α-酮戊二酸二甲酯预处理对犬脂肪源间充质干细胞免疫调节能力的影响

Immunomodulatory Effects of Dimethyl Alpha-ketoglutarate Pretreatment on Canine Adipose-derived Mesenchymal Stem Cells

【目的】探究α-酮戊二酸二甲酯(dimethyl alpha-ketoglutarate,DMKG)预处理对犬脂肪源间充质干细胞(canine adipose-derived mesenchymal stem cells,cAD-MSCs)免疫调节的影响。【方法】利用不同浓度的DMKG培养基,探索DMKG对cAD-MSCs增殖的影响。用1 mmol/L DMKG预处理cAD-MSCs后,采用ELISA方法检测培养上清液中炎性细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6、IL-10及转化生长因子-β(TGF-β)的水平。以含100、250、500、1000、2000 ng/mL的脂多糖(lipopolysaccharide,LPS)培养基培养RAW264.7细胞,24 h后采用CCK-8法检测细胞存活率,筛选最适LPS浓度以建立RAW264.7细胞炎症模型。把RAW264.7细胞分为空白对照组、LPS组、MSCs组及DMKG-MSCs组,将DMKG预处理后的cAD-MSCs与250 ng/mL LPS处理的RAW264.7细胞共培养24 h后,采用CCK-8法检测细胞增殖活性,Transwell法检测细胞迁移能力,Griess法检测培养上清液中一氧化氮(NO)含量,实时荧光定量PCR检测一氧化氮合成酶(iNOs)、IL-1β、IL-6、IL-10和精氨酸酶-1(Arg-1)的mRNA表达水平,流式细胞仪检测M1巨噬细胞表面标记物CD80+、CD86+。【结果】与MSCs组相比,DMKG预处理cAD-MSCs后培养上清液中TGF-β、IL-10的分泌显著升高(P<0.05),而IL-1β、IL-6的分泌受到显著抑制(P<0.05),TNF-α分泌无显著差异(P>0.05)。LPS浓度为250 ng/mL时,RAW264.7细胞增殖活性达到最高。与LPS组相比,MSCs组和DMKG预处理后的cAD-MSCs均能极显著抑制RAW264.7细胞的增殖活性和迁移能力(P<0.01),抑制细胞NO的释放量并下调iNOs的mRNA表达水平(P<0.01);此外,下调RAW264.7细胞IL-1β(P<0.01)、IL-6 mRNA表达水平,上调IL-10 mRNA表达水平(P<0.05;P<0.01),M2巨噬细胞表面标记物Arg-1的mRNA表达水平极显著上调(P<0.01),M1巨噬细胞表面标记物CD80+、CD86+的表达极显著降低(P<0.01);与MSCs组相比,DMKG预处理的cAD-MSCs抑制巨噬细胞激活效果更好。【结论】DMKG能有效增强cAD-MSCs的免疫调节作用,表现营养因子TGF-β和抑炎细胞因子IL-10表达增多,促炎细胞因子IL-1β和IL-6表达减少,抑制LPS刺激条件下RAW264.7细胞的激活,减轻细胞炎症反应。

【Objective】The aim of the study was to explore the effect of dimethyl alpha-ketoglutarate(DMKG)on immunomodulatory effect of canine adipose-derived mesenchymal stem cells(cAD-MSCs).【Method】In this experiment,DMKG medium with different concentrations was used to explore the effects of DMKG on the proliferation of cAD-MSCs.After cAD-MSCs were pretreated with 1 mmol/L DMKG,the levels of inflammatory cytokines of tumor nerosis factor-α(TNF-α),interleakin-1β(IL-1β),IL-6,IL-10 and transforming growth factor-β(TGF-β)in culture supernatant were determined by ELISA.RAW264.7 cells were cultured in medium containing 100,250,500,1000 and 2000 ng/mL lipopolysaccharide(LPS),respectively.After 24 h,the survival rate of cells was detected by CCK-8 assay to screen the optimal concentration of LPS was selected to establish RAW264.7 inflammation model.The cells were divided into blank control group,LPS group,MSCs group and DMKG-MSCs group,the cAD-MSCs pretreated with DMKG were co-cultured with RAW264.7 cells under 250 ng/mL LPS condition for 24 h,and the proliferation activity of the cells was detected by CCK-8 assay,the cell migration capacity was detected by Transwell assay,the content of NO in culture supernatant was determined by Griess assay,the mRNA expression levels of iNOs,IL-1β,IL-6,IL-10 and Arg-1 genes were detected by Real-time quantitative PCR,the surface markers CD80+and CD86+of M1 macrophages were detected by flow cytometry.【Result】Compared with MSCs group,the secretion of TGF-βand IL-10 in culture supernatant after DMKG pretreatment of MSCs was significantly increased(P<0.05),while the secretion of IL-1βand IL-6 were significantly inhibited(P<0.05),and the secretion of TNF-αwas not significantly different(P>0.05).The proliferation activity of RAW264.7 cells reached the highest when LPS concentration was 250 ng/mL.Compared with LPS group,MSCs and DMKG pretreated cAD-MSCs extremely significantly inhibited the proliferation activity and migration ability of RAW264.7 cells(P<0.01),inhibited NO release and down-regulated the mRNA expression level of iNOs(P<0.01).In addition,mRNA expression levels of IL-1β(P<0.01)and IL-6 in RAW264.7 cells were down-regulated,IL-10 was up-regulated(P<0.05 or P<0.01),the mRNA expression level of M2 macrophage surface marker Arg-1 was extremely significantly up-regulated(P<0.01),and the expressions of surface markers CD80+and CD86+in M1 macrophages were extremely significantly decreased(P<0.01).Compared with MSCs group,DMKG pretreated cAD-MSCs inhibited macrophage activation better.【Conclusion】DMKG could effectively enhance the immunomodulatory effect of cAD-MSCs and increased expression of trophic factor TGF-βand anti-inflammatory cytokine IL-10,and the decreased expression of proinflammatory cytokines IL-1βand IL-6,inhibit the activation of RAW264.7 cells under the condition of LPS stimulation,and reduce the cellular inflammatory response.