LPS对大鼠骨髓MSC NF-κB p65及抗炎性细胞因子mRNA表达的影响

EFFECTS OF LPS ON NF- κB p65 AND ANTI- INFLAMMATORY CYTOKINES mRNA EXPRESSION IN RAT BONE- MARROW MSC

目的观察LPS模拟的炎性刺激对体外培养大鼠BM-MSC细胞的NF-κB活化与抗炎性因子IL-10、TGF-β3及IDO mRNA表达的影响。方法以体外培养P2代大鼠BM-MSC为受试细胞,按空白对照、PDTC(100μmol/L)、PDTC(100μmol/L)﹢LPS(5μg/mL)、LPS(1μg/mL)、LPS(5μg/mL)、LPS(10μg/mL)分组处理4 h。提取细胞总RNA,RT-PCR两步法测定NF-κB亚基p65与抗炎性因子IL-10、TGF-β3及IDO的mRNA表达。结果与对照组及PDTC﹢LPS组相比,LPS处理组NF-κB p65亚基、抗炎因子IL-10和TGF-β3 mRNA表达升高(P<0.05),而LPS处理组中IDO的mRNA表达降低(P<0.05);抗炎因子IL-10、TGF-β3表达变化与NF-κB p65亚基表达变化间呈正相关,相关系数分别为0.844(P<0.05)、0.837(P<0.05);而IDO与NF-κB p65亚基在表达变化上呈高度负相关,相关系数为-0.942(P<0.05)。结论 (1)LPS作用于MSC,可通过活化NF-κB而诱导抗炎性细胞因子IL-10、TGF-β3的mRNA表达;(2)正常培养的MSC组成性表达IDO,LPS模拟的炎性刺激对IDO表达具有抑制作用。提示在炎性和非炎性微环境下,MSC发挥负性免疫调节的机制有所不同。

Objective To investigate the effects of inflammatory stimulation by lipopolysaccharide （ LPS ） on mRNA expression of NF - κB p65, anti - inflammatory cytokines IL - 10, TGF - β3 and indoleamine 2,3 - dioxyge- nase （IDO） in rat bone - nmrrow mesenchymal stem cells （BM - MSC）. Methods The MSC cultured in vitro was divided into 6 groups： The normal group, the pyrrolidinothiocarbamate （PDTC）（100μmoL/L） treatment group, the PDTC （ 1 00 μmol/L） plus LPS （5 μg/mL） treatment group and the 3 LPS dose （ 1 μg/mL, 5μg/mL, 10μg/mL） treatment groups. After LPS supplement for 4 h, the total RNA of the MSC was extracted and the mRNA expression of p65, IL - 10 ,TGF - β and IDO were detected by RT -PCR. The gray scale was used to compare the mean lev- els （x ＋ s） of PCR product within groups, and the correlation coefficient of p65 with IL - 10, TGF - β3, and IDO were determined. Results Compared with the control group, the mRNA expression of NF - κB Subunit p65 and anti - inflammatory cytokines IL - 10, TGF - βwere significant increased in the LPS treated groups （P 〈 0.05 ）. The correlation coefficient of p65 with IL - 10 and TGF - 133 were 0. 844 （ P 〈 0.05 ） ,0. 837 （ P 〈 0.05 ） respectively. The correlation coefficient of p65 with IDO was - 0. 942 （ P 〈 0.05 ）. Conclusion （ 1 ） LPS stimulation can induce MSC producing more anti - inflammatory cytokines through the activation of NF - κB. （2） IDO is constitutively ex- pressed in normal cultured MSC, and its expression may be down regulated by inflammatory stimulation. This result indicates that there are different immune -modulation pathways in MSC under different microenvironments.