本试验旨在研究大豆异黄酮对奶牛脾脏与肠系淋巴结淋巴细胞增殖、活化及细胞因子分泌的影响.采用单因素试验设计,不同浓度的大豆异黄酮[0.00(对照)、0.25、1.00、5.00、25.00和100.00μg/mL]与淋巴细胞于37℃、5％CO2下分别共育4、24和48h,采用双抗夹心酶联免疫法测定培养上清液中的干扰素γ、白介素2和白介素4的浓度,用实时定量PCR法测定脾脏和肠系淋巴结的淋巴细胞中雌激素受体βmRNA表达量.结果表明:1)0.25和1.00 μg/mL的大豆异黄酮与脾脏淋巴细胞共育一定时间后均能显著或极显著提高培养上清液中干扰素γ和白介素2的浓度(P＜0.05或P＜0.01),48 h后,5.00和25.00 μg/mL大豆异黄酮能显著抑制脾脏淋巴细胞干扰素γ和白介素2分泌(P＜0.05).2)1.00和5.00 μg/mL大豆异黄酮与肠系淋巴结淋巴细胞共育一定时间后干扰素γ和白介素2浓度均显著或极显著提高(P＜0.05或P＜0.01);0.25 μg/mL大豆异黄酮趋于增加二者浓度,但差异不显著(P＜0.05);48 h后,25.00 μg/mL大豆异黄酮抑制了干扰素γ(P＜0.05)和白介素2(P＜0.05)的分泌.3)与对照组相比,试验组脾脏淋巴细胞白介素4浓度无显著变化(P＞0.05),而肠系淋巴结淋巴细胞白介素4浓度显著或极显著降低(P ＜0.05或P＜0.01).4)大豆异黄酮能够提高脾脏淋巴细胞干扰素γ/白介素4,但趋于降低肠系淋巴结淋巴细胞干扰素γ/白介素4.5)大豆异黄酮显著或极显著提高了奶牛脾脏和肠系淋巴结中淋巴细胞雌激素受体βmRNA的表达量(P＜0.05或P＜0.01),且二者间存在正比的剂量关系.总之,大豆异黄酮能够促进奶牛脾脏和肠系淋巴结中淋巴细胞分泌干扰素γ和白介素2,提高干扰素γ/白介素4,促进雌激素受体βmRNA表达,降低白介素4浓度;低剂量(0.25～5.00 μg/mL)的大豆异黄酮能够获得较好免疫促进效果,而高剂量(25.00～100.00μg/mL)更能促进雌激素受体βmRNA表达.%This paper aims to investigate the effects of soy isoflavones on proliferation, activation and production of cytokines in lymphocytes of spleen and intestinal lymph nodes of dairy cows in vitro. A single factor design was used, and lymphocytes were incubated with different levels of soy isoflavones [ 0. 00 (control) , 0.25, 1.00, 5.00, 25.00 and 100.00 μg/mL] at37℃5% CO2 for 4, 24 and 48 h, respectively. Concentrations of interferon γ, interleukin 2 and interleukin 4 in culture supernatants were determined by double-antibody sandwich enzyme-linked immunosorbent assay, and expression level of estrogen receptors β mRNA in lymphocytes of spleen and intestinal lymph nodes was determined by real-time PCR. The results showed as follows : 1) after incubated with soy isoflavones (0. 25 and 1. 00 μg/mL) for a while, concentrations of interferon y and interleukin 2 in culture supernatants of lymphocytes of spleen were increased at different levels (P < 0. 05 or P <0. 01) , and 48 h later, 5. 00 and 25. 00 μg/mL soy isoflavones reduced interferon γ and interleukin 2 concentrations significantly (P <0.05). 2) In lymphocytes of intestinal lymph nodes, after incubated with soy isoflavones (1. 00 and 5. 00 μg/mL) for a while, concentrations of interferon γ and interleukin 2 were increased at different levels (P < 0. 05 or P < 0. 01); the concentrations tended to be increased by 0.25 μg/mL soy isoflavones (P>0.05) ; 48 h later, concentrations of interferon γ (P<0.05) and interleukin 2 (P <0. 01) were significantly decreased. 3) Compared with that in the control group, interleukin 4 concentration in lymphocytes of spleen in experimental groups were not change significantly (P > 0. 05) , and the concentration in lymph nodes of intestinal lymph nodes in experimental groups was significantly reduced (P < 0. 05 or P < 0. 01). 4) Ratios of interferon 7 and interleukin 4 in lymphocytes of spleen tended to be increased , but that of intestinal lymph nodes tended to be opposite. 5) Expression levels of estrogen receptors β mRNA in lymphocytes of spleen and intestinal lymph nodes were significantly increased (P < 0. 05 or P < 0.01) , and there was positive correlation between levels of estrogen receptors (5 mRNA expression and soy isoflavones. In conclusion, soy isoflavones can increase concentrations of interferon γ and interleukin 2, ratio of interferon γ and interleukin 2, as well as expression level of estrogen receptors β mRNA, but can decrease interleukin 4 concentration; low dose (0. 25 to 5. 00 (μg/mL) soy isoflavones can get better effects on immunity promotion, but high dose (25. 00 to 100.00 (μg/mL) can stimulate expression of estrogen receptors β mRNA
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