石鲽外周血细胞的培养及染色体制备条件的探讨

Study on Peripheral Blood Cell Cultivation and Chromosome Preparation in Stone flounder(Kareius bicoloratus)

以石鲽(Kareius bicoloratus)为试验材料研究探讨其外周血细胞培养及染色体制备的条件方法。用RPMI1640全血培养基进行培养,从血液保存时间、刺激源PHA(植物血球凝集素)和ConA(刀豆蛋白A)等方面对外周血淋巴细胞的培养及其染色体制备条件进行探讨,建立较成熟的石鲽外周血淋巴细胞培养及染色体制备的方法。结果表明:血液于4℃保存3d后用全血培养基进行培养,细胞仍可正常生长;在16℃培养温度下,ConA和PHA两种刺激源均能促进石鲽外周血淋巴细胞的分裂增殖,培养时间以72h最好;从培养的血细胞形态和白细胞数目来看,以ConA为刺激源的作用效果好于以PHA为刺激源的作用效果;5ml培养基中加入0.2ml全血,16℃下用终浓度为0.1mg/ml的ConA为刺激源培养72h,在结束培养前4h加入终浓度0.6μg/ml秋水仙素,0.075mol/L的氯化钾低渗35min可获得较好的染色体分裂相。

Stone flounder （Kareius bicoloratus） was used to explore the condition of the peripheral blood cell cultivation and chromosome preparation. The peripheral blood cell was cultured with RPMI1640 whole culture medium. The conditions of the peripheral blood cell cultivation and chromosome preparation were searched from preservation time of blood, stimulation （ PHA and ConA ） , and so on.. The result showed that the cells of whole blood which preserved for 3 days under 4℃ could grow nomally. Under 16℃, both ConA and PHA could facilitate Kareius bicoloratus＇ peripheral blood cell proliferating, and the best culturing time was 72h . The effect of ConA was better than that of PHA from the shape of hematocyte and the number of leukocyte. Better chromosome samples could be obtained under follow conditions ： adding 0.2 ml whole blood and ConA （ final concentration 0.1 mg/ml ） in 5 ml whole blood medium, cultivating for 72 hours under 16℃, adding colchicines （ final concentration 0.6 μg/ml ） for 4 hours before cultivation finished , doing hypotonie treatment with 0. 075mol/L KCl solution for 35minutes.