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目的探讨小干扰RNA(siRNA)对体外人CD4+CD25-T淋巴细胞MLL1基因表达的抑制效果及体外转化生长因子-β1(TGF-β1)诱导调节性T淋巴细胞(iTreg)的影响,为治疗与Treg失衡相关的临床疾病提供理论基础。方法采用免疫磁珠分选方法从健康人外周血中分离出CD4+CD25-T淋巴细胞(纯度>85%)。MLL1基因的SET区对组蛋白3上的第4个赖氨酸位点的甲基化起重要作用,针对其设计并合成了不同的siRNAs,分别为带绿色荧光的siRNA(siRNA-FAM)、3个实验组(siRNA-1、siRNA-2、siR-NA-3)与1个阴性对照组(siRNA-NC)。采用脂质体法转染分选后培养24 h的人CD4+CD25-T淋巴细胞。先转染siRNA-FAM,于转染后的6 h,用荧光显微镜观察siRNA的转染效率,寻找最佳的转染条件。按照上述转染条件转染siRNA-1、siRNA-2、siRNA-3、siR-NA-NC。于转染后的48 h,每组取2×106个细胞,提取细胞内RNA,然后反转录为cDNA,用聚合酶链反应扩增该片段,用琼脂糖凝胶检测扩增后的DNA。在MLL1基因表达受到抑制的情况下,采用流式细胞术检测TGF-β1诱导72 h iTreg的比例。结果当siR-NA为100 pmol,脂质体为5×10-6 L时,siRNA的转染效率最高为(50.80±0.61)%。该实验条件下,发现实验组siRNA-2对MLL1基因mRNA较对照组有明显削弱作用(F=5.820,P<0.05)。在MLL1基因表达削弱的情况下,TGF-β1诱导72 h iTreg比例有明显降低(F=4.046,P<0.05)。结论 siRNA可以有效抑制人CD4+CD25-T淋巴细胞中MLL1基因的表达,找到了具有较高转染效率的siRNA。TGF-β1体外诱导的iTreg受核因子MLL1的调节。
Objective To investigate the effect of small interfering RNA (siRNA) on MLL1 gene expression in human CD4 + CD25-T lymphocytes in vitro and the effect of transforming growth factor-β1 (TGF-β1) on regulatory T lymphocytes (iTreg) Provide a theoretical basis for clinical disease associated with imbalance of Tregs. Methods CD4 + CD25-T lymphocytes (purity> 85%) were isolated from healthy human peripheral blood by immunomagnetic bead sorting. The SET region of the MLL1 gene plays an important role in the methylation of the fourth lysine site on histone 3, and different siRNAs were designed and synthesized, namely siRNA with green fluorescence (siRNA-FAM) Three experimental groups (siRNA-1, siRNA-2, siR-NA-3) and one negative control group (siRNA-NC). The human CD4 + CD25-T lymphocytes cultured for 24 h were transfected by lipofectamine. First transfection siRNA-FAM, 6 h after transfection, using a fluorescence microscope to observe the transfection efficiency of siRNA, looking for the best transfection conditions. SiRNA-1, siRNA-2, siRNA-3, siR-NA-NC were transfected as described above. At 48 h after transfection, 2 × 106 cells in each group were taken and RNA was extracted from the cells and reverse transcribed into cDNA. The fragment was amplified by polymerase chain reaction (PCR) and the amplified DNA was detected by agarose gel . When MLL1 gene expression was inhibited, the proportion of iTreg induced by TGF-β1 at 72 h was detected by flow cytometry. Results The siRNA transfection efficiency was the highest (50.80 ± 0.61)% when siR-NA was 100 pmol and liposome was 5 × 10-6 L. Under the experimental conditions, it was found that siRNA-2 of the experimental group significantly attenuated MLL1 gene mRNA (F = 5.820, P <0.05) compared with the control group. When the expression of MLL1 was impaired, the proportion of iTreg induced by TGF-β1 at 72 h was significantly decreased (F = 4.046, P <0.05). Conclusion siRNA can effectively inhibit the expression of MLL1 gene in human CD4 + CD25-T lymphocytes and find siRNA with high transfection efficiency. ITreg induced by TGF-β1 in vitro is regulated by nuclear factor MLL1.