TGFβ1参与视黄酸诱导的HL-60细胞分化

为研究内源性TGF-β１对全反式视黄酸（ATRA）作用HL60细胞的影响，应用定量RT-PCR和ELISA方法，研究ATRA诱导HL-60细胞分化过程中TGF-β１ 表达的变化.并构建TGF-β１ RNA干扰表达质粒，抑制HL-60细胞内源性TGF-β１表达，进而研究ATRA诱导内源性TGF-β１表达下降的HL-60细胞分化的情况．结果发现,ATRA诱导HL-60细胞分化过程中,TGF-β１ 表达明显增高．得到4个针对不同靶位点的RNA干扰表达质粒.其中,针对起始编码区的质粒转染48 h后对HL-60细胞的TGF-β１蛋白抑制率为73~2％．内源性TGF-β１表达下降后，ATRA作用的HL-60细胞NBT还原试验的光密度值降低，CD33抗原阳性的细胞比例较对照组升高，CD11b抗原阳性的细胞比例较对照组降低.表明内源性TGF-β１表达下降后，ATRA诱导HL-60细胞分化的作用有所减弱，提示内源性TGF-β１在ATRA诱导HL-60细胞分化中起一定的作用．

TGF-β1Involved in All-trans Retinoic Acid-Stimulated Differentiation of HL60 Cells

To investigate the role of endogenous transforming growth factor beta1(TGF-β１) on the effects of all trans-retinoic acid (ATRA) in HL-60 cells, the mRNA level of TGF-β１ were detected by real-time quantitative RT-PCR and the protein level were determined by ELISA. Small RNA oligonucleotides targeted to different regions of TGF-β１ gene were designed by Ambion’s siRNA Designer software and expression plasmids for TGF-β１ RNA interference (RNAi) were constructed with pSilencer 2.1-U6 neo vectors, then transfected into HL-60 cells. Among four TGF-β１ shRNA (small hairpin RNA) plasmids constructed, one targeted particular to the beginning of TGF-β１ coding region presented the best gene expression inhibition by 73~2%. The effects of ATRA in TGF-β１ knockdown HL-60 cells were evaluated by nitro blue tetrazolium (NBT) staining and FACS (Fluorescence Activated Cell Sorter). The results showed that the ATRA-induced expression of TGF-β１ in HL-60 cells was upregulated, and the NBT staining was reduced. The CD33 positive cell population was increased, while the CD11b positive population was reduced compared to the controls with normal TGF-β１ levels. We concluded that the knockdown of endogenous TGF-β１ attenuates ATRA-induced effects in HL-60 cells, which indicated that endogenous TGF-β１ played a significant role downstream of ATRA treatments.