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Programmed cell death in rheumatoid arthritis peripheral blood T-cell subpopulations determined by laser scanning cytometry.
Laboratory Investigation ( IF 5 ) Pub Date : 2003-12-01 , DOI: 10.1097/01.lab.0000101703.80133.99
Peter Szodoray 1 , Stig Jellestad , Britt Nakken , Johan G Brun , Roland Jonsson
Affiliation  

Because peripheral blood mononuclear cells play an important role in the perpetuation of the autoimmune process in rheumatoid arthritis (RA) and because the maintenance of these cells might be caused by the dysregulation of apoptosis, we investigated the apoptosis susceptibility of peripheral blood mononuclear cells from patients with RA. Freshly separated peripheral blood lymphocytes were stained for apoptosis markers (CD95, Bax, Bcl-2, TNF receptor) and for an activation marker (CD45-RO), and the apoptosis frequency of cells bearing these markers were assessed by the terminal-deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling method and nuclear condensation analysis with laser scanning cytometry. Also, the ability of CD4(+) and CD8(+) T-cell populations to undergo apoptosis was investigated with 24-hour culture in medium alone or with different apoptosis inducers (anti-CD3, anti-CD95, anti-TNF receptor). Laser scanning cytometry analysis was used to enumerate the phenotype and apoptosis ratios of both freshly isolated and cultured lymphocytes. Quantitative ELISA was performed to detect plasma levels of TNF-alpha and soluble Fas ligand. Furthermore, we studied the relationship between marked apoptotic defects in patients with RA and the severity of clinical disease. CD4(+) T-cell counts in patients with RA were elevated compared with controls. A decreased rate of anti-CD95-mediated apoptosis was found within the CD4(+) and CD8(+) lymphocytic subpopulations. In patients with RA, decreased Bax expression and decreased apoptosis rate within the Bax-positive cells were found, whereas Bcl-2 expression was elevated. The CD45-RO expression was higher, whereas the apoptosis within CD45-RO(+) cells were decreased in RA. Evaluation of plasma soluble Fas ligand revealed significantly decreased levels in patients compared with controls. The reduced susceptibility to CD95-mediated apoptosis may contribute to the expansion of an activated CD4(+) lymphocyte subpopulation and thus to the maintenance of peripheral autoreactive T-cell clones in RA. We also revealed a relationship between in vitro demonstrated lymphocyte apoptosis defects and clinical disease activity.

中文翻译:

通过激光扫描细胞术测定类风湿性关节炎外周血 T 细胞亚群的程序性细胞死亡。

由于外周血单核细胞在类风湿性关节炎 (RA) 自身免疫过程的延续中起着重要作用,并且由于这些细胞的维持可能是由细胞凋亡失调引起的,我们研究了患者外周血单核细胞的凋亡易感性与风湿性关节炎。对新鲜分离的外周血淋巴细胞进行凋亡标记物(CD95、Bax、Bcl-2、TNF 受体)和激活标记物(CD45-RO)染色,并通过末端脱氧核苷酸转移酶评估带有这些标记物的细胞的凋亡频率介导的 dUTP 洋地黄毒苷缺口末端标记方法和激光扫描细胞术核浓缩分析。还,CD4(+) 和 CD8(+) T 细胞群接受细胞凋亡的能力是通过在培养基中单独培养 24 小时或使用不同的细胞凋亡诱导剂(抗 CD3、抗 CD95、抗 TNF 受体)进行研究的。激光扫描细胞术分析用于计算新鲜分离和培养的淋巴细胞的表型和凋亡率。进行定量 ELISA 以检测 TNF-α 和可溶性 Fas 配体的血浆水平。此外,我们研究了 RA 患者明显的细胞凋亡缺陷与临床疾病严重程度之间的关系。与对照组相比,RA 患者的 CD4(+) T 细胞计数升高。在 CD4(+) 和 CD8(+) 淋巴细胞亚群中发现了抗 CD95 介导的细胞凋亡率降低。在 RA 患者中,发现 Bax 阳性细胞内 Bax 表达降低和细胞凋亡率降低,而 Bcl-2 表达升高。CD45-RO 表达较高,而 RA 中 CD45-RO(+) 细胞内的细胞凋亡减少。血浆可溶性 Fas 配体的评估显示,与对照组相比,患者的水平显着降低。对 CD95 介导的细胞凋亡的易感性降低可能有助于激活的 CD4(+) 淋巴细胞亚群的扩大,从而有助于维持 RA 中外周自身反应性 T 细胞克隆。我们还揭示了体外证实的淋巴细胞凋亡缺陷与临床疾病活动之间的关系。血浆可溶性 Fas 配体的评估显示,与对照组相比,患者的水平显着降低。对 CD95 介导的细胞凋亡的易感性降低可能有助于激活的 CD4(+) 淋巴细胞亚群的扩大,从而有助于维持 RA 中外周自身反应性 T 细胞克隆。我们还揭示了体外证实的淋巴细胞凋亡缺陷与临床疾病活动之间的关系。血浆可溶性 Fas 配体的评估显示,与对照组相比,患者的水平显着降低。对 CD95 介导的细胞凋亡的易感性降低可能有助于激活的 CD4(+) 淋巴细胞亚群的扩大,从而有助于维持 RA 中外周自身反应性 T 细胞克隆。我们还揭示了体外证实的淋巴细胞凋亡缺陷与临床疾病活动之间的关系。
更新日期:2019-11-01
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