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目的 :研究与系统性红斑狼疮(systemic lupus erythematosus,SLE)患者调节性T细胞(regulatory T cell,Treg)功能密切相关的分子转化生长因子(transforming growth factor,TGF-β)的转录、表达、分泌水平的异常。方法:分离正常人和SLE患者的外周血单个核细胞(peripheral blood mononuclear cell,PBMC)和CD4+CD25+T细胞,以抗-CD3和抗-CD28刺激培养48 h,获取细胞,以三色流式细胞术检测CD4+CD25+LAP+T及CD8+CD25+LAP+T细胞,分析各亚群细胞比值;提取总m RNA,q RT-PCR测定TGF-β1的表达;收取上清用ELISA法检测总TGF-β1和游离的活化TGF-β1水平。结果 :活动性SLE患者CD4+T细胞中LAP+/CD4+T、CD25+LAP+/CD4+T、LAP+/CD4+CD25+比值较正常人显著升高,抗-CD3、抗-CD28抗体刺激后LAP+/CD4+T、CD25+LAP+/CD4+T、LAP+/CD4+CD25+比值进一步升高,与正常人比较差异有统计学意义(P<0.05);但均与疾病活动度无显著相关性;无论刺激与未刺激,SLE患者CD4+CD25+LAP+细胞中LAP染色的荧光强度亦高于相应正常组;而抗-CD3、抗-CD28抗体刺激后SLE患者PBMC及CD4+CD25+T细胞培养上清中总TGF-β1和游离的活化TGF-β1水平均显著低于正常人(P<0.05);q RT-PCR证实,受刺激后CD4+CD25+T细胞内TGF-β1的m RNA转录水平低于正常人。结论:尽管活动性SLE患者CD4+CD25+T细胞膜表面潜伏态TGF-β(LAP)的表达较正常人明显增高,但其TGF-β表达依然存在缺陷,主要表现为TGF-β1的转录、分泌水平降低,游离的活化TGF-β1产生障碍,从而可能削弱了Treg细胞的免疫抑制作用。新鲜分离的以及受刺激后的CD4+CD25+T细胞LAP表达增高可能反映了活动性SLE患者T细胞亚群的活化和CD4+CD25+LAP+Treg细胞的反应性扩增。
OBJECTIVE: To investigate the transcription, expression and secretion of transforming growth factor (TGF-β), which is closely related to the regulatory T cell (Treg) function in patients with systemic lupus erythematosus (SLE) Abnormal level. Methods: Peripheral blood mononuclear cells (PBMCs) and CD4 + CD25 + T cells from normal and SLE patients were isolated and cultured with anti-CD3 and anti-CD28 for 48 h. The percentage of CD4 + CD25 + LAP + T cells and CD8 + CD25 + LAP + T cells were detected by the cytometry method. The total RNA was extracted and the expression of TGF-β1 was detected by q RT-PCR. Total TGF-β1 and free activated TGF-β1 levels were measured. Results: The ratio of LAP + / CD4 + T, CD25 + LAP + / CD4 + T and LAP + / CD4 + CD25 + in CD4 + T cells in active SLE patients was significantly higher than that in healthy people. The ratio of CD4 + T, CD25 + LAP + / CD4 + T and LAP + / CD4 + CD25 + increased further compared with that of normal subjects (P <0.05), but no significant correlation with disease activity The fluorescence intensity of LAP staining in CD4 + CD25 + LAP + cells of unstimulated SLE patients was also higher than that of normal corresponding groups. However, in the supernatant of PBMC and CD4 + CD25 + T cells stimulated by anti-CD3 and anti-CD28 antibodies The levels of total TGF-β1 and free activated TGF-β1 in CD4 + CD25 + T cells were significantly lower than those in normal controls (p <0.05) Normal people. CONCLUSIONS: Although the expression of latent-state TGF-β (LAP) on the surface of CD4 + CD25 + T cells in active SLE patients is significantly higher than that in normal people, the expression of TGF-β is still deficient. The main manifestations are the transcription and secretion of TGF- Decreased levels of free activated TGF-β1 produce disorders that may impair the immunosuppressive effects of Treg cells. Increased LAP expression of freshly isolated and stimulated CD4 + CD25 + T cells may reflect activation of T cell subsets and reactive expansion of CD4 + CD25 + LAP + Treg cells in patients with active SLE.