IFN-γ预处理骨髓间充质干细胞及其抑制小胶质细胞活化作用的影响

Inhibitory effects of IFN-γ pretreated bone marrow mesenchymal stem cells on lipopolysaccharide-induced microglia activation

目的探讨干扰素γ(interferon-γ,IFN-γ)预处理大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)及其抑制脂多糖(lipopolysaccharide,LPS)诱发的小胶质细胞(microglia,MG)活化作用的影响。方法采用全骨髓贴壁法分离培养大鼠BMMSCs,利用含100 ng/ml的IFN-γ完全培养液对BMMSCs进行预处理;利用LPS(10μg/ml)诱导大鼠小胶质细胞活化。小胶质细胞分离纯化后,以1×105/孔种植于6孔板中,按以下分组将小胶质细胞和BMMSCs以直接接触的方式进行共培养24h:B组(单纯BMMSCs组)、L+B组(LPS+BMMSCs组)、I+B组(IFN-γ预处理BMMSCs组)、L+I+B组(LPS+IFN-γ预处理BMMSCs组)、M组(单纯MG组)、L+M组(LPS+MG组)、B+L+M组(BMMSCs+LPS+MG组)、I+B+L+M组(IFN-γ预处理BMMSCs+LPS+MG组)。采用ELISA技术检测培养液上清TNF-α、IL-1β水平,免疫荧光法检测小胶质细胞表面标记物CD68表达。结果 L+M组小胶质细胞CD68表达增高,分泌TNF-α、IL-1β含量明显增多;B+L+M组以及I+B+L+M组的小胶质细胞CD68表达水平、TNF-α、IL-1β分泌量低于L+M组,且B+L+M组与I+B+L+M组相比有统计学差异(P<0.05)。结论 BMMSCs和IFN-γ预处理的BMMSCs均可以直接接触的方式抑制LPS诱发的小胶质细胞的活化且经IFN-γ预处理后的BMMSCs对小胶质细胞活性抑制作用更强。

Objective To explore the inhibitory effect of pre-treatment of one marrow mesenchymal stem cells （BMMSCs） with interferon-γ and IFN-γ on the activation of microglia（MG） induced by lipopolysaccharide（LPS）.Methods BMMSCs were isolated and cultured using the whole bone marrow adherence method. Then, 100ng/ml IFN-γ was used to induce the activation of BMMSCs and10μg/ml lipopolysaccharide was used as the inflammatory stimulating factor to induce the activation of microglia. After isolation and purification, microglia were seeded in 6-well plates at the density of 1×105/well and BMMSCs were co-cultured with microglia with the cell contact cell method for 24 h in the following groups： B group（BMMSCs group）, L＋B group（LPS＋BMMSCs group）, I＋B group（IFN-γ pretreatment BMMSCs group）, L＋I＋B group（LPS＋IFN-γ pretreatment BMMSCs group）, M group（microglia group）, L＋M group（LPS＋ microglia group）, B＋L＋M group（BMMSCs＋ LPS＋ microglia group）, and I＋B＋L＋M group（BMMSCs pretreated with IFN-γ＋LPS＋ microglia group）. 24 hours later, the expressions of TNF-α、IL-1βin cell culture supernatants were detected by ELISA kit and the expression of microglia surface marker CD68 was detected by immunofluorescence.Results In the L＋M group,LPS promoted the expression of CD68 and the production of TNF-α and IL-1β in microglial cells. The expression of CD68 and the production of TNF-α and IL-1β in B＋L＋M group and I＋B＋L＋M group were lower than in L＋M group, and there was statistically significant difference between the B＋L＋M group and the I＋B＋L＋M group（P＜0.05）.Conclusions Both BMMSCs and IFN-γ pretreatment of BMMSCs can inhibit the activation of LPS-induced microglia by direct cell contact. After IFN-γ pretreatment ,BMMSCs could more strongly inhibit the activity of microglia.