简介:PUBs蛋白调控了作物的生长发育及响应非生物胁迫和生物胁迫的过程。为探讨棉花中U-box类型泛素连接酶家族,本研究利用生物信息学方法分析了二倍体雷蒙德氏棉中PUBs的数目、进化、基因结构、结构域分布及基因表达模式。结果表明,雷蒙德氏棉中有93个GrPUBs基因家族成员,基因长度为1101~11191bp。亚细胞定位预测结果表明,GrPUBs编码产物大部分定位在细胞质和细胞核中,少数定位在胞外基质线粒体中。根据除U-box以外所含结构域将该家族成员分为7类,分别含有UFD2结构域、ARM结构域、激酶结构域、只含U-box、WD-40、TPR以及异构酶结构域。GrPUBs基因家族染色体定位显示,基因在13条染色体上均有分布,但分布不均匀。在第5条染色体最多,有11条基因序列,第4、第12和Scaffold染色体上的基因最少,仅有2条。基因结构分析表明,基因内含子的数目变化较大,在0~17之间,且具有相似结构的基因,其编码蛋白聚为一类。基因表达模式分析发现。几乎1/3的基因在花中优势表达,个别基因在开花后10d、20d、30d和40d的种子中表达量高于在叶和花中的表达,如Gorai.006G251300。本试验为深入研究U-box类型泛素连接酶在棉花生长发育及抗逆的机理提供了理论指导。
简介:AbstractBackground:Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear.Methods:To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification.Results:PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs.Conclusions:As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.
简介:目的克隆人HMGBlA—box的cDNA,构建高效稳定的大肠杆菌(E.coli)表达菌株并对其诱导表达纯化.同时探讨其对间充质干细胞(Mesenchymalstemcells,MSC)的促分化作用。方法根据优选合成的HMGBl基因序列设计引物,PCR扩增目的基因片段,插入克隆载体pGEM—T并进行序列测定。重组克隆载体经BamHI和xhoI酶切.琼脂糖凝胶电泳分离目的基冈,插入含His标签的表达载体pET24a。将构建的质粒转染BL21大肠杆菌,经IPTG诱导后.SDS—PAGE观察表达量。HIS—link层析柱纯化重组人HMGBlA—box.蛋白印迹鉴定重组蛋白。将重组蛋白加入hBMSC培养体系中培养一周后,碱性磷酸酶染色检测其促MSC成骨分化作用。结果经RT—PCR扩增得到了261bD的DNA片段,经测序分析.与GenBank中报道的已知序列完全一致,构建了含融合蛋白的重组表达质粒。经诱导后细菌高表达A—boX,表达量为总蛋白的45%。经层析柱纯化后,蛋白印迹证实目的蛋白为高纯度的A—box。将A—box加入MSC培养体系中培养后,细胞活性无明显改变.但细胞碱性磷酸酶表达明显增高。结论成功构建了重组人HMGBlA—Box的表达载体.纯化的重组蛋白能有效促进MSC向成骨细胞分化。
简介:摘要:文章探讨了Box-Jenkins模型(ARIMA模型)在汽车行业经济订货批量(EOQ)管理中的应用。选择了一家具有代表性的汽车制造企业作为研究实例,详细描述了Box-Jenkins模型在该企业EOQ管理中的应用过程。通过收集和分析历史销售数据、市场需求等时间序列信息,识别出数据中的关键特征。基于Box-Jenkins模型的基本原理,构建了适合该企业的ARIMA预测模型,并进行了参数估计和诊断。基于模型的预测结果,制定了优化的EOQ决策方案。
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简介:摘要: 本文旨在探讨上海莲花路地铁站直连商业室内设计项目的设计实践与创新。上海地铁1号线莲花路车站通过改造更新后建成的LaLa station不仅重焕了莲花路地铁站的活力,还成功打造了一个集公共交通换乘与综合性商业服务于一体的多功能空间。本文分析了项目的设计策略、主题空间创造以及细节体验,旨在为类似地铁直连商业空间及地铁更新改造项目的设计提供参考与启示。
简介:Rapidexpansionofbig-boxstoreindevelopingcountrycausedtypicalarchetypalchangeinmarketstructure:SuccesstotheSuccessful,becausebig-boxstoresarmedwithmodernizedinfrastructureandmanagementcapabilityareabsorbingtheoncecustomersofthetraditionalmarketlikeablackhole.Facingrapidchangeinmarketstructureandsurmountingpleasfromtraditionalmarketmerchants,governmenttookaninevitableinterventionwithlawregulatingthebig-boxstore’sbusinessandimprovingtraditionalmarket’scompetencebuilding.Notsolong,however,didgovernmentconfrontpolicyresistancefrombothsides:Stillongoingpolarizationofbothside’ssales.Thisstudyarticulatesbehaviorovertimeofmarketstructurewithcausalloopdiagramsofwhichcausalitiesareextractedfromliteratures.Thisstudyprovidessignificantcontributiontopolicymakersandtraditionalmarkets’merchantsinotherdevelopingcountrieslikeIndiaandChina,aswellasKorea.
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简介:摘要目的探讨微RNA(miR)-214靶向性别决定区Y-box 4(SOX4)对RA滑膜成纤维细胞增殖、凋亡的影响。方法收集2017—2019年商丘市第一人民医院就诊的45例RA患者的滑膜组织样本和30例接受关节置换手术的关节创伤患者的滑膜组织样本,分别作为RA组和对照组。通过实时荧光定量PCR(RT-qPCR)检测滑膜组织中miR-214和SOX4表达水平;ELISA法检测患者血清RF、CRP浓度,采用血沉动态分析仪检测ESR浓度,Pearson相关分析miR-214及SOX4与患者血清RF、CRP、ESR相关性。采用Lipofectamine 3000将人成纤维样滑膜细胞(HFLS)-RA细胞分为对照组、miR-214 mimic组、mimic-NC组、miR-214 mimic+pcDNA3.1-SOX4组。通过CCK8检测各组细胞活力,流式细胞仪检测各组细胞凋亡情况,蛋白质印迹法检测相关指标蛋白表达水平。2组比较采用独立样本t检验,多组比较采用单因素方差分析,两两比较采用LSD-t检验。结果与对照组(4.6±0.7,1.9±0.7)和HFLS细胞(1.00±0.06,1.00±0.09)比,miR-214在RA患者组织(2.6±0.9)和HFLS-RA(0.30±0.05)中低表达(t=10.026,P<0.05;t=15.815,P<0.05);SOX4在RA患者组织(4.6±0.9)和HFLS-RA(3.89±0.41)中高表达(t=14.772,P<0.05;t=12.020,P<0.05)。RA组患者血清RF、ESR、CRP表达水平[(46±7)U/ml、(46.4±9.6)mm/1 h、(34.8±9.8)mg/ml]较对照组[(16±4)U/ml、(9.2±2.0)mm/1 h、(2.1±0.7)mg/ml]显著升高(t=22.906,P<0.05;t=25.338,P<0.05;t=22.314,P<0.05)。患者血清RF、CRP、ESR与miR-214呈负相关(r=-0.574,P<0.05;r=-0.448,P<0.05;r=-0.549,P<0.05),与SOX4、ESR呈正相关(r=0.492,P<0.05;r=0.369,P<0.05;r=0.325,P<0.05)。转染miR-214 mimic 24 h后细胞活力、Ki-67和p-p65/p65蛋白表达量较对照组和mimic-NC组显著下降(F24 h=16.980、F48 h=42.735、F72 h=164.448、FKi-67=290.112、Fp-p65/p65=223.548,P<0.05),细胞凋亡率和Bax蛋白表达量显著升高(F=344.360,P<0.05;F=106.376,P<0.05)。过表达SOX4可逆转miR-214的作用。结论miR-214靶向SOX4抑制RA滑膜成纤维细胞增殖,并促进凋亡,其机制可能与miR-214抑制NF-κB信号通路有关。
简介:AbstractBackground:Myocardial infarction occurs due to insufficient (ischemia) blood supply to heart for long time; plasmacytoma variant translocation 1 (PVT1) is a long non-coding RNAs (lncRNAs) involved in the pathogenesis of various diseases, including heart disease; However, few studies have explored its role. The present study evaluated the effects of lncRNA PVT1 on hypoxic rat H9c2 cells.Methods:Hypoxic injury was examined by measuring cell viability and apoptosis by using cell counting kit-8 activity and flow cytometry assays. Gene expressions after hypoxia were estimated by quantitative real time polymerase chain reaction and the signaling pathway were explored by Western blot analysis. RNA immunoprecipitation and luciferase reporter assays were applied to examine the interactions among genes. Data were analyzed using t-test with one-way or two-way analysis of variance.Results:The lncRNA PVT1 is up-regulated in hypoxia-stressed H9c2 cells and knockdown of PVT1 mitigates hypoxia-induced injury in H9c2 cells. PVT1 acts as a sponge for miR-135a-5p and knockdown of PVT1 attenuated the increased hypoxia-induced injury by up-regulating miR-135a-5p. Forkhead box O1 (FOXO1) was identified as a target of miR-135a-5p, and the expression was negatively regulated by miR-135a-5p. The exploration of the underlying mechanism demonstrated that knockdown of FOXO1 reversed PVT1/miR-135a-5p mediated hypoxia-induced injury in H9c2 cells.Conclusions:PVT1 plays a crucial role in hypoxia-injured H9c2 cells through sponging miR-135a-5p and then positively regulating FOXO1.
简介:摘要目的探讨微小RNA(miR)-215-5p对白细胞介素(IL)-1β诱导的骨关节软骨细胞凋亡的影响及其机制。方法将骨关节炎软骨细胞ATDC5随机分为磷酸盐缓冲液(PBS)处理对照组和IL-1β处理模型组。为了分析miR-215-5p对IL-1β诱导细胞凋亡的影响,进一步的将IL-1β组分为:模型组+miR对照组,模型组+miR-215-5p抑制剂组。细胞转染后采用10 ng/ml IL-1β处理软骨细胞。采用流式细胞术检测细胞凋亡水平。采用实时定量反转录聚合酶链反应(RT-qPCR)检测miR-215-5p和锌指结构E-box同源结合框2(ZEB2)的表达水平。蛋白质印迹法(Western blot)检测ZEB2、p65、磷酸化p65、裂解的半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved Caspase-3)蛋白水平。将野生型ZEB2的3’-非编码区(pmiR-ZEB2-WT)或者突变型ZEB2的3’-非编码区(pmiR-ZEB2-Mut)分别与miR-215-5p模拟物(miR-215-5p mimic)和对照物(miR-NC)共转染细胞,双荧光素酶报告基因测定实验研究miR-215-5p与ZEB2的相互作用。结果与对照组比较,miR-215-5p在IL-1β诱导软骨细胞凋亡模型中表达上调(2.87±0.07)倍。双荧光素酶报告实验发现ZEB2是miR-215-5p的直接作用靶点。miR-215-5p mimic处理可使转染pmiR-ZEB2-WT的细胞荧光素酶活性降低为miRNA对照组的40.2%,而在转染pmiR-ZEB2-Mut的细胞中miR-215-5p mimic组与对照组比较,差异无统计学意义。miR-215-5p抑制剂使IL-1β诱导的软骨细胞凋亡水平降低为对照组的50.9%,并且可降低软骨细胞凋亡标志物cleaved Caspase-3水平和核因子-κB(NF-κB) p65的磷酸化水平为对照组的68.2%和75.4%。结论miR-215-5p通过靶向ZEB2促进白细胞介素-1β诱导软骨细胞凋亡。