目的:明确辛伐他汀对氧化应激下人原代角质形成细胞(KC)分泌趋化因子CXCL9、CXCL10、CXCL11和CCL22的影响。方法:常规培养KC,H2O2组给予1 m M H2O2模拟白癜风KC氧化应激模型,实验组予不同浓度辛伐他汀(0.1μmol/L、0.5μmol/L、1.0μmol...目的:明确辛伐他汀对氧化应激下人原代角质形成细胞(KC)分泌趋化因子CXCL9、CXCL10、CXCL11和CCL22的影响。方法:常规培养KC,H2O2组给予1 m M H2O2模拟白癜风KC氧化应激模型,实验组予不同浓度辛伐他汀(0.1μmol/L、0.5μmol/L、1.0μmol/L)预处理后加入H2O2;采用Real-time PCR、ELISA及Western blot检测CXCL9、CXCL10、CXCL11和CCL22的mRNA表达及蛋白分泌。结果:辛伐他汀组CXCL9、CXCL10、CXCL11水平低于H2O2组,CCL22水平高于且呈H2O2组,呈剂量依赖方式。结论:辛伐他汀能通过应激的角质形成细胞调控分泌Th1型趋化因子CXCL9、CXCL10、CXCL11及CCL22的水平。展开更多
Patients with pemphigus vulgaris (PV) have circulating antidesmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic...Patients with pemphigus vulgaris (PV) have circulating antidesmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantlyby Dsg3,we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting,and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PVsera(n =10,77.4 ±21.4)thancontrols(n=11,16.0±9.6;p = 0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n = 6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.展开更多
目的：人乳头瘤病毒16型（human papillomavirus，HPV 16）与包括宫颈癌在内的多种肿瘤的发生明确相关。诱导宿主基因组不稳定性可能是HPV16致瘤的重要机制之一。本研究对HPV16型E6蛋白诱导人原代角质形成细胞（primary human keratinoc...目的：人乳头瘤病毒16型（human papillomavirus，HPV 16）与包括宫颈癌在内的多种肿瘤的发生明确相关。诱导宿主基因组不稳定性可能是HPV16致瘤的重要机制之一。本研究对HPV16型E6蛋白诱导人原代角质形成细胞（primary human keratinocytes，PHK）形成多倍体以及对PHK有丝分裂纺锤体检查点的影响进行初步的探索。方法：取正常人包皮组织，分离表皮，常规方法培养PHK。脂质体介导法将pBabe-16E6质粒转染逆转录病毒包装细胞PA317。挑选G418抗性克隆，检测病毒滴度，将表达HPV16型E6蛋白的高滴度逆转录病毒感染PHK。通过逆转录PCR法和免疫印迹法证实HPV16E6成功感染PHK。PHK经Nocodazole处理后，利用流式细胞仪分析多倍体形成，于不同时间点收集细胞、固定，进行抗磷酸化组蛋白染色，利用流式细胞仪分析细胞有丝分裂指数差异。结果：HPV16型E6成功感染PHK，并呈功能性表达。HPV16型E6可诱导PHK形成多倍体，表达E6蛋白的PHK和对照细胞以相似的动力学进入和退出有丝分裂。结论：HPV16型E6对PHK有丝分裂过程中的纺锤体检查点无直接影响，鉴于有丝分裂后期检查点在细胞有丝分裂过程中较持久和严格的作用，本研究结果提示，对有丝分裂后期检查点的作用可能是HPV16型E6蛋白诱导宿主细胞多倍体形成的重要机制。为进一步探讨HPV相关肿瘤的分子发生机制奠定了一定基础。展开更多
文摘目的:明确辛伐他汀对氧化应激下人原代角质形成细胞(KC)分泌趋化因子CXCL9、CXCL10、CXCL11和CCL22的影响。方法:常规培养KC,H2O2组给予1 m M H2O2模拟白癜风KC氧化应激模型,实验组予不同浓度辛伐他汀(0.1μmol/L、0.5μmol/L、1.0μmol/L)预处理后加入H2O2;采用Real-time PCR、ELISA及Western blot检测CXCL9、CXCL10、CXCL11和CCL22的mRNA表达及蛋白分泌。结果:辛伐他汀组CXCL9、CXCL10、CXCL11水平低于H2O2组,CCL22水平高于且呈H2O2组,呈剂量依赖方式。结论:辛伐他汀能通过应激的角质形成细胞调控分泌Th1型趋化因子CXCL9、CXCL10、CXCL11及CCL22的水平。
文摘Patients with pemphigus vulgaris (PV) have circulating antidesmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantlyby Dsg3,we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting,and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PVsera(n =10,77.4 ±21.4)thancontrols(n=11,16.0±9.6;p = 0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n = 6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.
文摘目的：人乳头瘤病毒16型（human papillomavirus，HPV 16）与包括宫颈癌在内的多种肿瘤的发生明确相关。诱导宿主基因组不稳定性可能是HPV16致瘤的重要机制之一。本研究对HPV16型E6蛋白诱导人原代角质形成细胞（primary human keratinocytes，PHK）形成多倍体以及对PHK有丝分裂纺锤体检查点的影响进行初步的探索。方法：取正常人包皮组织，分离表皮，常规方法培养PHK。脂质体介导法将pBabe-16E6质粒转染逆转录病毒包装细胞PA317。挑选G418抗性克隆，检测病毒滴度，将表达HPV16型E6蛋白的高滴度逆转录病毒感染PHK。通过逆转录PCR法和免疫印迹法证实HPV16E6成功感染PHK。PHK经Nocodazole处理后，利用流式细胞仪分析多倍体形成，于不同时间点收集细胞、固定，进行抗磷酸化组蛋白染色，利用流式细胞仪分析细胞有丝分裂指数差异。结果：HPV16型E6成功感染PHK，并呈功能性表达。HPV16型E6可诱导PHK形成多倍体，表达E6蛋白的PHK和对照细胞以相似的动力学进入和退出有丝分裂。结论：HPV16型E6对PHK有丝分裂过程中的纺锤体检查点无直接影响，鉴于有丝分裂后期检查点在细胞有丝分裂过程中较持久和严格的作用，本研究结果提示，对有丝分裂后期检查点的作用可能是HPV16型E6蛋白诱导宿主细胞多倍体形成的重要机制。为进一步探讨HPV相关肿瘤的分子发生机制奠定了一定基础。
