大豆异黄酮对褐牙鲆头肾巨噬细胞免疫相关基因表达的体外研究

In Vitro Effects of Soybean Isoflavones on Expression Level of Genes Related to Immunity in Head Kidney Macrophages of Olive Flounder Paralichthys olivaceus

大豆异黄酮作为免疫增强剂对机体具有免疫调节作用。在离体条件下以不同质量浓度大豆异黄酮细胞培养液(0、0.1、0.5、1.0、1.5、2.0mg/mL)培养褐牙鲆头肾巨噬细胞,分别在培养至6、12、24h收集细胞并进行细胞RNA提取,通过荧光定量PCR测定巨噬细胞中免疫相关基因IL-1"、IFN-#、IgM及HSP70的相对表达量。试验结果表明,在体外培养下,大豆异黄酮显著影响褐牙鲆头肾巨噬细胞中4种免疫相关基因的相对表达,且大豆异黄酮质量浓度为0.5~1.5mg/mL时,显著上调IL-1"、IFN-#和HSP70基因的相对表达(P<0.05),0.5~1.0mg/mL的大豆异黄酮显著上调IgM基因的相对表达(P<0.05)。选取大豆异黄酮质量浓度为1.0mg/mL时,研究脂多糖刺激下大豆异黄酮对4种基因相对表达量的影响,试验结果显示,在脂多糖刺激下,褐牙鲆头肾巨噬细胞4种免疫相关基因随刺激时间的变化趋势与未添加大豆异黄酮组基本一致,但是在特定取样时间点,其相对表达量显著增加。IL-1"在脂多糖刺激后6h和24h表达量极显著高于对照组(P<0.01);IFN-#在脂多糖刺激后24h表达量显著高于对照组(P<0.05);IgM则在刺激后6h极显著高于对照组(P<0.01);而HSP70自脂多糖刺激后3h开始,其相对表达量均极显著高于对照组(P<0.01)。综上所述,在本试验条件下,培养液中大豆异黄酮质量浓度为0.5~1.5mg/mL时,能够显著上调体外培养条件下褐牙鲆头肾巨噬细胞IL-1"、IFN-#、IgM和HSP70基因的相对表达,从而增强褐牙鲆头肾巨噬细胞的免疫力。

The RNA was extracted from the head kidney macrophages isolated from head kidney of olive flounder Paralichthys olivaceus cultured in vitro in medium supplemented with different concentrations of soy isoflavones(0,0.1,0.5,1.0,1.5 and 2.0 mg/mL)as immunostimulant and the expression levels of interleukin-1(IL-1),interferon(IFN),immunoglobulin M(IgM)and heat shock protein(HSP70)were measured by RT-PCR in 6,12,and 24 h.The soy isoflavones was shown to significantly affect the relative expression of these four immune genes,significantly higher expression of IL-1,IFN-and HSP70 at dose of 0.5-1.5 mg/mL(P<0.05).The significant increase in relative expression level of IgM gene was observed in the olive flounder at soy isoflavone dose of 0.5-1.0 mg/mL(P<0.05).Then the relative expression of the four genes under the stimulation of lipopolysaccharide(LPS)revealed that the four genes from head kidney macrophages cultured with 1.0 mg/mL soybean isoflavone showed as the same trend as in the control under the stimulation of LPS.However,at the specific time,the relative expression was significantly increased(P<0.05).The expression of IL-1 F was very significantly higher at 6 h and 24 h after LPS stimulation than that in the control group(P<0.01);the expression of IFN-F was significantly higher at 24 h after LPS stimulation than that in the control group;the expression of IgM was significantly higher at 6 h than that in the control group(P<0.01).The expression of HSP70 was significantly higher from 3 h after LPS stimulation than that in the control group(P<0.01).Under the conditions of this test,the relative expressions of IL-1β,IFN-γ,IgM and HSP70 genes in the head kidney macrophages of olive flounder under culture conditions were up-regulated in the culture medium supplemented with soybean isoflavone at a dose of 0.5-1.5 mg/mL,thus enhancing the immunity of the macrophages in the head kidney of olive flounder.