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流式细胞术检测脂多糖对罗氏沼虾血细胞活性和免疫功能的影响
Flow cytometric analysis of viability and immune response of Macrobrachium rosenbergii haemocytes activated by lipopolysaccharide
投稿时间:2009-04-19  修订日期:2010-08-06
DOI:
中文关键词:  脂多糖  罗氏沼虾  血细胞  免疫反应  流式细胞术
英文关键词:Lipopolysaccharide  Macrobrachium rosenbergii  Haemocyte  Immune response  Flow cytometry
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)
作者单位E-mail
冼健安 华南师范大学生命科学学院 xian-ja@qq.com 
王安利 华南师范大学生命科学学院 wanganl@scnu.edu.cn 
苗玉涛 华南师范大学生命科学学院  
伍娟 华南师范大学生命科学学院  
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中文摘要:
      以脂多糖(LPS)刺激罗氏沼虾(Macrobrachium rosenbergii)离体血细胞,应用流式细胞术检测血细胞大小、活性、吞噬功能和呼吸爆发的变化,并检测细胞培养液中的酚氧化酶活力。根据前向角散射光(FSC)和侧向角散射光(SSC)特征的不同,可把罗氏沼虾的血细胞分为三个亚群:透明细胞、小颗粒细胞和大颗粒细胞。血细胞经LPS 刺激20-40min后,大型血细胞(小颗粒细胞和大颗粒细胞)的比例开始迅速下降,而细胞的大量死亡从40-60min开始。添加胰蛋白酶抑制剂(TI)能显著减缓LPS导致的细胞变小和死亡。3和5µg/ml LPS能显著提高血细胞的吞噬活力、呼吸爆发水平和培养液中的酚氧化酶活力。结果表明,LPS是罗氏沼虾血细胞的强免疫刺激物和毒物,它刺激血细胞脱颗粒释放酚氧化酶,提高了血细胞的吞噬能力,促进了大量活性氧的产生;其毒性致血细胞死亡可能是血细胞被诱导产生的过量活性氧带来的氧化伤害所造成的。
英文摘要:
      Cell size, viability, phagocytosis activity and respiratory burst (RB) of lipopolysaccharide (LPS) stimulated haemocytes from Macrobrachium rosenbergii were measured by flow cytometry. Phenoloxidase (PO) activity of the cell supernatant was also determined. According to the forward light scatter (FSC) and the side light scatter (SSC) signals of each individual, haemocytes could be divided into three subpopulations: hyaline cells, semigranular cells and granular cells. After 20-40 min of incubation with LPS, the percentage of large cells (semigranular and granular cells) decreased sharply, while the number of live cells began to reduce after 40-60 min. Trypsin inhibitor (TI) further delayed these changes induced by LPS. Phagocytosis activity, RB of haemocytes and PO activity of haemocyte supernatants significantly increased in the 3 and 5µg/ml LPS treatments. These results indicated that LPS was a stronge stimulator and toxicant for the M. rosenbergii haemocytes. The immune activity of haemocytes was activated by LPS. Semigranular and granular cells were activated to degranulation to release PO. LPS was also an efficient activator of phagocytosis and RB. It’s toxicity on the haemocytes may resulted from the damage of excessive ROS.
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