| 摘要: |
| 【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（0，5，10，15，20 μg/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（0，20，40，60，80 μg/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 10 μg/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。 |
| 关键词: 流式细胞术 二十碳五烯酸（EPA） B淋巴细胞 Ca2+浓度 |
| DOI： |
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| 基金项目:西北农林科技大学科研启动费项目（01140517）；陕西省“13115”科技创新工程项目（2009K01-02,2010ZDGC-02） |
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| Establishment of the method to detect Ca2+ of intestinal B lymphocytes of broiler chickens with flow cytometry |
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WANG Yibing, YAO Junhu, QIN Dingkui,et al
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College of Animal Science and Technology, Northwest A&F University
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| Abstract: |
| 【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 (P＜0.01).【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. |
| Key words: flow cytometry EPA B lymphocytes Ca2+concentration |
