目的:探究黑色素瘤中膜联蛋白A5(annexinA5,ANXA5)表达与铁死亡的相关性。方法:利用The Human Protein Atlas数据分析ANXA5在黑色素瘤中的表达情况,并采用人黑素瘤组织芯片进行免疫组化验证,再以黑色素瘤细胞B16F10为研究对象,通过CRISP...目的:探究黑色素瘤中膜联蛋白A5(annexinA5,ANXA5)表达与铁死亡的相关性。方法:利用The Human Protein Atlas数据分析ANXA5在黑色素瘤中的表达情况,并采用人黑素瘤组织芯片进行免疫组化验证,再以黑色素瘤细胞B16F10为研究对象,通过CRISPR-Cas9技术构建ANXA5敲除株,通过CCK-8及乳酸脱氢酶(lactate dehydrogenase,LDH)检测敲除ANXA5对B16F10铁死亡效应的影响,进一步通过RNA-seq寻找ANXA5调节铁死亡可能的靶分子并验证,最后在不同的黑色素瘤细胞中检测内源性ANXA5表达水平,并确认ANXA5对铁死亡敏感性的调节作用。结果:ANXA5在黑色素瘤组织中异常高表达,敲除或敲低ANXA5能促进黑色素瘤细胞对铁死亡的敏感性。酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long-chain family member 4,ACSL4)作为铁死亡关键调节分子,与ANXA5表达呈负相关性,过表达ANXA5能降低ACSL4水平,表现出对铁死亡的抵抗性。结论:在黑色素瘤细胞中,敲低ANXA5能够上调ACSL4水平,增强黑色瘤细胞对铁死亡的敏感性,提示ANXA5有望成为黑色素瘤铁死亡治疗的潜在靶点。展开更多
目的建立快速检测CYP3A5基因型的新方法及其临床应用。方法针对CYP3A5不同基因型特异性位点设计引物,采用等位基因特异性PCR方法(Allele specific PCR,AS-PCR)进行扩增,通过琼脂糖凝胶电泳分辨不同基因型,并检测临床标本100例,与一代测...目的建立快速检测CYP3A5基因型的新方法及其临床应用。方法针对CYP3A5不同基因型特异性位点设计引物,采用等位基因特异性PCR方法(Allele specific PCR,AS-PCR)进行扩增,通过琼脂糖凝胶电泳分辨不同基因型,并检测临床标本100例,与一代测序法比对两个方法的一致率。结果新方法检测100例临床标本中,CYP3A5基因型*1/*1型15例;*1/*3型26例;*3/*3型59例,结果与一代测序法一致,一致率100%。结论通过AS-PCR检测CYP3A5基因型,操作简便,结果易于判断,可应用于临床。展开更多
目的:急性脑梗死病情进展迅速,常导致神经不可逆损伤,预后较差,需寻找相关预后标志物以了解预后情况。本研究旨在分析细胞色素P450(cytochrome P450,CYP)3A5基因多态性联合血清微RNA-543(microRNA-543,miR-543)预测急性脑梗死患者预后...目的:急性脑梗死病情进展迅速,常导致神经不可逆损伤,预后较差,需寻找相关预后标志物以了解预后情况。本研究旨在分析细胞色素P450(cytochrome P450,CYP)3A5基因多态性联合血清微RNA-543(microRNA-543,miR-543)预测急性脑梗死患者预后不良的价值。方法:选取2021年5月至2024年1月在郑州市第七人民医院诊断为急性脑梗死的176例患者作为研究对象,依据患者出院90 d后的门诊改良Rankin量表评分结果,将评分≤2的患者纳入预后良好组(n=92),评分>2的纳入预后不良组(n=84)。采用实时荧光定量PCR法检测治疗前血清miR-543水平,采用PCR法检测治疗前CYP3A5基因型,采用多因素Logistic回归分析影响急性脑梗死患者预后不良的因素,采用受试者操作特征(receiver operating characteristic,ROC)曲线分析CYP3A5基因rs776746位点GG基因型联合血清miR-543水平对急性脑梗死患者预后不良的预测价值。结果:CYP3A5基因rs776746位点上检出AA型、GA型、GG型3种基因型。预后不良组发病至溶栓时间长于预后良好组,血清miR-543水平、携带CYP3A5基因rs776746位点GG基因型及G等位基因、入院时美国国立卫生研究院卒中量表(National Institutes of Health Stroke Scale,NIHSS)评分≥15和高血压患者比例均显著高于预后良好组(均P<0.001)。携带CYP3A5基因rs776746位点GG基因型和高miR-543水平是急性脑梗死患者预后不良的危险因素(均P<0.05)。CYP3A5基因rs776746位点GG基因型和血清miR-543水平单独及联合预测急性脑梗死患者预后不良的曲线下面积(area under the curve,AUC)分别为0.702、0.833、0.932,敏感度分别为52.38%、75.68%、92.86%,特异度分别为88.04%、78.33%、93.48%。结论:携带CYP3A5基因rs776746位点GG基因型和高miR-543水平均与急性脑梗死患者预后不良有关,二者联合可显著提高预测效能。展开更多
Ischemic stroke remains a leading cause of neurological disability worldwide.Effective therapeutic interventions for stroke treatment are still critically needed.Annexin A5,a calcium-dependent phospholipid-binding pro...Ischemic stroke remains a leading cause of neurological disability worldwide.Effective therapeutic interventions for stroke treatment are still critically needed.Annexin A5,a calcium-dependent phospholipid-binding protein,has been reported to possess a wide range of biological functions,including anti-inflammation,anticoagulation and antithrombotic,and apoptosis regulation.It also has protective effects on the ischemic stroke.Previous studies have demonstrated that Annexin A5 has neuroprotective effects during ischemic stroke.However,there is still a lack of systematic research on the exact mechanism.Our study aims to clarify the related signaling pathways of Annexin A5 after ischemic stroke.In the present study,we validated that Annexin A5 administration significantly enhanced neuronal survival and attenuated apoptotic processes in both in vitro and in vivo models.As expected,Annexin A5 treatment led to a reduction in infarct volume and improved neurological function scores in tMCAO mouse models.Notably,RNA-seq identified 1430 differentially expressed genes(DEGs)between OGD/R and Annexin A5-treated groups.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed involvement in multiple signaling cascades,such as apoptosis,TGF-βpathway,NOD-like receptor pathway,phagosome,PI3K-Akt pathway,necroptosis,NF-kB pathway.These findings were subsequently validated through qRT-PCR analysis,showing high concordance with RNA-seq results.Our integrated findings propose the multi-target mechanism positions Annexin A5 as a potential therapeutic candidate for ischemic stroke management.展开更多
基金National Natural Science Foundation of China(No.81600249,82470055)Natural Science Foundation of Hunan Province(No.2022JJ30823)+1 种基金General Funding Project of Hunan Provincial Health Commission(No.202103010961,W20243119)Changsha Natural Science Foundation,China(No.kq2202392,kq2502197)。
文摘目的:探究黑色素瘤中膜联蛋白A5(annexinA5,ANXA5)表达与铁死亡的相关性。方法:利用The Human Protein Atlas数据分析ANXA5在黑色素瘤中的表达情况,并采用人黑素瘤组织芯片进行免疫组化验证,再以黑色素瘤细胞B16F10为研究对象,通过CRISPR-Cas9技术构建ANXA5敲除株,通过CCK-8及乳酸脱氢酶(lactate dehydrogenase,LDH)检测敲除ANXA5对B16F10铁死亡效应的影响,进一步通过RNA-seq寻找ANXA5调节铁死亡可能的靶分子并验证,最后在不同的黑色素瘤细胞中检测内源性ANXA5表达水平,并确认ANXA5对铁死亡敏感性的调节作用。结果:ANXA5在黑色素瘤组织中异常高表达,敲除或敲低ANXA5能促进黑色素瘤细胞对铁死亡的敏感性。酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long-chain family member 4,ACSL4)作为铁死亡关键调节分子,与ANXA5表达呈负相关性,过表达ANXA5能降低ACSL4水平,表现出对铁死亡的抵抗性。结论:在黑色素瘤细胞中,敲低ANXA5能够上调ACSL4水平,增强黑色瘤细胞对铁死亡的敏感性,提示ANXA5有望成为黑色素瘤铁死亡治疗的潜在靶点。
文摘目的建立快速检测CYP3A5基因型的新方法及其临床应用。方法针对CYP3A5不同基因型特异性位点设计引物,采用等位基因特异性PCR方法(Allele specific PCR,AS-PCR)进行扩增,通过琼脂糖凝胶电泳分辨不同基因型,并检测临床标本100例,与一代测序法比对两个方法的一致率。结果新方法检测100例临床标本中,CYP3A5基因型*1/*1型15例;*1/*3型26例;*3/*3型59例,结果与一代测序法一致,一致率100%。结论通过AS-PCR检测CYP3A5基因型,操作简便,结果易于判断,可应用于临床。
文摘目的:急性脑梗死病情进展迅速,常导致神经不可逆损伤,预后较差,需寻找相关预后标志物以了解预后情况。本研究旨在分析细胞色素P450(cytochrome P450,CYP)3A5基因多态性联合血清微RNA-543(microRNA-543,miR-543)预测急性脑梗死患者预后不良的价值。方法:选取2021年5月至2024年1月在郑州市第七人民医院诊断为急性脑梗死的176例患者作为研究对象,依据患者出院90 d后的门诊改良Rankin量表评分结果,将评分≤2的患者纳入预后良好组(n=92),评分>2的纳入预后不良组(n=84)。采用实时荧光定量PCR法检测治疗前血清miR-543水平,采用PCR法检测治疗前CYP3A5基因型,采用多因素Logistic回归分析影响急性脑梗死患者预后不良的因素,采用受试者操作特征(receiver operating characteristic,ROC)曲线分析CYP3A5基因rs776746位点GG基因型联合血清miR-543水平对急性脑梗死患者预后不良的预测价值。结果:CYP3A5基因rs776746位点上检出AA型、GA型、GG型3种基因型。预后不良组发病至溶栓时间长于预后良好组,血清miR-543水平、携带CYP3A5基因rs776746位点GG基因型及G等位基因、入院时美国国立卫生研究院卒中量表(National Institutes of Health Stroke Scale,NIHSS)评分≥15和高血压患者比例均显著高于预后良好组(均P<0.001)。携带CYP3A5基因rs776746位点GG基因型和高miR-543水平是急性脑梗死患者预后不良的危险因素(均P<0.05)。CYP3A5基因rs776746位点GG基因型和血清miR-543水平单独及联合预测急性脑梗死患者预后不良的曲线下面积(area under the curve,AUC)分别为0.702、0.833、0.932,敏感度分别为52.38%、75.68%、92.86%,特异度分别为88.04%、78.33%、93.48%。结论:携带CYP3A5基因rs776746位点GG基因型和高miR-543水平均与急性脑梗死患者预后不良有关,二者联合可显著提高预测效能。
基金supported by the National Natural Science Foundation of China(82071466 and 82371470).
文摘Ischemic stroke remains a leading cause of neurological disability worldwide.Effective therapeutic interventions for stroke treatment are still critically needed.Annexin A5,a calcium-dependent phospholipid-binding protein,has been reported to possess a wide range of biological functions,including anti-inflammation,anticoagulation and antithrombotic,and apoptosis regulation.It also has protective effects on the ischemic stroke.Previous studies have demonstrated that Annexin A5 has neuroprotective effects during ischemic stroke.However,there is still a lack of systematic research on the exact mechanism.Our study aims to clarify the related signaling pathways of Annexin A5 after ischemic stroke.In the present study,we validated that Annexin A5 administration significantly enhanced neuronal survival and attenuated apoptotic processes in both in vitro and in vivo models.As expected,Annexin A5 treatment led to a reduction in infarct volume and improved neurological function scores in tMCAO mouse models.Notably,RNA-seq identified 1430 differentially expressed genes(DEGs)between OGD/R and Annexin A5-treated groups.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed involvement in multiple signaling cascades,such as apoptosis,TGF-βpathway,NOD-like receptor pathway,phagosome,PI3K-Akt pathway,necroptosis,NF-kB pathway.These findings were subsequently validated through qRT-PCR analysis,showing high concordance with RNA-seq results.Our integrated findings propose the multi-target mechanism positions Annexin A5 as a potential therapeutic candidate for ischemic stroke management.
