CD4^+ CD25^+调节性T细胞在血吸虫可溶性虫卵抗原影响哮喘中的作用研究

A study of the effects of Schistosoma japonicum soluble egg antigen on CD4^+ CD25^+ regulatory T cells in patients with asthma

目的研究血吸虫可溶性虫卵抗原(SEA)与哮喘发生的关系及其对CD4+CD25+调节性T细胞(CD4+CD25+Treg)及相关基因Foxp3表达的影响。方法 BALB/C小鼠腹腔及足垫共注射SEA 50μg/只,每周1次,共4次。对照组注射生理盐水。然后用卵清白蛋白(OVA)诱导小鼠产生过敏性哮喘。剖杀小鼠,取肺组织,做病理学检查;收集支气管肺泡灌洗液(BALF),涂片、染色后进行细胞分类计数;分离脾细胞,用流式细胞仪检测CD4+CD25+调节性T细胞(CD4+CD25+Treg)占CD4+T细胞的比例;提取脾脏RNA,以逆转录得到的cDNA为模板扩增Foxp3基因,检测脾脏Foxp3mRNA表达水平。结果 SEA免疫并OVA致哮喘组小鼠肺组织炎症较OVA单纯哮喘对照组轻,只有少量炎性细胞浸润。BALF涂片染色检查SEA免疫组BALF中的细胞密度低于对照组,其中嗜酸粒细胞占总细胞数的(2.22±1.52)%,对照组占(19.93±4.08)%,差异有统计学意义(P<0.05);流式细胞仪检测SEA免疫组小鼠脾细胞中CD4+CD25+Treg占CD4+T细胞总数的(32.24±2.19)%,对照组占(27.41±2.87)%,差异有统计学意义(P<0.05);PCR检测SEA免疫组脾细胞Foxp3mRNA表达水平高于对照组。结论 SEA对哮喘的发生有一定抑制作用,可能通过CD4+CD25+Treg影响机体的免疫调节机制。

Objective To study the relationship between Schistosoma japonicum soluble egg antigen（SEA） and asthma and the effects of SEA on CD4＋CD25＋ regulatory T cells（CD4＋CD25＋Treg） and expression of the Foxp3 gene.Methods BALB/C mice were each injected with 50 μg SEA peritoneally and through the foot pad once a week for 4 weeks.In the control group,all injections were with normal saline.Then asthma was induced with ovalbumin（OVA） in all mice.After mice were sacrificed,the lungs were subjected to pathologic examination;the bronchoalveolar lavage fluid（BALF） was collected and different cells were classified and counted after smearing and staining.Spleen cells were separated and the percentage of CD4＋CD25＋Treg out of total CD4＋ T cells was determined using flow cytometry.Total spleen RNA was prepared and synthesized into cDNA through reverse transcription;cDNA was subjected to PCR amplification to determine the level of Foxp3 mRNA expression.Results Mild pulmonary inflammation was observed in the SEA immunization group,whereas severe inflammation was observed in the control group.Staining of the BALF revealed that the SEA immunization group had a much lower BALF cell density than did the control group.In the SEA immunization group,the percentage of eosinophils out of total cells was（2.22±1.52）% while it was（19.93±4.08）% in the control group.The difference between the 2 groups was statistically significant（P0.05）.Flow cytometry revealed that the percentage of CD4＋CD25＋Treg out of total CD4＋ T cells was（32.24±2.19）% in the SEA immunization group while it was（27.41±2.87）% in the control group.The difference between the 2 groups was statistically significant（P0.05）.The level of Foxp3 mRNA expression was higher than that in the control group as well.Conclusion SEA inhibits the development of asthma to some extent and it seems influence immune regulation through its effect on CD4＋CD25＋Treg.