Effects of exosomes derived from hypoxia-preconditioned umbilical cord mesenchymal stem cells on function of endothelial cells

AIM To investigate the effect of exosomes derived from hypoxia-preconditioned human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs). METHODS hUCMSCs and HUVECs were isolated, cultured and identified. Exosomes derived from hUCMSCs were extracted by ultracentrifugation. The morphological change of exosomes was observed under transmission electron microscope. The particle size and concentration of exosomes were detected by nanoparticle tracking analysis, and the surface specific marker proteins of exosomes were determined by Western blot. hUCMSCs were divided into normoxia group and hypoxia group. The viability of hUCMSCs was measured by CCK-8 assay. HUVECs were divided into control group, normoxic exosome group and hypoxic exosome group. The proliferation of HUVECs was detected by EdU assay. The migration ability was detected by cell scratch assay and Transwell experiment. Tube formation ability was evaluated by tube formation experiment. RESULTS Compared with normoxia group, hypoxia pretreatment enhanced the viability and exosome release of hUCMSCs. Compared with normoxic exosome group, hypoxic exosomes enhanced the proliferation, migration and tube formation of HUVECs. CONCLUSION Exosomes derived from hUCMSCs under hypoxia enhances the proliferation, migration and tube formation of HUVECs.     

High‐level exogenous trans10, cis12 conjugated linoleic acid plays an anti‐lipogenesis role in bovine mammary epithelial cells

Primary bovine mammary epithelial cells (BMECs) were treated by 0, 37.5, 75, 112.5, 150 μmol/L trans10, cis12 conjugated linoleic acid (CLA) to evaluate the effects of different level trans10, cis12 CLA on lipogenesis in BMEC. Addition of 75–150 μmol/L trans10, cis12 CLA reduced significantly the triacylglycerol (TAG) content (P < 0.05), but did not have inhibiting action on cell proliferation (P > 0.05). Treatment with 150 μmol/L trans10, cis12 CLA for 48 h resulted in a 17.1% reduction (P < 0.0001) of medium chain fatty acids (MCFA, C14 < C < C16), a 26.5% reduction (P < 0.0001) of unsaturated fatty acids (UFA) and a corresponding reduction of the mRNA abundance of acetyl coenzyme A (acetylCoA) carboxylase (ACC) (P = 0.046), fatty acid synthase (FAS) (P = 0.017) and stearoylCoA desaturase1 (SCD1) (P = 0.002). Another finding was that trans10, cis12 CLA elevated expression of diacylglycerol acyltransferase2 (DGAT2) (P = 0.020) and long chain acylCoA synthetases (ACSL) (P = 0.032). In conclusion, higher trans10, cis12 CLA, not low trans10, cis12 CLA, inhibited milk fat synthesis and changed fatty acid composition by regulating the expression of FAS, ACC, SCD1, DGAT2 and ACSL.     

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Cn...     

新城疫活疫苗对控制鸡大肠杆菌病的效果观察

为了研究鸡新城疫活疫苗紧急免疫控制鸡大肠杆菌病的效果,通过大肠杆菌人工感染30日龄蛋鸡,待出现病症后,紧急免疫新城疫La-sota弱毒苗(Ⅳ系)4羽份.免疫后5～17 d,通过血凝抑制试验测定血清新城疫抗体水平,试验组新城疫抗体水平逐步提高,对照组抗体略有下降,免疫后17 d两组抗体水平差异显著(P＜0.05);对照组...     

Bovine Cells Infected in Vivo with Theileria Annulata Express CD11b,the C3bi Complement Receptor

Forsyth, L.M.G., Jackson, L.A., Wilkie, G., Sanderson, A., Brown, C.G.D. and Preston, P.M., 1997. Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor. Veterinary Research Communications, 21 (4), 249-263Bovine cells from cattle infected with Theileria annulata were phenotyped with monoclonal antibodies recognizing bovine leukocyte antigens. Macroschizont-infected, transformed cell lines prepared from peripheral blood mononuclear cells of cattle, infected with sporozoites, were assessed by flow cytometry; parasitized cells in tissues from infected cattle were examined by immunocytochemical techniques. Co-expression of markers for different cell lineages by the cell lines precluded a definite conclusion as to their phenotypic origins. For, while the pattern of leukocyte antigens expressed by these in vivo-derived schizont-infected cells, which included CD11b, was indicative of a myeloid origin, the possibility that they were NK cells could not be excluded. The monoclonal antibody (MAb) IL-A15, which recognizes CD11b, reacted with a high proportion of parasitized cells in sections of tissues from infected cattle at all stages of acute disease. Mononuclear cells infected with parasites at all stages of differentiation, from macroschizont to microschizont, expressed CD11b. Such parasitized cells occurred throughout the lymphoid tissues, being found in the thymus, spleen and lymph nodes, particularly the prescapular node draining the site of infection, the hepatic, mesenteric and precrural nodes, as well as in the reticulo-endothelial tissue of the liver, kidney, lung, abomasum, adrenal and pituitary glands. These observations provided the first evidence for a myeloid origin for the parasitized T. annulata cells found in infected bovine tissues and blood and suggested a mechanism whereby schizonts could transfer from cell to cell during mechanical infection with schizont-infected cells.     

鸡溶菌酶基因3＇端MAR在同源细胞系对基因表达调控的研究

用带有鸡溶菌酶基因增强子（Ｅ）和启动子（Ｐ）的编码细菌氯霉素乙酰基转移酶（ＣＡＴ）的融合基因（ＥＰＣ），在其上游区、下游区或在上游和下游区同时插入鸡溶菌酶基因３＇端核基质附着区（ＭＡＲ）的不同表达载体ＭＥＰＣ、ＥＰＣＭ和ＭＥＰＣＭ，稳定转染鸡ＨＤ１１Ｐｒｏｍａｃｒｏｐｈａｇｅ同源细胞系，筛选不同表达载体稳定整合后的细胞株，检测ＣＡＴ表达水平，确定其表达基因拷贝数，从而分析鸡溶菌酶基因３＇端ＭＡＲ对基因表达的影响。稳定整合表达和瞬时表达实验结果表明，鸡溶菌酶基因３＇端ＭＡＲ在同源细胞系对基因表达没有激活作用，与该基因５＇端ＭＡＲ在同源或异源细胞系中能激活基因表达形成鲜明对照，说明５＇瑞ＭＡＲ和３＇端ＭＡＲ在调节鸡溶菌酶基因表达上存在一种协调关系。     

ST贴壁细胞悬浮驯化及应用

为将ST贴壁细胞通过自主驯化,使其能在ST-S细胞无血清培养基中悬浮生长且能稳定传代,在摇瓶中实现高密度生长,并应用于猪伪狂犬病毒(PRV)悬浮培养,经ST-A低血清培养基适应培养,对一株贴壁的ST细胞进行了无血清的全悬浮驯化,并将PRV在悬浮细胞中连续盲传培养。结果显示:ST悬浮细胞能在无血清培养基中传代,培养48 h至第3代后,所能达到的最终细胞密度为3.00×10^(6 )cells/mL以上;PRV连续培养至第5代,毒价可达到109.0 TCID50/mL。结果表明,用专用培养基可使ST贴壁细胞实现悬浮驯化,并可应用于PRV悬浮培养。本研究为获得高密度ST悬浮细胞和提高PRV增殖效率奠定了技术基础。     

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

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.     

Qualitative tear film and conjunctival goblet cell assessment of cats with corneal sequestra

OBJECTIVES: To evaluate the tear film qualitatively and conjunctival goblet cell numbers in cats with and without corneal sequestra. ANIMALS STUDIED AND PROCEDURES: This was a prospective evaluation of 11 cats with corneal sequestra and 14 control eyes that were either the contralateral normal eye when the sequestrum was unilateral or from control cats of similar age with no ocular disease. All cats in this study were examined by a veterinary ophthalmologist. The ophthalmic examinations included a neuro-ophthalmic evaluation, Schirmer tear tests, fluorescein staining, tear film break-up times, applanation tonometry, biomicroscopy, and indirect ophthalmoscopy. The palpebral conjunctiva at the dorsal nasal, ventral nasal, dorsal temporal and ventral temporal fornices were biopsied after topical anesthetic was applied to the cornea and conjunctiva. The conjunctival biopsies were fixed in formalin and sectioned routinely and stained with hematoxylin and eosin, and periodic acid-Schiff. These slides were examined by light microscopy by a blinded examiner. Goblet cell numbers were compared to conjunctival basal epithelial cell numbers by region. The goblet cell numbers by region from the eyes with sequestra was statistically compared to those from eyes without sequestra, with a student's paired t-test. Conjunctival swabs were collected from the cats with corneal sequestra and submitted for polymerase chain reaction for Herpes felis, Chlamydia psiitticia, and Mycoplasma felis. The corneal sequestra were removed by surgical keratectomy and fixed and stained routinely, and examined by light microscopy. RESULTS: No neurologic abnormalities were detected in any of the cats. The Schirmer tear tests (eyes with sequestra 14+/-5.1 mm/min; normal eyes 15+/-6.8 mm/min) and intraocular pressures (eyes with sequestra 21+/-6.6; normal eyes 22+/-5.8) were within normal reference ranges for cats. Biomicroscopic examinations revealed varied sizes and depths of brown- and amber-colored corneal sequestra. No abnormalities were noted on indirect ophthalmoscopic examinations. The tear film break-up time was 21 s (+/-12) for the normal eyes (n=14) and 14 s (+/-13) in eyes with corneal sequestra (n=11). The average goblet/epithelial cell ratios by region for the normal eyes and the eyes with sequestra respectively were 0.66, 0.56 for the dorsal nasal fornix, 0.68, 0.57 for the ventral nasal fornix, 0.63, 0.48 for the temporal dorsal fornix, and 0.55, 0.49 for the temporal ventral fornix. There were no significant differences in tear film break-up times and goblet cell numbers in eyes with corneal sequestra and those without sequestra. Three conjunctival swabs from two of 11 cats with sequestra were positive with PCR for Herpes felis virus. These included one cat with bilateral sequestra and one cat with unilateral corneal sequestrum. CONCLUSIONS: The pathogenesis of feline corneal sequestra does not appear to be linked primarily to abnormal goblet cell numbers, qualitative tear film abnormalities, and accelerated tear film break-up time.     

牛卵丘细胞对卵母细胞体外成熟与孤雌发育的影响

试验以牛卵泡内卵丘-卵母细胞复合体(COCs)为材料,探讨了牛卵丘细胞包裹程度对卵母细胞体外成熟(IVM)和孤雌胚胎(PAEs)发育的影响。试验1,将COCs随机分为2组,在开展IVM前,将其中一组COCs的卵丘细胞机械吹打去除成为机械裸卵(DOs),研究卵丘细胞层存在与否对卵母细胞体外核成熟(排出第一极体)和孤雌激活后发育能力的影响;试验2,根据COCs卵丘细胞包裹层数将COCs分为3组,即卵丘细胞层少(1～4层)的COCs为A组、卵丘细胞层多(5层以上)的COCs为B组及包裹5层以上卵丘细胞且附带卵泡壁颗粒细胞层(FSP)的COCs为C组,研究卵丘细胞层包裹程度对卵母细胞的体外核成熟和孤雌激活后胚胎发育能力的影响。结果表明:卵丘细胞的存在更有利于卵母细胞体外成熟和孤雌胚胎发育;COCs中卵丘细胞包裹层数对卵母细胞体外核成熟率没有显著影响,但包裹卵丘细胞层数多且附带FSP的卵母细胞,其PAEs的囊胚率显著高于包裹卵丘细胞少的卵母细胞PAEs。