肉仔鸡肠道B淋巴细胞Ca^(2+)浓度流式细胞术测定方法的建立

Establishment of the method to detect Ca^(2+) of intestinal B lymphocytes of broiler chickens with flow cytometry

【目的】建立以流式细胞术结合Fluo-3-AM测定肉仔鸡肠道B淋巴细胞胞内Ca2+浓度的方法。【方法】分离培养21 d肉仔鸡肠道B淋巴细胞,以不同质量浓度(0,5,10,15,20μg/mL)脂多糖(LPS)刺激B淋巴细胞,测定B淋巴细胞刺激指数(SI),确定B淋巴细胞增殖的最适LPS质量浓度,建立肉仔鸡B淋巴细胞体外增殖试验模型。以最佳质量浓度LPS刺激培养72 h的肉仔鸡肠道B淋巴细胞为研究对象,应用Fluo-3-AM作为钙指示剂,建立肉仔鸡肠道B淋巴细胞胞内Ca2+浓度的流式细胞术测定方法,并用该测定方法探讨不同质量浓度(0,20,40,60,80μg/mL)二十碳五烯酸(EPA)对肉仔鸡肠道B淋巴细胞胞内的Ca2+浓度的影响。【结果】LPS的最佳质量浓度为10μg/mL。建立了以Fluo-3-AM为标记的肉仔鸡肠道B淋巴细胞胞内Ca2+浓度流式细胞仪测定方法。EPA可极显著降低B淋巴细胞胞内Ca2+浓度(P<0.01)。【结论】Fluo-3-AM结合流式细胞术可检测肉仔鸡肠道B淋巴细胞胞内的Ca2+浓度,且EPA极显著降低了B淋巴细胞胞内的Ca2+浓度。

【Objective】 The study was done to establish a method of detecting intracellular Ca2＋ in intestinal B lymphocytes of broiler chickens with Fluo-3-AM by flow cytometry.【Method】 Healthy 21 d broiler chickens were used for our experiments.The 3 cm jejunum was removed from 21 d broilers,the intestinal B lymphocytes were isolated and cultured in vitro.LPS was used at different concentrations（0,5,10,15,20 μg/mL） for stimulation of intestinal B cell cultures.The optimal conditions for LPS stimulation of in vitro B lymphocyte proliferation was determined by choosing the LPS concentration that gave the maximal stimulation index.Intestinal B lymphocytes were stimulated by LPS,while the system was added different concentrations of EPA（0,20,40,60,80 μg/mL）,and then the cell was loaded with Fluo-3-AM as a calcium indicator.The intracellular Ca2＋ in intestinal B lymphocytes of broilers was examined with Fluo-3-AM by flow cytometry.【Result】 The optimal concentration of LPS was 10 μg/mL.The fluorescence intensity of B lymphocytes with Fluo-3-AM was increased significantly.The method was established to detect the intracellular Ca2＋ in intestinal B lymphocytes of broiler chickens.EPA can significantly reduce the Ca2＋ of B lymphocytes of broilers（P0.01）.【Conclusion】 EPA significantly decreased the intracellular Ca2＋ of B lymphocytes,and using Fluo-3-AM and flow cytometry could detect the intracellular Ca2＋ of B lymphocyte.