两种方法测定羊外周血淋巴细胞增殖的研究

Determination of cell proliferation of peripheral blood lymphocytes from sheep by different assays

本试验比较3H胸腺嘧啶核苷(3H-TdR)渗入法和BrdU-ELISA方法测定羊外周血淋巴细胞增殖.分离健康羊外周血淋巴细胞,与不同浓度的非特异刺激因子刀豆素A(concanavalinA,1.0,0.5,0.25,0.125,0.0625,0.0313,0.0156,0.0078,0.0039 μg/孔)培养,分别用3H胸腺嘧啶核苷(3H-TdR)和5-溴-2′-脱氧尿苷(BrdU)标记细胞,测定刺激细胞和对照细胞的每分钟脉冲数cpm或光吸收值OD,计算刺激指数SI和光吸收值差ΔOD,统计学方法分析SI和ΔOD的相关程度.同样的方法测定羊试验感染肝片吸虫(Fasciola hepatica)后,在感染早期阶段(PIW0-PIW4)其外周血淋巴细胞在特异性抗原-片形吸虫分泌排泄产物(ESP,5 μg/孔)刺激下的增殖.结果表明,刺激指数SI和光吸收值差ΔOD在两个试验中的相关系数分别为0.946和0.924,SI和*OD均呈强正线性相关.BrdU-ELISA方法无同位素放射物,且敏感、快速、方便,和3H-TdR渗入法一样可以用于测定淋巴细胞增殖.

Cell proliferations in vitro of peripheral blood lymphocytes (PBLs) from sheep determinated by 3H-Thymidine (3H-TdR) incorporation assay and BrdU-ELISA were compared. PBLs from non-infected sheep were incubated with concanavalinA (ConA) at several doses (1. 0 to 0. 0039 pig/well) and PBLs from sheep infected experimentally by Fasciola hepatica with the fluke excretory-secretory products (ESP) at 5 /ig/well from PIWO to PIW4. The cultivated cells were labeled by 3H-Thymidine or 5-bromo-2'-deoxyuridine (BrdU) respectively after 5 days cultivation. Cpm was counted in 3H-TdR labeled group and OD was measured in BrdU labeled group for stimulated cells and control cells,and stimulation index (SI) and difference of OD (AOD) were calculated. Statistics analysis indicated that correlation between SI and AOD is significant at the 0. 01 level (2-tailed) in both cell proliferations. The coefficient of correlation is 0. 946 in non-infected sheep stimulated by ConA and 0. 924 in infected sheep stimulated by ESP from PIWO to PIW4. BudU-ELISA offers a number of key advantages:non-radioactive,sensitive,fast and convenient,and can be used for the determination of lymphocyte proliferation in vitro.