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采用rmGM-CSF和rmIL-4联合诱导培养小鼠骨髓树突状细胞(bone marrow dendritic cells),经免疫磁珠方法纯化;利用慢病毒载体制备RelB shRNA慢病毒,与小鼠骨髓树突状细胞共培养,经流式细胞术观察树突状细胞表面分子MHCⅡ、CD86和CD40的表达;采用CD3+免疫磁珠分离纯化小鼠T细胞;分别采用四唑蓝(MTT)比色法和液相芯片法检测异基因T细胞增殖的程度以及Th1/Th2细胞因子的分泌水平。此外,实验设LPS-DC组、未处理组和LPS RNAi RelB DC组。发现核因子RelB抑制的树突状细胞表面分子MHCⅡ、CD86和CD40均低水平表达,显著低于LPS-DC表面分子的表达(P<0.05),且该DC经LPS刺激后(LPS RNAi RelB DC)表面上述3类分子的表达水平仍显著低于LPS-DC组(P<0.05),与未处理组(immature DC)表面分子表达水平相当。其刺激异基因小鼠T细胞增殖的能力和Th1/Th2分子的分泌水平与未成熟DC组相当。表明抑制核因子RelB的骨髓树突状细胞在体外具有诱导T细胞免疫耐受的能力,是一种新型的致耐受树突状细胞研究方法。
The murine bone marrow dendritic cells were induced by combination of rmGM-CSF and rmIL-4 and purified by immunomagnetic beads. RelB shRNA lentivirus was prepared by lentiviral vector and incubated with mouse bone marrow dendritic cells The expression of MHCⅡ, CD86 and CD40 on dendritic cells was observed by flow cytometry. The T cells were isolated and purified by CD3 + immunomagnetic beads. MTT assay and liquid chromatography Method to detect the degree of proliferation of allogeneic T cells and the secretion of Th1 / Th2 cytokines. In addition, LPS-DC group, untreated group and LPS RNAi RelB DC group were set up. The results showed that the expression of MHCⅡ, CD86 and CD40 on the surface of dendritic cells inhibited by RelB was lower than that on LPS-DC (P <0.05), and the expression of LPS RNAi RelB DC ) On the surface of these three kinds of molecules is still significantly lower than the expression of LPS-DC group (P <0.05), and untreated group (immature DC) surface molecule expression levels. Its ability to stimulate allogeneic mouse T cell proliferation and Th1 / Th2 secretion levels were comparable to those in immature DCs. It is indicated that bone marrow-derived dendritic cells that inhibit nuclear factor RelB have the ability of inducing T cell immune tolerance in vitro and is a novel research method of tolerogenic dendritic cells.