选,筛选结束后更换正常培养基继续培养,观察绿色荧光的表达并提取阳性细胞基因组,针对ROSA26位点设计上下游两对引物对其进行PCR检测其整合情况。【结果】（1）针对绵羊Rosa26位点设计3对PCR引物,利用点突变法将sgRNA 克隆至px330的BbsⅠ酶切位点上,成功构建sgRNA/Cas9载体px330-sgRNA1/2/3,将其分别转染sUMSCs,T7E1酶切结果表明px330-sgRNA2/3出现脱靶现象,未在靶位点发生编辑,px330-sgRNA1的编辑效率最高,约为20%;（2）基于sgRNA1,PCR法获得打靶载体的左、右同源臂,经一系列分子生物学方法先后连接到载体DC-DON-SH02 ROSA26上,经酶切和PCR鉴定,成功获得绵羊ROSA26位点的重组载体sROSA26-HA;（3）筛选得到sUMSCs最佳抗性浓度时间,利用Lipofectamine2000共转染sgRNA/Cas9载体和Donor重组载体到sUMSCs,1.5 μg·mL^-1嘌呤霉素筛选15d至对照组细胞全部死亡,获得的阳性克隆并扩大培养。显微镜下可观察到明显的绿色荧光,且与对照组相比,阳性细胞克隆的基因组PCR均检测到特异条带,表明sUMSCs的ROSA26位点发生同源重组,GFP基因被成功敲入到基因组中并能正常表达,该细胞可以用于动物疾病模型中追踪sUMSCs的去向和分化方向的研究。【结论】成功利用CRISPR/Cas9系统在sUMSCs内实现外源GFP基因的定点敲入,获得绵羊示踪脐带间充质干细胞系,为间充质干细胞进一步的临床转化奠定了基础。  相似文献   

In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood     

Ki-Soo Park  Yong-Soon Lee  Kyung-Sun Kang 《Journal of veterinary science (Suw?n-si, Korea)》2006,7(4):343-348

Mesenchymal stem cells (MSCs) have the capabilities for self-renewal and differentiation into cells with the phenotypes of bone, cartilage, neurons and fat cells. These features of MSCs have attracted the attention of investigators for using MSCs for cell-based therapies to treat several human diseases. Because bone marrow-derived cells, which are a main source of MSCs, are not always acceptable due to a significant drop in their cell number and proliferative/differentiation capacity with age, human umbilical cord blood (UCB) cells are good substitutes for BMCs due to the immaturity of newborn cells. Although the isolation of hematopoietic stem cells from UCB has been well established, the isolation and characterization of MSCs from UCB still need to be established and evaluated. In this study, we isolated and characterized MSCs. UCB-derived mononuclear cells, which gave rise to adherent cells, exhibited either an osteoclast or a mesenchymal-like phenotype. The attached cells with mesenchymal phenotypes displayed fibroblast-like morphologies, and they expressed mesenchym-related antigens (SH2 and vimentin) and periodic acid Schiff activity. Also, UCB-derived MSCs were able to transdifferentiate into bone and 2 types of neuronal cells, in vitro. Therefore, it is suggested that the MSCs from UCB might be a good alternative to bone marrow cells for transplantation or cell therapy.  相似文献   

马脐带不同部位间充质干细胞的分离培养及成软骨诱导分化     

唐小云  周桂珍  周正娜  邓雅迪  张慧  张欣如  王旭光 《中国畜牧兽医》2022,49(1):70-80

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基于图像处理与GUI的大蒜切脐的研究     

赵丽清  陈兆文  刘向征  郭栋 《青岛农业大学学报(自然科学版)》2011,(4):305-308

本课题采用数字图像处理技术,通过研究适合于大蒜切脐定位的专用算法,通过GUI界面编程编写出大蒜切脐软件系统,实现对切脐位置的自动、精确定位。结果表明,该程序能够很好的将大蒜根须与蒜体区分,为大蒜切脐机械化的实现提供了技术支持。  相似文献   

Hepatic myelolipoma incarcerated in a peritoneopericardial diaphragmatic hernia in a cat     

RM Wouda  MD Chalkley  AR Fraser  PAE Moses 《Australian veterinary journal》2010,88(6):231-235

Hepatic myelolipoma incarcerated in a peritoneopericardial diaphragmatic hernia was diagnosed in an 11-year-old, desexed female Persian cat. The cat was initially referred for investigation of tachypnoea and dyspnoea. Peritoneopericardial diaphragmatic hernia is a common incidental finding in cats and is usually asymptomatic. Myelolipoma is an extremely rare benign tumour, composed of extramedullary haematopoietic cells and adipose tissue. Myelolipomas are hypothesised to result from metaplastic alteration, rather than a neoplastic process, although this theory cannot be substantiated. The present case is only the fourth report of such an unusual occurrence in cats and displays significant differences to previous reports. Hepatic entrapment and burgeoning of the mass within the pericardial sac resulted in cardiac tamponade and overt signs of right-sided cardiac failure. Surgical intervention was successful and despite concerns regarding the cat's clinical presentation and the gross appearance of the lesion(s), a good long-term outcome is anticipated.  相似文献   

Protective effect of BNDF on vascular endothelial cells with H₂O₂-induced oxidative injury     

WANG Shi-cai  CHEN Tai-jun  HUANG Mei-song  ZHU Shao-ming 《园艺学报》2015,31(8):1384-1394

AIM: To study the protective effect of brain-derived neurotrophic factor (BDNF) on vascular endothelial cells with H₂O₂-induced oxidative injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and the oxidation injury model of HUVECs was established by treatment with H₂O₂. The oxidatively injured HUVECs were cultured with different concentrations (1, 10 and 100 μg/L) of BDNF. At the same time, the control group (no injury), PBS treatment after H₂O₂ injury group and TrkB inhibitor group (with 100 μg/L BDNF and 1: 1 000 TrkB inhibitor) were also set up. The viability of the HUVECs was detected by MTT assay. The levels of LDH, MDA, SOD and GSH were measured. The releases of NO, ET-1 and ICAM-1 were analyzed by ELISA. The changes of ROS production and cell apoptosis were evaluated by flow cytometry. The protein levels of TrkB, p-TrkB, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with uninjured control group, in H₂O₂ oxidative injury plus PBS treatment group, the viability of the cells was decreased significantly, the LDH and MDA levels were increased significantly and the activities of SOD and GSH were decreased significantly. The NO secretion was decreased, and the ET-1 and ICAM-1 concentrations were increased significantly. The ROS content and apoptotic rate were increased significantly. The protein levels of cleaved caspase-3 and Bax were increased but Bcl-2 protein expression was decreased significantly. Compared with PBS treatment group, in H₂O₂-injured HUVECs treated with different concentrations of BDNF, the cell viability was gradually increased, the LDH and MDA levels were decreased and the activities of SOD and GSH were increased gradually. The secretion of NO was increased but ET-1 and ICAM-1 were decreased gradually. The ROS content and apoptotic rate were decreased significantly. The TrkB and p-TrkB levels were significantly increased significantly, the protein expression of cleaved-caspase 3 and Bax was decreased gradually and the Bcl-2 protein expression increased gradually. The role of BDNF was inhibited by TrkB inhibitor. CONCLUSION: BDNF protects HUVECs from oxidative injury by binding with TrkB to activate the BDNF-TrkB signaling pathways.  相似文献