小干扰RNA干扰人CD4^+CD_(25)-T淋巴细胞MLL1基因的表达

Expression of Mixed-Lineage Leukemia 1 Gene in Human CD4^+CD_(25)-T Cells Inhibited by Specific Small Interfering RNA

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 assess the expression state of mixed - lineage leukemia 1 （ MLL1 ） gene in human CD4^＋ CD25^ - T cells trans- fected by MLL1 - specific small interfering RNA （ siRNAs）, explore the influence on regulatory T cell（iTreg） and provide theoretical basis for curing clinical diseases associated with Treg unbalance. Methods CD4^＋ CD25 - T cells （ purity 〉 85 % ） were isolated with magnetic cell sorting （MACS） from healthy human peripheral blood. Specific siRNAs of MLL1 SET domain, which was important for the histone（ H3 ） lysine 4 （K4） methylation, were chemically synthesized, and they contained the following groups ：A green fluorescent siRNA（ siRNA -FAM） ,5 experimental groups（ siRNA - 1, siRNA - 2, siRNA - 3 ）, and a negative control （ siRNA - NC ）. Firstly, human CD4^＋ CD25^ - T cells isolated after 24 hours were transfected by siRNA - FAM. After 6 hours, transfection efficiency of siRNA was detected by fluorescence microscopy in order to find the optimal transfection condition. Based on the above condition, siRNA - 1, siRNA - 2, siRNA - 3, siRNA - NC were transfected to hu- man CD4^＋ CD25^ - T cells. After 48 hours,intracellular RNA was extracted from 2 ~ 106 ceils of each group and reversed the cDNA. Then, the cDNA fragments were amplified using polymerase chain reaction and the amplified DNA were detected by agarose gel. It was found that siRNA -2 significantly inhibited MLL1 gene expression. The ratio of iTreg induced with transforming growth factor - β1 was measured by flow cytometry in the MLL1 - silencing cells. Results When the siRNA was 100 pmol and liposome was 5 · 10^-s L,the transfection efficiency was highest （50.80 ± 0.61 ）%. The level of MLL1 mRNA was decreased significantly in transfected with MLL1 - specific siRNA -2 （F = 5. 820, P 〈 0.05 ）. The radio of iTreg was significantly lower in the MLL1 - silencing ceils than the control groups （ F = 4. 046, P 〈 0.05 ）. Conclusions The MLL1 - specific siRNA - 2 can significantly inhibite the expression of MLL1 gene on CD4^＋ CD25^ - T cells, and the optimal siRNA sequence segment is identified. The inducing of iTreg is regulated by nuclear factor MLL1.