目的探讨中期因子(midkine,MK)在人乳腺癌MDA-MB-231细胞体外血管生成中的作用及其机制。方法采用shRNA干扰技术降低MDA-MB-231细胞MK表达,应用Western blot技术检测肿瘤细胞中内皮蛋白C受体(endothelial protein C receptor,EPCR)的表...目的探讨中期因子(midkine,MK)在人乳腺癌MDA-MB-231细胞体外血管生成中的作用及其机制。方法采用shRNA干扰技术降低MDA-MB-231细胞MK表达,应用Western blot技术检测肿瘤细胞中内皮蛋白C受体(endothelial protein C receptor,EPCR)的表达;干扰MK和EPCR表达或通过抗体阻断活化蛋白酶激活受体1(protease-activated receptor 1,PAR1)作用后,制备肿瘤条件培养基作用于人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),通过CCK-8试剂盒检测HUVECs增殖、Transwell小室检测迁移以及Matrigel表面培养检测脉管形成能力。结果干扰MK表达后,EPCR表达随之降低。干扰MK和EPCR低表达后,HUVECs增殖、迁移及脉管形成能力均低于对照组(P<0.05),EPCR干扰组低于MK干扰组(P<0.05)。应用抗PAR1作用后,HUVECs增殖、迁移及脉管形成能力低于对照组和EPCR干扰组(P<0.05)。结论 MK可通过EPCR/PAR1通路促进乳腺癌MDA-MB-231细胞体外血管生成。展开更多
2024年7月12日,浙江大学基础医学院张岩/毛春友教授团队联合上海药物研究所徐华强团队在《细胞研究》(Cell Research)上发表了题为“Structural basis of tethered agonism and G protein coupling of protease-activated receptor”的...2024年7月12日,浙江大学基础医学院张岩/毛春友教授团队联合上海药物研究所徐华强团队在《细胞研究》(Cell Research)上发表了题为“Structural basis of tethered agonism and G protein coupling of protease-activated receptor”的研究论文(DOI:10.1038/s41422-024-00997-2),报道了人源PAR1独特的栓配体结合激活机制和G蛋白选择性机制,为开发新型靶向蛋白酶激活受体(PAR)1的G蛋白选择性药物提供了结构基础。展开更多
Astrocytes are important cellular centers of cholesterol synthesis and metabolism that help maintain normal physiological function at the organism level.Spinal cord injury results in aberrant cholesterol metabolism by...Astrocytes are important cellular centers of cholesterol synthesis and metabolism that help maintain normal physiological function at the organism level.Spinal cord injury results in aberrant cholesterol metabolism by astrocytes and excessive production of oxysterols,which have profound effects on neuropathology.25-Hydroxycholesterol(25-HC),the main product of the membrane-associated enzyme cholesterol-25-hydroxylase(CH25H),plays important roles in mediating neuroinflammation.However,whether the abnormal astrocyte cholesterol metabolism induced by spinal cord injury contributes to the production of 25-HC,as well as the resulting pathological effects,remain unclear.In the present study,spinal cord injury-induced activation of thrombin was found to increase astrocyte CH25H expression.A protease-activated receptor 1 inhibitor was able to attenuate this effect in vitro and in vivo.In cultured primary astrocytes,thrombin interacted with protease-activated receptor 1,mainly through activation of the mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway.Conditioned culture medium from astrocytes in which ch25h expression had been knocked down by siRNA reduced macrophage migration.Finally,injection of the protease activated receptor 1 inhibitor SCH79797 into rat neural sheaths following spinal cord injury reduced migration of microglia/macrophages to the injured site and largely restored motor function.Our results demonstrate a novel regulatory mechanism for thrombin-regulated cholesterol metabolism in astrocytes that could be used to develop anti-inflammatory drugs to treat patients with spinal cord injury.展开更多
Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and invest...Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and investigated the co-relationship between PAR expression and clinic-pathological data for esophageal cancer.The methylation of PAR4 gene promoter involved in esophageal carcinoma was also analyzed.By comparing the mRNA expressions of normal esophageal tissue and human esophageal epithelial cells(HEEpiC),we found that among the 28 cases of esophageal squamous cell carcinoma,PAR1(60%) and PAR2(71%) were elevated in 17 and 20 cases,respectively,and PAR4(68%) expression was lowered in 19 cases.Whereas,in human esophageal squamous cells(TE-1 and TE-10),PAR1 and PAR2 expression was increased but PAR4 was decreased.Combined with clinical data,the expression of PAR1 in poorly differentiated(P=0.016) and middle and lower parts of the esophagus(P=0.016) was higher; expression of PAR4 in poorly differentiated carcinoma was lower(P=0.049).Regarding TE-1 and TE-10 protein expression,we found that in randomized esophageal carcinoma,PAR1(P=0.027) and PAR2(P=0.039) expressions were increased,but lowered for PAR4(P=0.0001).In HEEpiC,TE-1,TE-10,esophageal and normal esophagus tissue samples(case No.7),the frequency of methylation at the 19 CpG loci of PAR4 was 35.4%,95.2%,83.8%,62.6% and 48.2%,respectively.Our results indicate that the expression of PAR1 and PAR2 in esophageal squamous cell carcinoma is increased but PAR4 is decreased.Hypermethylation of the promoter of the PAR4 gene may contribute to reduced expression of PAR4 in esophageal squamous cell carcinoma.展开更多
文摘2024年7月12日,浙江大学基础医学院张岩/毛春友教授团队联合上海药物研究所徐华强团队在《细胞研究》(Cell Research)上发表了题为“Structural basis of tethered agonism and G protein coupling of protease-activated receptor”的研究论文(DOI:10.1038/s41422-024-00997-2),报道了人源PAR1独特的栓配体结合激活机制和G蛋白选择性机制,为开发新型靶向蛋白酶激活受体(PAR)1的G蛋白选择性药物提供了结构基础。
基金supported by the National Natural Science Foundation of ChinaNo.81971826 (to AG)+5 种基金the China Postdoctoral Science FoundationNo.2020M681 689 (to YH)the Scientific Research Project of The Health Commission of Jiangsu ProvinceNo.ZDB2020003 (to AG)the Basic Scientific Research Projects of NantongNo.JC2020041 (to YH)
文摘Astrocytes are important cellular centers of cholesterol synthesis and metabolism that help maintain normal physiological function at the organism level.Spinal cord injury results in aberrant cholesterol metabolism by astrocytes and excessive production of oxysterols,which have profound effects on neuropathology.25-Hydroxycholesterol(25-HC),the main product of the membrane-associated enzyme cholesterol-25-hydroxylase(CH25H),plays important roles in mediating neuroinflammation.However,whether the abnormal astrocyte cholesterol metabolism induced by spinal cord injury contributes to the production of 25-HC,as well as the resulting pathological effects,remain unclear.In the present study,spinal cord injury-induced activation of thrombin was found to increase astrocyte CH25H expression.A protease-activated receptor 1 inhibitor was able to attenuate this effect in vitro and in vivo.In cultured primary astrocytes,thrombin interacted with protease-activated receptor 1,mainly through activation of the mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway.Conditioned culture medium from astrocytes in which ch25h expression had been knocked down by siRNA reduced macrophage migration.Finally,injection of the protease activated receptor 1 inhibitor SCH79797 into rat neural sheaths following spinal cord injury reduced migration of microglia/macrophages to the injured site and largely restored motor function.Our results demonstrate a novel regulatory mechanism for thrombin-regulated cholesterol metabolism in astrocytes that could be used to develop anti-inflammatory drugs to treat patients with spinal cord injury.
基金supported by the National Natural Foundation of China(81160302)the Major Research Project of Yunnan Province(2011FZ109)Research project of Yunnan Education Bureau(2014Y153)
文摘Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and investigated the co-relationship between PAR expression and clinic-pathological data for esophageal cancer.The methylation of PAR4 gene promoter involved in esophageal carcinoma was also analyzed.By comparing the mRNA expressions of normal esophageal tissue and human esophageal epithelial cells(HEEpiC),we found that among the 28 cases of esophageal squamous cell carcinoma,PAR1(60%) and PAR2(71%) were elevated in 17 and 20 cases,respectively,and PAR4(68%) expression was lowered in 19 cases.Whereas,in human esophageal squamous cells(TE-1 and TE-10),PAR1 and PAR2 expression was increased but PAR4 was decreased.Combined with clinical data,the expression of PAR1 in poorly differentiated(P=0.016) and middle and lower parts of the esophagus(P=0.016) was higher; expression of PAR4 in poorly differentiated carcinoma was lower(P=0.049).Regarding TE-1 and TE-10 protein expression,we found that in randomized esophageal carcinoma,PAR1(P=0.027) and PAR2(P=0.039) expressions were increased,but lowered for PAR4(P=0.0001).In HEEpiC,TE-1,TE-10,esophageal and normal esophagus tissue samples(case No.7),the frequency of methylation at the 19 CpG loci of PAR4 was 35.4%,95.2%,83.8%,62.6% and 48.2%,respectively.Our results indicate that the expression of PAR1 and PAR2 in esophageal squamous cell carcinoma is increased but PAR4 is decreased.Hypermethylation of the promoter of the PAR4 gene may contribute to reduced expression of PAR4 in esophageal squamous cell carcinoma.
