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靶向Nectin1的DNA适配体筛选及其对伪狂犬病病毒体外感染细胞的吸附阻断效应
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作者 乔岩 赵家富 +3 位作者 常潇天 陈征 韩莹倩 张超 《畜牧兽医学报》 北大核心 2026年第2期979-993,共15页
本研究旨在可溶性原核表达伪狂犬病病毒(PRV)受体Nectin1蛋白的N端胞外结构域,并以此为靶标,筛选出高亲和力、高特异性的DNA适配体,为阻断伪狂犬病病毒吸附宿主细胞提供新的分子工具。将猪源Nectin1基因克隆至带有麦芽糖结合蛋白(MBP)... 本研究旨在可溶性原核表达伪狂犬病病毒(PRV)受体Nectin1蛋白的N端胞外结构域,并以此为靶标,筛选出高亲和力、高特异性的DNA适配体,为阻断伪狂犬病病毒吸附宿主细胞提供新的分子工具。将猪源Nectin1基因克隆至带有麦芽糖结合蛋白(MBP)促溶标签的原核表达载体pET28a中,构建重组质粒pET28a-MBPNectin1,将双酶切鉴定和测序正确的重组质粒转入大肠杆菌BL21(DE3)进行诱导表达,使用Western blot和Pulldown试验验证重组蛋白。采用基于磁珠偶联的系统进化适配体筛选技术(SELEX)筛选特异性靶向rMBPNectin1的DNA适配体,并利用磁珠ELASA检测筛选所得适配体Apt-Nectin1-6的亲和力与特异性。以敲减Nectin1蛋白的细胞系(sh-Nectin1-1、sh-Nectin1-2)为对照,应用流式细胞术验证Apt-Nectin1-6对细胞表面Nectin1蛋白的结合能力,同时通过流式细胞术和基因组拷贝数试验评估其对PRV吸附细胞的抑制作用。Western blot和Pull-down结果表明重组MBP-Nectin1蛋白正确表达且具有生物学活性。通过SELEX技术筛选获得的适配体Apt-Nectin1-6对rMBP-Nectin1具有高亲和力,平衡解离常数(Kd)为(0.11±0.02)nmol,且不与rMBP-gD、rMBP-S1、rMBP-CD163及rMBP蛋白发生交叉反应。分子对接结果显示,Apt-Nectin1-6通过氢键与Nectin1结合。进一步流式细胞术、基因组拷贝数检测和病毒滴度测定结果显示,Apt-Nectin1-6可剂量依赖性地抑制PRV对细胞的吸附(P<0.05)。本研究成功筛选得到可高特异性、高亲和力结合Nectin1蛋白的DNA适配体Apt-Nectin1-6,并证实其可有效阻断PRV对细胞的吸附,为开发新的抗PRV策略奠定基础。 展开更多
关键词 伪狂犬病病毒 nectin1 SELEX DNA适配体 病毒吸附阻断
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NECTIN1缺失在食管鳞癌发生和发展中的作用及其机制
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作者 杨航 潘英杰 +6 位作者 龙元凤 张佳怡 乔彦 刘云 杨思芸 宋桂芹 刘康 《基因组学与应用生物学》 北大核心 2025年第1期92-106,共15页
本研究旨在探究黏连蛋白细胞黏附分子1(nectin cell adhesion molecule 1,NECTIN1)缺失对食管鳞癌(esophageal squamous cell carcinoma,ESCC)细胞发生和发展的影响及其分子作用机制。利用UCSC数据库分析食管癌(esophageal carcinoma,ES... 本研究旨在探究黏连蛋白细胞黏附分子1(nectin cell adhesion molecule 1,NECTIN1)缺失对食管鳞癌(esophageal squamous cell carcinoma,ESCC)细胞发生和发展的影响及其分子作用机制。利用UCSC数据库分析食管癌(esophageal carcinoma,ESCA)中NECTIN1基因的线性拷贝数,通过Western blot和免疫组化染色分析NECTIN1在食管鳞癌细胞、食管鳞癌组织和癌旁组织中的蛋白水平。利用TISIDB数据库分析NECTIN1表达与食管癌临床特征的相关性。采用shRNA敲低NECTIN1在KYSE150和KYSE510细胞中的表达,利用CCK-8实验、克隆形成实验、细胞划痕和Transwell实验以及流式细胞术检测敲低NECTIN1对细胞功能的影响。对敲低NECTIN1的KYSE510细胞进行转录组测序,筛选差异表达基因;通过GO、KEGG富集分析,探讨NECTIN1可能影响的信号调控网络。结果表明,NECTIN1在部分食管癌患者中拷贝数减少,蛋白水平显著低于癌旁组织的(P<0.0001),NECTIN1低表达的食管鳞癌患者分化较差(P<0.0001),与临床晚期分期呈负相关。敲低NECTIN1基因后KYSE150和KYSE510细胞增殖、迁移和侵袭能力均增强(P<0.05),细胞S期增多,并使细胞凋亡减少(P<0.01)。在KYSE150细胞中敲低NECTIN1,进行转录组测序分析后发现115个基因显著上调、209个基因显著下调。KEGG分析表明,差异表达基因富集于肿瘤坏死因子(tumor necrosis factor,TNF)信号通路(P<0.01),通过GEPIA数据库分析发现,在ESCA患者中,该通路中的3个基因(IL1B、TNFAIP3、CSF2)的表达水平与NECTIN1呈显著正相关(P<0.05),与转录组测序结果相符。NECTIN1的缺失促进了ESCC细胞增殖、迁移和侵袭,其机制可能是通过TNF信号通路发挥抑癌作用,调控ESCC的发生与发展。 展开更多
关键词 黏连蛋白细胞黏附分子1(nectin1) 食管鳞癌(ESCC) 增殖 侵袭 肿瘤坏死因子(TNF)信号通路
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Research progress in mulberry leaf polysaccharide in treating diabetic nephropathy 被引量:1
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作者 ZHANG Shi-lin JING Yong-shuai +2 位作者 ZHANG Dan-shen ZHENG Yu-guang WU Lan-fang 《中国药理学与毒理学杂志》 CAS 北大核心 2021年第10期778-779,共2页
Diabetic nephropathy(DN)is one of the most common complications of diabetes.It is an important cause of diabetes disability and death.DN is a systemic metabolic syndrome.In its pathogenesis,the interaction of various ... Diabetic nephropathy(DN)is one of the most common complications of diabetes.It is an important cause of diabetes disability and death.DN is a systemic metabolic syndrome.In its pathogenesis,the interaction of various cell activities and a large number of cytokine biological activities,the activation of signal pathways and so on are involved in the development of DN.At present,the clinical treatment of DN is mainly Western medicine,but it has limitations such as strong toxicity,high side effects and poor compliance.Therefore,the discovery of natural anti-DN substances has also become an important means to treat DN.Mulberry leaves are the dry leaves of Morus alba L.It is not only a traditional Chinese medicine,but also a dual-purpose medicinal material for medicine and food.It has the effects of dispelling wind and clearing heat,cooling blood and brightening eyes,tonifying and so on.Mulberry leaf polysaccharide(MLP)is a kind of high molecular compound in mulberry leaves.It has many pharmacological effects,such as hypoglycemic,antioxidant,anti-stress,anti-virus and so on.Therefore,the pharmacological effects of mulberry leaf polysaccharides on diabetic nephropathy are reviewed in this paper,so as to provide references for further research and application.The pathogenesis of DN is complex,and the mechanism of renal injury has not been completely clarified.The current studies believe that DN is closely related to heredity,abnormal glucose metabolism,abnormal lipid metabolism,microcirculation disorder,cytokine action,oxidative stress and so on.Relevant studies show that the pharmacological effects of mulberry leaf polysaccharide in the prevention and treatment of DN mainly include:①Effect on transforming factor-β1(TGF-β1):TGF-β1 has become an important cytokine involved in the formation of renal fibrosis by regulating cell proliferation and differentiation and the production of extracellular matrix(ECM).MLP can significantly inhibit TGF-β1 protein,and then inhibit the synthesis of extracellular matrix by renal interstitial fibroblasts and inhibit the realization of fibrosis.②Effect on insulin receptor substrate(IRS-1):IRS-1 is an important signal molecule at the beginning of IR signal transduction.The decrease of IRS-1 gene expression or the decrease of expression can affect the effective transmission of IR signal and lead to the development and deterioration of diabetes. MPL can significantly increase the expression of IRS-1 mRNA in liver tissue of DN rats, so as to prevent and treat DN. ③ Effect on the expression of resistin protein in adipose tis sue. Resistin is a secretory polypeptide derived from adipose tissue and is specifically expressed in white adipose tissue and is closely related to type 2 diabetes mellitus (T2DM). Experimental studies show that MLP can effectively reduce the expression of resistin protein in white adipose tissue of T2DM rats, indicating that MLP may reduce the level of IR by inhibiting the expression of resistin in adipose tissue, thereby reducing the insulin resistance state of T2DM rats, so as to achieve the goal of treating diabetes. ④ Effect on adiponectin receptor 1 (AdipoR1): adiponectin can improve insulin resistance, reduce blood glucose and lipid. AdipoR1 is mainly expressed in skeletal muscle and kidney. Studies have shown that AdipoR1 is closely related to the occurrence and development of DN. The results showed that MLP could reduce the blood glucose and blood lipid level and up regulate the expression of AdipoR1 mRNA in DN rats, suggesting that MLP may delay the occurrence and development of DN. This article reviewed the pharmacological effects of mulberry leaf polysaccharides on diabetic nephropathy, and provided a useful basis for further development and utilization of mul berry leaf polysaccharides in the treatment of DN. 展开更多
关键词 mulberry leaf polysaccharide diabetic nephropathy TGF-β1 insulin receptor substrate RESISTIN adipo⁃nectin receptor 1
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