CFSE标记结合流式细胞术检测肝星状细胞抑制CD4^+/CD8^(+)T淋巴细胞增殖的研究

Application of CFSE-based flow cytometric approaches on the detection of the proliferation of CD4^+/CD8^(+)T cells inhibited by hepatic stellate cells

目的研究羟基荧光素二醋酸琥珀酰亚胺脂(carboxyfluorescein diacetate succinimidyl ester,CFSE)标记结合流式细胞术在检测体外共培养体系中T淋巴细胞亚群增殖中的应用,并利用此方法研究小鼠肝星状细胞(hepatic stellate cells,HSCs)对T淋巴细胞亚群增殖的影响。方法采用磁珠分选雄性C57/B6小鼠脾脏T淋巴细胞,经CFSE标记后与脂多糖(1ipopolysaccharide,LPS)刺激或不刺激的C57/B6来源的小鼠肝星状细胞系JSl共培养3 d,流式细胞术检测T淋巴细胞亚群的增殖,flowjo软件分析增殖动力模型。结果JSl可以抑制CD4^+和CD8^(+)T淋巴细胞增殖的能力,并且当JSl/T细胞比值增加时,抑制作用更强。LPS的刺激不影响JSl对CD4^+和CD8^(+)T细胞增殖的抑制作用。结论肝星状细胞JS1具有免疫抑制能力,CFSE标记结合流式细胞术是一种可靠的分析淋巴细胞增殖的工具,可以有效检测出共培养体系中细胞亚群的增殖情况,在研究体外淋巴细胞功能中具有重要的应用价值。

Objective To establish the CFSE-based flow cytometric approaches in the detection of T cell subsets pro- liferation response in the co-culture system, and to explore its role for the investigation of the inhibition effect of HSCs on the proliferation of CD4^＋ and CD8^＋ T cells. Methods Naive T lymphocytes were isolated from the spleen of C57/B6 mice by MACS pan T cell isolation kit and labeled by CFSE, then wild type mouse hepatic stellate cell line JS1 was cultured with or without the stimulation of LPS before co-cultured with naive T lymphocytes T cell. After 3 days of stimulation and co- culture, T cells were harvested and stained by CD4^＋/CD8^＋T specific antibodies. The proliferation dynamics of CFSE labeled T cells were detected by flow cytometry and analyzed with flowjo. Results JS1 could inhibit both CD4^＋ and CD8^＋ T cells proliferation, and the inhibition effect was in a cell number-dependent manner. LPS stimulation did not affect the inhibition effect of JS1. Conclusion The flow cytometric method presented in this study is a reliable tool which can effectively detect the proliferation of lymphocyte subsets in the co-culture system and has important application value in the in vitro study of lymphocyte function.