血浆外泌体样小体免疫调节作用的初步研究

目的 鉴定血浆中外泌体(exosomes)样小体,研究其生物学特性及免疫调节作用.方法 利用多次超速离心结合膜超滤的方法,从健康供者血浆中分离纯化外泌体样小囊泡.采用透射电镜从形态学方面鉴定此囊泡为外泌体,用流式细胞术检测其生物学特性.通过磁珠分选从健康供者外周血单个核细胞中分选CD4+T细胞及CD4+CD25+CD127low调节性T细胞(Treg),外泌体样小体与CD4+T细胞或Treg细胞共孵育,通过增殖和凋亡实验分析其免疫调节作用.采用流式细胞术检测外泌体样小体与Treg细胞共孵育后,Treg细胞中Wnt经典信号转导通路中磷酸化β-catenin的变化.结果 人血浆中的外泌体样小体与外泌体在形状和大小方面均相似,表达外泌体的标志性蛋白CD63和CD81,还表达MHC-Ⅱ类分子和共刺激分子CD86等.血浆外泌体样小体与CD4+T细胞孵育后能抑制CD4+T细胞的增殖,且呈剂量依赖性.血浆外泌体样小体与Treg细胞孵育后,Treg细胞的生存时间延长,培养14 d后与血浆外泌体样小体共孵育的Treg细胞存活率为57.07%,而对照Treg细胞存活率为30.91%.RT-PCR检测出Treg细胞有Frizzled受体FRZ2、3、4及LRP6 mRNA表达(相对灰度值分别为48.50、34.84、23.85、49.73),而血浆中外泌体样小体携带Wnt分子.与血浆外泌体孵育后,Treg细胞磷酸化β-catenin平均荧光强度降低(由20.06±2.99降低到12.41±2.08),抗凋亡基因bcl-2明显上调(相对灰度值由0.45上调到84.97).结论 血浆中存在外泌体样小体,表达免疫调节分子,并且体外能够抑制活化的CD4+T细胞增殖,通过Wnt信号转导途径延长Treg细胞生存时间.     

血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

CD4+CD25+调节性T细胞免疫抑制作用机制的研究进展

CD4+CD25+调节性T细胞是一群具有免疫抑制作用的负性调控细胞,在人体自身免疫、维持免疫系统稳态及许多疾病中发挥重要作用.目前有关CD4+CD25+调节性T细胞分化发育的确切机制及行使功能的作用机制尚不清楚.本文就其免疫抑制作用机制的研究进展作一综述.     

荧光染色法和KOH湿片法检测浅部真菌感染的效果比较

目的 对比荧光染色法和KOH湿片法检测浅部真菌感染的效果。方法 收集2017年7月至2018年2月同济大学附属第十人民医院皮肤科门诊600例临床拟诊为浅部真菌感染及102例拟诊为马拉色菌感染（包括花斑糠疹54例,马拉色菌毛囊炎48例）的标本,分别采用荧光染色法和KOH湿片法直接镜检,比较检出阳性率及平均阅片时间。以培养为金标准,比较两种方法的漏诊情况。计数资料的比较采用卡方检验或Fisher精确检验,计量资料的比较采用配对t检验。结果 荧光染色法与KOH湿片法检测600例浅部真菌感染标本时检出阳性分别为546例（91.00%）和489例（81.50%）,阳性率差异有统计学意义（χ2 = 22.83,P < 0.05）,平均阅片时间分别为（73.67 ± 13.56） s和（87.12 ± 15.83） s（t = 14.60,P < 0.05）;54例花斑糠疹标本检出阳性分别为51例（94.44%）、50例（92.59%）（校正χ2 ＝ 0,P > 0.05）,平均阅片时间分别为（38.36 ± 8.79） s、（41.25 ± 15.67） s（t = 1.14,P > 0.05）;48例马拉色菌毛囊炎检出阳性分别为43例（89.58%）、11例（22.92%）（χ2 = 43.34,P < 0.05）,平均阅片时间分别为（42.14 ± 12.61） s、（103.56 ± 9.48） s（t = 17.83,P < 0.05）。600例浅部真菌感染标本中,479例培养阳性,其中荧光染色法检测阳性476例,阴性3例（0.63%）,KOH湿片法检测阳性465例,阴性14例（2.92%）,漏诊率差异有统计学意义（χ2 = 7.25,P < 0.05）。结论 荧光染色法相比于KOH湿片法可以提高检出阳性率,减少漏诊,缩短阅片时间。     

甲真菌病400例致病真菌分析

目的:确定上海地区甲真菌病的致病菌种。方法:对本院皮肤科门诊就诊的直接镜检阳性的400例甲真菌病患者的甲标本做真菌分离培养和分析。结果:分离出致病真菌233株,其中皮肤癣菌120株(红色毛癣菌104株,须癣毛癣菌10株,犬小孢子菌3株,絮状表皮癣菌3株),酵母菌68株,非皮肤癣菌11株(曲霉6株,青霉5株),其余为丝状真菌。结论:上海地区甲真菌病的致病真菌以皮肤癣菌为主,酵母菌中非白念珠菌占有一定比例。