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关键词： 金环蛇   多克隆抗体   胃蛋白酶   F(ab′)_(2)片段   层析纯化  

摘要： 为制备高纯度马源金环蛇毒多克隆抗体F(ab′)_(2),以金环蛇毒高免马血浆为原料,采用Protein G亲和层析捕获马血浆中的IgG,经胃蛋白酶酶解后制得F(ab′)_(2),分别通过亲和层析、凝胶过滤层析和混合模式层析对F(ab′)_(2)进行精制纯化,并以间接ELISA法测定F(ab′)_(2)对金环蛇毒的中和活性。结果表明:Protein G亲和层析捕获的IgG纯度为83.6%,较传统的盐析纯化法有明显提高;在3种层析精制方法中,通过凝胶过滤层析精制的F(ab′)_(2)纯度最高,为89.3%,远高于《中华人民共和国药典》标准(60%以上),且收率为56.0%;间接ELISA法测定结果显示,F(ab′)_(2)的效价高于1∶4096。该研究为马源抗毒素多克隆抗体F(ab′)_(2)的工业化制备提供了工艺参考。

关键词： 淋巴瘤   多发性骨髓瘤   铁代谢   铁死亡  

摘要： 铁作为一种重要的微量元素,参与机体的多种生理过程。研究发现,肿瘤的发生发展与铁代谢异常密切相关,且作用方式呈现明显异质性。肿瘤细胞需要更多的铁来促进自身存活和增殖,但过量的铁也会对肿瘤细胞产生不利的影响,如铁死亡等。铁死亡是一种特殊的调节性细胞死亡机制,不同于其他的细胞调节死亡途径,其主要通过铁依赖的过氧化脂质和活性氧(ROS)过度积累,从而诱导细胞死亡。在血液系统中,淋巴瘤及多发性骨髓瘤(MM)的肿瘤细胞都对铁死亡较敏感,且通过多种机制影响疾病进展。本文就淋巴瘤的部分亚型及MM中的铁死亡机制进行阐述,揭示血液肿瘤细胞铁死亡与血液肿瘤发生发展的相关性,旨在为治疗这些血液系统疾病提供新的思路。

关键词： 后发性白内障   白介素-8(IL-8)   晶状体上皮细胞   细胞迁移   单核细胞趋化蛋白1(MCP-1)   NF-κB/p65信号通路  

摘要： 目的:探究炎症因子白介素-8(IL-8)影响后发性白内障(PCO)细胞迁移的过程中对晶状体上皮细胞(LEC)分泌的单核细胞趋化蛋白1(MCP-1)的调控作用。方法:构建大鼠晶状体囊袋模型,采用10%胎牛血清培养撕囊后的晶状体囊袋使LEC迁移至后囊30%-50%面积后,撤去血清,将囊袋分为对照组和15 ng/mL IL-8组,并在不同时间点进行拍照观察囊袋LEC迁移。ELISA法和RT-qPCR检测不同组MCP-1的分泌和信使RNA(mRNA)相对表达水平。免疫荧光检测不同组MCP-1表达水平。将SRA01/04细胞分为对照组、15 ng/mL IL-8组和15 ng/mL IL-8+200μmol/L Bindarit(BND)组,用Transwell法检测不同组迁移细胞数量。将SRA01/04细胞分为阴性对照(NC)组、NC+15 ng/mL IL-8组及15 ng/mL IL-8+p65 siRNA组,ELISA法和RT-qPCR检测不同组MCP-1的分泌和mRNA相对表达水平。结果:体外培养的大鼠晶状体囊袋LEC迁移水平显示,在48、72、96 h时15 ng/mL IL-8组囊袋内细胞迁移明显增加(均P<0.05)。ELISA的结果显示,15 ng/mL IL-8组囊袋和SRA01/04细胞与对照组相比较,在12、24 h分泌的MCP-1的水平升高(均P<0.05);RT-qPCR的结果同样显示15 ng/mL IL-8组SRA01/04细胞在12、24 h分泌的MCP-1 mRNA的相对表达水平增高(均P<0.05)。免疫荧光的结果显示,与对照组相比,24 h时15 ng/mL IL-8组的囊袋上皮细胞MCP-1表达水平升高(P=0.007)。Transwell的检测结果显示,与对照组相比,15 ng/mL IL-8组迁移数量增加(P=0.001);与15 ng/mL IL-8组相比,15 ng/mL IL-8+200μmol/L BND组迁移数量减少(P=0.003)。ELISA和RT-qPCR的结果显示,与NC组相比,NC+15 ng/mL IL-8组在12、24 h时的MCP-1分泌和mRNA的相对表达增加(均P<0.01);与NC+15 ng/mL IL-8组比较,15 ng/mL IL-8+p65 siRNA组在12、24 h时的MCP-1分泌和mRNA的相对表达减少(均P<0.01)。结论:IL-8可促进囊袋内残留上皮细胞的迁移,调控晶状体上皮细胞MCP-1的分泌和表达水平,推测其作用机制与NF-κB/p65信号通路有关。

关键词： 血液恶性肿瘤   血流感染   T淋巴细胞亚群   诊断价值  

摘要： 目的探讨外周血T淋巴细胞亚群检测对血液恶性肿瘤患者合并血流感染(BSI)的诊断价值。方法前瞻性选取2023年1月至2023年11月四川省隆昌市人民医院收治的120例血液恶性肿瘤合并BSI患者作为BSI组,选取同期收治的135例未合并BSI的血液恶性肿瘤患者作为非BSI组,另选取同期139例健康志愿者作为对照组。采用流式细胞仪检测三组患者血清CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞百分比。采用多因素logistic回归分析探讨血液恶性肿瘤患者合并BSI的影响因素。受试者操作特征(ROC)曲线分析CD3^(+)T淋巴细胞百分比、CD4^(+)T淋巴细胞百分比及CD4^(+)/CD8^(+)比值对血液恶性肿瘤患者合并BSI的诊断价值。结果BSI组120例患者血培养共检出菌株132株,其中革兰氏阴性菌占65.91%,革兰氏阳性菌占28.79%,真菌占5.30%。BSI组、非BSI组CD3^(+)T淋巴细胞百分比、CD4^(+)T淋巴细胞百分比及CD4^(+)/CD8^(+)比值均明显低于对照组,且BSI组低于非BSI组,差异有统计学意义(P<0.05)。多因素logistic回归分析结果显示,CD3^(+)T淋巴细胞百分比(OR=0.773)、CD4^(+)T淋巴细胞百分比(OR=0.745)、CD4^(+)/CD8^(+)比值(OR=0.714)、降钙素原(PCT)(OR=1.874)、白细胞计数(OR=1.742)、经外周静脉置入中心静脉导管留置时间(OR=1.581)、抗生素使用种类(OR=1.396)均为血液恶性肿瘤患者合并BSI的影响因素(P<0.05)。ROC曲线分析结果显示,CD3^(+)T淋巴细胞百分比、CD4^(+)T淋巴细胞百分比、CD4^(+)/CD8^(+)比值联合诊断恶性血液肿瘤患者合并BSI的曲线下面积(0.913,95%CI 0.872~0.945)高于CD3^(+)T淋巴细胞(Z=3.716,P<0.001)、CD4^(+)T淋巴细胞(Z=4.093,P<0.001)、CD4^(+)/CD8^(+)比值(Z=4.764,P<0.001)单独诊断。结论血液恶性肿瘤合并BSI患者的T淋巴细胞亚群失衡,CD3^(+)T淋巴细胞百分比、CD4^(+)T淋巴细胞百分比、CD4^(+)/CD8^(+)比值联合应用对血液恶性肿瘤患者合并BSI具有一定诊断价值。

关键词： 鸡传染性贫血病毒   转移因子   免疫抑制   细胞因子  

摘要： 为探索转移因子的免疫增强作用及其对鸡传染性贫血病毒(Chicken infectious anemia virus,CIAV)感染的抑制作用,试验研究了高、低两种剂量转移因子对SPF鸡CD4^(+)和CD8^(+)T淋巴细胞水平,鸡白细胞介素-12(IL-12)、γ-干扰素(IFN-γ)等6种细胞因子和Mx抗病毒蛋白表达的影响,以及对SPF鸡体重、器官发育指数、红细胞压积(HCT)值和血液、肝脏、胸腺中CIAV增殖的影响。结果显示:转移因子可引起CD4^(+)和CD8^(+)T细胞比重显著增加并上调IFN-γ、Mx蛋白表达量(P<0.01);CIAV感染组的脾脏发育指数相对于空白对照组显著增加(P<0.05),胸腺发育指数极显著降低(P<0.01),转移因子可以缓解感染CIAV带来的脾脏肿大和胸腺萎缩;转移因子可显著抑制CIAV在血液、肝脏和胸腺中的复制(P<0.01),且高剂量组抑制作用强于低剂量组;CIAV感染组血浆HCT值极显著低于空白对照组(P<0.01),高剂量转移因子使用后缓解了CIAV感染引起的HCT值下降。研究表明,转移因子对缓解CIAV感染引起的危害具有良好的应用价值,为防控鸡传染性贫血提供新的辅助措施。

摘要： Corticotomy is a clinical procedure to accelerate orthodontic tooth movement characterized by the regional acceleratory phenomenon (RAP). Despite its therapeutic effects, the surgical risk and unclear mechanism hamper the clinical application. Numerous evidences support macrophages as the key immune cells during bone remodeling. Our study discovered that the monocyte-derived macrophages primarily exhibited a pro-inflammatory phenotype that dominated bone remodeling in corticotomy by CX3CR1CreERT2;R26GFP lineage tracing system. Fluorescence staining, flow cytometry analysis, and western blot determined the significantly enhanced expression of binding immunoglobulin protein (BiP) and emphasized the activation of sensor activating transcription factor 6 (ATF6) in macrophages. Then, we verified that macrophage specific ATF6 deletion (ATF6f/f;CX3CR1CreERT2 mice) decreased the proportion of pro-inflammatory macrophages and therefore blocked the acceleration effect of corticotomy. In contrast, macrophage ATF6 overexpression exaggerated the acceleration of orthodontic tooth movement. In vitro experiments also proved that higher proportion of pro-inflammatory macrophages was positively correlated with higher expression of ATF6. At the mechanism level, RNA-seq and CUT&Tag analysis demonstrated that ATF6 modulated the macrophage-orchestrated inflammation through interacting with Tnf alpha promotor and augmenting its transcription. Additionally, molecular docking simulation and dual-luciferase reporter system indicated the possible binding sites outside of the traditional endoplasmic reticulum-stress response element (ERSE). Taken together, ATF6 may aggravate orthodontic bone remodeling by promoting Tnf alpha transcription in macrophages, suggesting that ATF6 may represent a promising therapeutic target for non-invasive accelerated orthodontics.

摘要： NF-kappa B mediates transcriptional regulation crucial to many biological functions, and elevated NF-kappa B activity leads to autoimmune and inflammatory diseases, as well as cancer. Since highly aggressive breast cancers have few therapeutic molecular targets, clarification of key molecular mechanisms of NF-kappa B signaling would facilitate the development of more effective therapy. In this report, we show that Tob, a member of the Tob/BTG family of antiproliferative proteins, acts as a negative regulator of the NF-kappa B signal in breast cancer. Studies with 35 human breast cancer cell lines reveal that Tob expression is negatively correlated with NF-kappa B activity. Analysis of The Cancer Genome Atlas (TCGA) database of clinical samples reveals an inverse correlation between Tob expression and NF-kappa B activity. Tob knockdown in human breast cancer cells promoted overactivation of NF-kappa B upon TNF-alpha treatment, whereas overexpression of Tob inhibited TNF-alpha stimulation-dependent NF-kappa B activation. Mechanistically, Tob associates with the TNF receptor complex I and consequently inhibits RIPK1 polyubiquitylation, leading to possible prevention of overwhelming activation of NF-kappa B.

关键词： 槲皮素   IL-17   内皮细胞炎症   单核-内皮细胞黏附   NF-κB  

摘要： 目的:探讨槲皮素是否通过NF-κB通路抑制IL-17诱导的单核-内皮细胞黏附。方法:体外培养人血脐静脉融合细胞株***926,加入不同浓度的IL-17,MTS法检测内皮细胞活力,筛选IL-17的最佳干预浓度,建立IL-17诱导的内皮细胞炎症模型。与人单核细胞株THP-1共同培养,建立单核-内皮细胞黏附模型,以槲皮素进行干预,MTS法筛选槲皮素的最佳干预浓度,免疫荧光法观察槲皮素抑制单核-内皮细胞的黏附及NF-κB p65核转位情况。Western Blot法检测NF-κB p65、p-NF-κB p65、IκBα、p-IκBα蛋白表达,RT-PCR及ELISA法检测vWF、ICAM-1、VCAM-1、MCP-1 mRNA及蛋白表达。结果:150 ng/mL IL-17可以诱导***926建立内皮细胞炎症模型,与单核细胞共培养,可以建立单核-内皮细胞黏附模型,0.5μmol/L是槲皮素的最佳干预浓度。槲皮素可抑制IL-17诱导的内皮细胞炎症模型vWF蛋白及mRNA表达;可减少单核-内皮细胞黏附数,但无浓度依赖性;可显著降低p-NF-κB p65/NF-κB p65、p-IκBα/IκBα比值,减少NF-κB p65入核数量,显著下调ICAM-1、VCAM-1、MCP-1的蛋白及mRNA表达水平。结论:槲皮素能有效抑制IL-17诱导的内皮细胞炎症反应,抑制单核细胞与内皮细胞黏附,其机制可能与抑制NF-κB通路及其下游炎症因子ICAM-1、VCAM-1、MCP-1的表达有关。

关键词： MitoTEMPOL   endothelium   inflammation   vascular dysfunction  

摘要： Chronic inflammation is a major contributor to the development of endothelial dysfunction. Circulating concentrations of the proinflammatory cytokine interleukin-6 (IL-6) have been shown to predict cardiovascular disease risk and are associated with the development of vascular dysfunction. However, the mechanisms that underlie inflammation-induced endothelial dysfunction are not fully understood. Vascular endothelial dysfunction is characterized by blunted nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS), with mitochondrial ROS suggested to play a primary role. Therefore, we tested the hypothesis that IL-6 induces mitochondrial ROS production and blunts NO bioavailability in endothelial cells. To study the effect of IL-6, we treated the human aortic endothelial cells (HAECs) with IL-6, MitoTEMPOL (MT; a mitochondria-targeted antioxidant), and/or a nitric oxide synthase (NOS) inhibitor (l-NAME) with and without ACh stimulation. Results are expressed as means ± SD ( = 4 replicates), one-way ANOVA, and Bonferroni's post hoc tests were performed. IL-6 treatment resulted in greater mitochondrial ROS (IL-6: 2.94 ± 0.93 a.u.) when compared with the untreated cells (Control: 1 ± 0; = 0.0021) and also blunted NO bioavailability at baseline (Control: 1 ± 0; IL-6: 0.57 ± 0.08 a.u. = 0.0008) and with acetylcholine stimulation (Control Ach: 1.27 ± 0.09; IL-6 Ach: 0.60 ± 0.13 a.u. < 0.0001). Scavenging mitochondrial ROS with MT restored NO bioavailability in the IL-6-treated cells (IL-6: 0.57 ± 0.08; IL-6 MT: 1.16 ± 0.20 a.u. < 0.0001). These findings indicate that IL-6 has a direct effect on mitochondrial ROS in human aortic endothelial cells, which leads to reduced nitric oxide bioavailability. In this study, we provide evidence that IL-6 induces mitochondrial ROS production, which impairs nitric oxide bioavailability in human aortic endothelial cells. This finding adds an important perspective to the existing literature on the mechanism by whic

关键词： VZV   等电点   毛细管等电聚焦电泳   脱糖酶  

摘要： 目的:建立重组带状疱疹gE抗原等电点(isoelectric point, pI)的全柱成像毛细管等电聚焦电泳(capillary isoelectric focusing, cIEF)检测方法。方法:通过优化重组带状疱疹gE抗原等电点检测过程中的两性电解质比例、蛋白质浓度、稳定剂比例参数,建立重组带状疱疹gE抗原的等电点检测方法(cIEF法)。在此基础上,对重组带状疱疹gE抗原进行脱糖酶处理后,采用cIEF法检测等电点。结果:优化后的检测条件为2 M尿素、0.35%甲基纤维素(methylcellulose, MC)、4%两性电解质、1%等电点标记物混合溶液作为样品缓冲液,聚焦条件为1500 V 1 min,3000 V 8 min。重组带状疱疹gE抗原经PNGase F酶脱糖后,采用该方法分析等电点,可得到峰型清楚且分离度高的图谱。结论:本研究建立的cIEF方法适用于重组带状疱疹gE抗原的pI检测及分析,可为重组带状疱疹gE抗原的表征以及相关疫苗研发和生产过程中的质量控制提供依据和参考。Objective: To establish a full column imaging capillary isoelectric focusing (cIEF) detection method for recombinant herpes zoster gE antigen isoelectric point (pI). Method: By optimizing the parameters such as reagent ratio, final sample concentration, and sample stabilizer in the process of detecting recombinant herpes zoster gE antigen, a cIEF method for determining recombinant herpes zoster gE antigen was established. Based on this, the recombinant herpes zoster gE antigen was subjected to enzymatic digestion before cIEF detection. Results: The optimized method uses a mixed solution of 2 M urea, 0.35% methylcellulose (MC), 4% zwitterionic electrolyte, and 1% isoelectric point marker as the sample buffer, with focusing conditions of 1500 V for 1 min and 3000 V for 8 min. After PNGase F enzyme deglycosylation, this method can be used to analyze the isoelectric point of recombinant herpes zoster gE antigen, and a clear peak pattern and high separation degree can be obtained. Conclusion: The established cIEF method is suitable for pI detection and analysis of recombinant herpes zoster gE antigen, which can provide a basis for the characterization of recombinant herpes zoster gE antigen and a reference for quality control in the development and production of related vaccines.