类风湿关节炎患者外周血淋巴细胞的钙内流流式分析

niwanmao

T淋巴细胞胞内钙离子水平的短暂升高在类风湿关节炎疾病（RA）自身免疫反应的发生和发展中起着一定作用，而Kv1.3和IKCal钾离子通道在淋巴细胞活化时的钙离子内流中起着重要作用。本文作用研究了9例初发和6例已确诊的RA患者外周血淋巴细胞，检测并比较了CD4、Th1、Th2、CD8等亚群中的钙离子动力学，分别采用FlowJo的平均荧光平滑方法和FacsKin方法。结果发现，确诊RA患者的Th2细胞活化时钙离子内流速度比初发患者慢，而CD8细胞的钙离子内流速度快于初发患者。RA患者的Th1细胞对Kv1.3和IKCal的抑制反应较不敏感，但随着疾病进展，敏感性逐渐增高。而两种计算方法中，FacsKin方法在统计学比较上更有优势。

FacsKin法

Analysis by Flow Cytometry of Calcium Influx Kinetics in Peripheral Lymphocytes of Patients with Rheumatoid Arthritis
Abstract:
The transient increase of the cytoplasmic free calcium level in T lymphocytes plays a key role in initiating and maintaining the autoimmune reaction in rheumatoid arthritis (RA). Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. We aimed to compare peripheral T lymphocyte calcium influx kinetics upon activation in patients with recently diagnosed and established RA, and to demonstrate the differences in analysis of kinetic flow cytometry data when using two different algorithms. We took peripheral blood samples from nine patients with recently diagnosed and six patients with established RA. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2, and CD8 cells applying an approach based on smoothing of median fluorescence values (FlowJo) and an algorithm based on function fitting (FacsKin). We assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. Th2 cells of patients with established RA react slower to activating stimuli, whereas CD8 cells show a faster reaction than in patients with recently diagnosed RA. While initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. With the algorithm of function fitting instead of smoothing, more statistically significant differences of potassium channel inhibition between the two RA groups could be demonstrated. The function fitting algorithm applied by FacsKin is suitable to provide a common basis for evaluating and comparing flow cytometry kinetic data.

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