间质干细胞培养上清对日本血吸虫SEA诱导活化的巨噬细胞株RAW264.7的抑制作用

Inhibition of Culture Supernatant of Mesenchymal Stem Cells on Macrophages RAW264.7 Activated by Soluble Egg Antigen of Schistosoma japonicum

目的观察大鼠骨髓间质干细胞(mesenchymal stem cell,MSC)培养上清对日本血吸虫可溶性虫卵抗原(soluble egg antigen,SEA)诱导活化的巨噬细胞的抑制作用。方法用5、10、20和40μg/ml SEA分别诱导巨噬细胞株RAW264.7 12 h,或用20μg/ml SEA分别诱导小鼠巨噬细胞株(RAW264.7)4、8、12和24 h后,用实时荧光定量PCR检测α肿瘤坏死因子(TNF-α)的mRNA水平,选择SEA的最佳作用浓度和作用时间。将巨噬细胞分成5组,分别为阴性对照组、SEA组、SEA+MSC上清组(MSC组)、SEA+大鼠肾小管上皮细胞株(NRK-52E)上清组(NRK-52E组)和SEA+DMEM细胞培养基组(DMEM组)。除阴性对照组外,其他各组给予20μg/ml SEA诱导巨噬细胞活化12 h后,MSC组、NRK-52E组和DMEM组换液撤SEA,分别给予MSC培养上清、NRK-52E细胞培养上清和DMEM培养液,继续培养。显微镜观察细胞上清培养12 h后各组细胞形态。实时荧光定量PCR检测细胞上清培养12 h和24 h后TNF-αmRNA水平。蛋白质印迹(Western blotting)分析检测细胞上清培养12 h后转化生长因子β1(TGF-β1)的蛋白表达水平。噻唑蓝比色法(MTT法)测定细胞上清培养24 h和48 h后巨噬细胞增殖情况。结果 SEA活化巨噬细胞的最佳浓度和时间分别为20μg/ml和12 h。镜下观察显示,MSC上清培养12 h后,MSC组与SEA组、NRK-52E组和DMEM组相比,细胞变圆,体积明显较小,伪足较少。MSC上清作用12 h和24 h后,MSC组TNF-αmRNA水平分别为阴性对照组的(1.0±0.4)和(1.0±0.5)倍,显著低于NRK-52E组[分别为(10.4±3.9)和(16.5±5.0)倍(12 h:P<0.05;24 h:P<0.01)]和DMEM组[分别为(6.0±2.1)和(2.4±0.7)倍(均P<0.05)]。MSC上清作用12 h后,MSC组蛋白TGF-β1/GAPDH为0.31±0.10,显著低于NRK-52E组(0.88±0.10,P<0.01)和DMEM组(0.58±0.06,P<0.05)。MSC上清作用48 h后,MSC组吸光度(A490值)为0.22±0.05,与NRK-52E组(0.53±0.02)和DMEM组(0.31±0.03)比较差异均有统计学意义(均P<0.05)。结论 MSC培养上清能抑制SEA诱导的巨噬细胞株RAW264.7活化。

Objective To observe the inhibitive effect of rat mesenchymal stem cells（MSC） culture supernatant on macrophages activated by soluble egg antigen（SEA） of Schistosoma japonicum.Methods To select optimal SEA effecting concentration and time,macrophages RAW264.7 were induced by 5,10,20 or 40 μg/ml SEA for 12 h,or by 20 μg/ml SEA for 4,8,12 or 24 h before examination of TNF-α mRNA by RT-PCR.Macrophages were divided into five groups,i.e.negative control group,SEA group,SEA＋MSC supernatant group（MSC group）,SEA＋NRK-52E supernatant group（NRK-52E group） and SEA＋DMEM group（DMEM group）.Except negative control group,macrophages in other four groups were induced by 20 μg/ml SEA for 12 h.SEA was then removed from MSC group,NRK-52E group and DMEM group and replaced with MSC supernatant,NRK-52E supernatant and DMEM,respectively.Morphology of macrophages in each group was observed by microscope after cultured with supernatant for 12 h.TNF-α mRNA in macrophages was detected by real-time quantitative PCR after cultured with supernatant for 12 h and 24 h.TGF-β1 in macrophages was observed by Western blotting analysis after cultured with supernatant for 12 h.Macrophage proliferation was tested by MTT method after cultured with supernatant for 24 h and 48 h.Results The optimal SEA concentration and time for macrophage activation was 20 μg/ml and 12 h,respectively.Compared with SEA group,NRK-52E group,and DMEM group,macrophages in MSC group were round and small with less pseudopodia after cultured with supernatant for 12 h.TNF-α mRNA after cultured with MSC supernatant for 12 h and 24 h was（1.0±0.4） and（1.0±0.5） fold of negative control group,respectively,significantly less than NRK-52E group [（10.4±3.9） and（16.5±5.0） fold]（12 h： P0.05;24 h：P0.01） and DMEM group [（6.0±2.1） and（2.4±0.7） fold]（P0.05）.The grey density image analysis of TGF-β1/GAPDH was 0.31±0.10 in MSC group,much lower than 0.88±0.10 in NRK-52E group（P0.01） and 0.58±0.06 in DMEM group（P0.05） after cultured with supernatant for 12 h.After 48 h culture,A490 of macrophages in MSC group was 0.22±0.05,much lower than 0.53±0.02 in NRK-52E group and 0.31±0.03 in DMEM group（P0.05）.Conclusion MSC supernatant can inhibit activation and proliferation of macrophages which were induced by SEA of S.japonicum.