关键词: 间质干细胞, 日本血吸虫, 巨噬细胞, 可溶性虫卵抗原

Abstract: 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: P<0.05；24 h：P<0.01） and DMEM group ［（6.0±2.1） and （2.4±0.7） fold］（P<0.05）. 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 （P<0.01） and 0.58±0.06 in DMEM group （P<0.05） 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 （P<0.05）.   Conclusion   MSC supernatant can inhibit activation and proliferation of macrophages which were induced by SEA of S. japonicum.

Key words: Mesenchymal stem cell, Schistosoma japonicum, Macrophage, Soluble egg antigen