摘要
从异育银鲫外周血中分离淋巴细胞,利用RPM I-1640培养基培养48 h后,采用流式细胞仪分析方法研究不同浓度的植物凝集素（phytohem agglutin in,PHA）（0μg/mL、80μg/mL、200μg/mL、400μg/mL、600μg/mL、800μg/mL、1 000μg/mL、1 200μg/mL）对外周血淋巴细胞转化率的影响,旨在探索异育银鲫外周血淋巴细胞体外转化最适刺激剂量。结果表明：（1）在PHA浓度0~400μg/mL的范围内,随着PHA浓度的增加,淋巴细胞转化率也增加,呈浓度依赖的关系,但当PHA的浓度增加到1 200μg/mL,淋巴细胞转化率明显下降。其中PHA浓度为400μg/mL时,淋巴细胞转化率最高,显著高于低浓度组（80μg/mL）和高浓度组（1000μg/mL、1200μg/mL）（P〈0.05）,略高于中浓度组（200μg/mL、600μg/mL、800μg/mL）,差异不显著（P〉0.05）。由此可见,在本试验条件下,PHA体外刺激异育银鲫淋巴细胞转化的最适剂量为400μg/mL。（2）用流式细胞仪检测细胞内DNA合成情况,Mu lticyc le AV软件进行细胞周期分析,通过DNA增殖指数测定淋巴细胞转化率是一种简单、可靠的方法。需要注意的是,采用流式细胞仪测定鲫鱼淋巴细胞转化率时,FS/SS参数设置为FS：Volts 62-gain 100;SS：Volts 1000-gain 500能够获得清晰的图像。
Lymphocytes were separated from peripheral blood of allogynogenetic crucian carp（Carassius auratus gibelio）,and incubated in RPMI-1640 medium for 48 h.The effect of different concentration of PHA（0 μg/mL,80 μg/mL,200 μg/mL,400 μg/mL,600 μg/mL,800 μg/mL,1 000 μg/mL,1 200 μg/mL） on the lymphocyte transformation ratio was determined by flow cytometry（FCM）.The result showed：（1） When the concentration of PHA was below 400 μg/mL,there was a positive-correlation between the concentration of PHA and the lymphocyte transformation ratio;When the concentration of PHA was 400 μg/mL,the lymphocyte transformation ratio reached a peak,and it was significantly higher than that of low concentration groups（80 μg/mL）and high concentration groups（1 000 μg/mL,1 200 μg/mL）（P0.05）,and a little higher than that of moderate groups（200 μg/mL,600 μg/mL,800 μg/mL）,no significant difference（P0.05）.Under our conditions of experiment,the optimum concentration of PHA was 400 μg/mL.（2） Flow cytometry was applied to determine the state of intracellular DNA synthesis and cell cycle was analyzed by Multicycle AV software.The results indicated that it was a simple and reliable method to obtain the lymphocyte transformation ratio by determining the proliferation index of DNA.What calls for special attention was that only when the parameters of flow cytometry were FS： Volts 62-gain 100;SS： Volts 1000-gain 500,the scatter diagram was clear.
出处
《上海海洋大学学报》
CAS
CSCD
北大核心
2010年第2期167-171,共5页
Journal of Shanghai Ocean University
基金
江苏省社会发展项目(BS2006021
BS2007140)
