扩张型心肌病患者血清中的β1-肾上腺素受体自身抗体通过β1-肾上腺素受体抑制CD4＋T淋巴细胞的增殖作用

Autoantibodies against the second extracellular loop of β1-adrenoceptor from patients with DCM in- hibit the proliferation of CD4＋ T lymphocytes by β1-adrenoceptor

目的探讨扩张型心肌病患者血清中针对β1-肾上腺素受体细胞外第二环（the second extracellular loop of β1-adrenoceptor，β1-AR-ECⅡ）功能表位肽段的自身抗体（β1—AA）对大鼠CD4＋ T淋巴细胞增殖能力的影响。方法采用亲和柱纯化试剂盒提纯扩张型心肌病（DCM）患者血清中的β1-AA；利用免疫磁珠分选技术分离出大鼠外周血单核细胞中的CD4＋ T淋巴细胞，流式细胞技术检测CD4＋ T细胞的阳性率；采用CCK-8试剂盒检测细胞增殖能力；流式细胞术检测CD4＋ ／CD8＋T淋巴细胞的比值。结果经免疫磁珠分选后CD4＋T淋巴细胞的阳性率为97．7％；β1-AA可浓度依赖性抑制CD3／CD28刺激的CD4＋T淋巴细胞增殖；β1-AA被β1-AR—ECⅡ中和后，该效应消失；β1-AR特异性阻断剂美托洛尔可完全阻断该抑制效应；β1-AA作用于总T淋巴细胞24h后，CD4＋／CD8＋T淋巴细胞比值没有发生明显改变。结论从DCM患者血清中提取的β1-AA可能通过CD4＋T淋巴细胞表面的β1-AR途径，导致该细胞增殖能力下降。提示β1-AA可能会直接减少T淋巴细胞的数量，使得T淋巴细胞功能下降，进而导致免疫系统紊乱，参与DCM的发生发展。

Objective To investigate the effects of autoantibodies （β1-AA） against second extra- cellular loop of the β1-adrenergic receptor （β1-AR-ECu ） in sera of patients with dilated eardiomyopathy （DCM） on proliferation of rat CD4＋ T lymphocytes. Methods 61-AA in the sera of patients with DCM was purified by affinity chromatography. CD4＋ T lymphocytes were isolated by immunomagnetic microbeads from peripheral blood mononuclear cells of rats and its positive rate was detected by flow cytometry. CCK-8 meth- od was used to detect the proliferation of CD4＋ T lymphocytes and flow cytometry was performed to measure the ratio of CD4＋/CD8＋ T lymphocyte. Results The purity of isolated rat CD4＋ T lymphocytes by immu- nomagnetic mierobeads reached 97.7%. The proliferation of CD4＋ T lymphocytes stimulated by CD3/CD28 was inhibited by β1-AA in a concentration-dependent manner. However, IgG antibodies extracted from sera of healthy controls did not suppress lymphocyte proliferation （P〉0.05）. The suppression effect of β1-AA was inhibited after binding to antigenic peptides corresponding to β1-AR-EC＋ and was completely blocked by metoprolol, a specific antagonist of β1-adrenergic receptor （β1-AR）. In addition, β1-AA had no effects on the ratio of CD4＋/CD8＋ T lymphocyte. Conclusion β1-AA isolated from DCM patients suppresses the pro- liferation of CD4＋ T lymphocytes through β1-AR pathway, which indicates that β1-AA can directly reduce the number of T lymphocytes and impair the function of T lymphocytes, resulting in immune system disorders and the development of DCM.