日本血吸虫可溶性虫卵抗原诱导小鼠巨噬细胞增殖及TNF-α、TGF-β1的表达

Proliferation and TNF-α、TGF-β1 Expression of Macrophage RAW264.7 Induced by Soluble Egg Antigen of Schistosoma Japonicum

目的探讨日本血吸虫可溶性虫卵抗原(SEA)对小鼠巨噬细胞株RAW264.7增殖及TNF-α、TGF-β1表达的影响。方法实验分三组:RAW264.7对照组、SEA组和细菌脂多糖(LPS)对照组。培养巨噬细胞株RAW264.7,用20μg/ml SEA分别作用巨噬细胞3、6、9、12和15 h后,荧光定量PCR检测炎症因子TNF-αmRNA和TGF-β1 mRNA的表达;倒置显微镜观察SEA作用12 h后巨噬细胞的形态;Western blot检测炎症相关蛋白TNF-α和TGF-β1的表达;MTT比色法检测SEA对细胞增殖的影响。结果 SEA作用RAW264.7细胞3 h起,TNF-αmR-NA和TGF-β1 mRNA的表达逐渐增高,在第12 h时,其相对表达倍数分别达23.07±3.172和9.83±1.45,与其他各时间点比较差异均有统计学意义(均P<0.05);SEA作用12 h巨噬细胞体积明显增大,细胞拉长且形态不规则;SEA作用巨噬细胞24、48、72 h时,A_(490)值分别与RAW264.7对照组相比,差异均有高度统计学意义(均P<0.01),而与LPS组的差异无统计学意义(P>0.05);在SEA作用RAW264.7细胞12 h时,TNF-α和TGF-β1蛋白表达均高于RAW264.7对照组(均P<0.05)。结论 SEA能促进巨噬细胞的增殖并诱导TNF-α和TGF-ββ1的表达。

Objective To explore the effect of proliferation and TNF-a,TGF-β1 expression of macrophage RAW264.7 induced by soluble egg antigen（SEA） of Schistosoma japonicum.Methods Macrophages in the experiment were divided into three groups;the RAW264.7 control gronp,SEA group and Lipopolysaccharide（LPS） control group.The macrophage RAW264.7 was cultured and stimulated with 20μg/ml SEA for 3,6,9,12 and 15 hours,then the expression of TNF-αmRNA and TGF-β1 mRNA were checked by quantitative PCR（qPCR）.After stimulated by SEA for 12 hours,the appearance of RAW264.7 cells was observed by microscope,the protein expression of TNF-αand TGF-β1 was tested by Western blot.Cell proliferation was examined with MTT method.Results The relative expression of TNF-αand TGF-β1 mRNA increased gradually from 3 hours of stimulation and reached the peak of 23.07±3.17 and 9.83±1.45 at 12 hours respectively.Compared with other time points,the difference was significant（P〈0.05 ）.Compared with the RAW264.7 control group,cells became longer and less regular after treated with SEA or LPS for 12 hours.After treated with SEA for 24,48 and 72 hours,there was statistical difference of A_（490） between the SEA group and the RAW364.7 control group（P 0.01）,but there was no significant difference between the SEA group and the LPS group（P〈0.05）.After treated with SEA for 12 hours,the protein expression of both TNF-a and TGF-β1 increased in the SEA group, compared with the RAW364.7 control group（P〈0.05）.Conclusion SEA can promote the proliferation of macrophage RAW264.7 and induce the expression of TNF-a and TGF-β1.