CAJ | 학술논문

从异育银鲫外周血中分离淋巴细胞,利用RPMI-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(phytohemagglutinin, 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)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0～400 μg/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1 200 μg/mL,淋巴细胞转化率明显下降.其中PHA浓度为400 μg/mL时,淋巴细胞转化率最高,显著高于低浓度组(80 μg/mL)和高浓度组(1 000 μg/mL、1 200 μg/mL)(P<0.05),略高于中浓度组(200 μg/mL、600 μg/mL、800 μg/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400 μg/mL.(2)用流式细胞仪检测细胞内DNA合成情况,Multicycle 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.
종이육은즉외주혈중분리림파세포,이용RPMI-1640배양기배양48 h후,채용류식세포의분석방법연구불동농도적식물응집소(phytohemagglutinin, 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)대외주혈림파세포전화솔적영향,지재탐색이육은즉외주혈림파세포체외전화최괄자격제량.결과표명:(1)재PHA농도0～400 μg/mL적범위내,수착PHA농도적증가,림파세포전화솔야증가,정농도의뢰적관계,단당PHA적농도증가도1 200 μg/mL,림파세포전화솔명현하강.기중PHA농도위400 μg/mL시,림파세포전화솔최고,현저고우저농도조(80 μg/mL)화고농도조(1 000 μg/mL、1 200 μg/mL)(P<0.05),략고우중농도조(200 μg/mL、600 μg/mL、800 μg/mL),차이불현저(P>0.05).유차가견,재본시험조건하,PHA체외자격이육은즉림파세포전화적최괄제량위400 μg/mL.(2)용류식세포의검측세포내DNA합성정황,Multicycle AV연건진행세포주기분석,통과DNA증식지수측정림파세포전화솔시일충간단、가고적방법.수요주의적시,채용류식세포의측정즉어림파세포전화솔시,FS/SS삼수설치위FS: Volts 62-gain 100;SS: Volts 1000-gain 500능구획득청석적도상.
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)(P<0.05), and a little higher than that of moderate groups(200 μg/mL, 600 μg/mL, 800 μg/mL), no significant difference(P>0.05). 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.