TGF-β1参与视黄酸诱导的HL-60细胞分化  

TGF-β_1 Involved in All-trans Retinoic Acid-Stimulated Differentiation of HL60 Cells

作　　者：黄美娟[1] 吴立德[2] 陈元仲[1] 李昊[3] 张臣青[1] 张昭秀[1] 陈显凌[1] 

机构地区：[1]福建医科大学附属协和医院血液病研究所,福州350001 [2]福建省泉州市第一医院,泉州362000 [3]福州大学生物工程研究所,福州350001

出　　处：《中国生物化学与分子生物学报》2008年第5期444-451,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：福建医科大学青年教师基金资助项目(No.FTGXQ04020);福建省自然科学基金高校专项基金资助项目(No.C0440004)~~

摘　　要：为研究内源性TGF-β1对全反式视黄酸(ATRA)作用HL-60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-β1RNA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48h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.To investigate the role of endogenous transforming growth factor betal （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 upregnlated, 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.

关 键 词：转化生长因子Β1 SIRNA 视黄酸 HL-60细胞 

分 类 号：Q74[生物学—分子生物学]