【摘要】 目的比较结缔组织生长因子(CTGF)反义寡核苷酸两种导入方法的优缺点,观察CTGF反义寡核苷酸对TGF-β1刺激的肾小管上皮细胞胶原分泌的影响。方法分别用脂质体包裹和未包裹的CTGF反义寡核苷酸(分高剂量和低剂量)处理NRK52E肾小管上皮细胞,观察异硫氰酸荧光素(FITC)荧光标记的反义寡核苷酸的导入情况和细胞的生长状态。采用RT-PCR和Western印迹方法检测CTGF和Ⅰ型胶原的表达。结果脂质体包裹有助于反义寡核苷酸的导入,于6h～9h细胞即出现生长抑制,形态改变。直接导入法反义寡核苷酸进入较慢,在96h内仍未出现明显细胞毒性。直接导入法中,反义寡核苷酸处理48h后,高、低剂量组CTGFmRNA和蛋白大部分受抑,72h时低剂量组CTGF的表达有少量的恢复。TGF-β1刺激能够显著增加Ⅰ型胶原分泌,加入CTGF反义寡核苷酸共同孵育48h,可以大部分取消TGF-β1的作用,而正义和错义的寡核苷酸没有作用。结论反义寡核苷酸直接导入细胞较慢,但仍然能够达到较好的抑制效果,且细胞毒性小,可以用于体外反义技术的研究。CTGF反义寡核苷酸能够抑制TGF-β1引起的肾小管上皮细胞胶原Ⅰ型分泌增多,表明阻断CTGF可能是延缓肾间质纤维化的有效手段。更多还原

【Abstract】 Objective To compare two transduction ways of connective tissue growth factor(CTGF) antisense oligonucleotide(ASON)into tubular epithelial cells,and investigate the influence of CTGF antisense oligonucleotide on collagen secretion induced by transforming growth factor β1(TGF β1). Methods CTGF ASON( high dose or low dose) was transduced into the normal rat kidney tubular epithelial cell line(NRK52E) by co transfection with DOTAP liposomal or direct ways. The transfection ratio of FITC labeled ASON was measured by fluoroscope, and the cytotoxicity was observed by light microscopy. The expression of CTGF and collagen Ⅰwas detected by RT PCR and Western blotting. Results DOTAP liposome could enhance ASON transfection, but within 6 h～9 h, cells growth was inhibited, and morphological changes were found. Direct ASON transfection was slow, and no obvious cytotoxity was observed within 96 h. ASDN treated for 48h, the expression of CTGF was mostly inhibited. For 72 h, CTGF expression was partly restored. Culture in TGF β1 10 ng/ml caused profound increase of collagen type Ⅰlevel. CTGF antisense oligonucleotide inhibited the expression of CTGF gene and protein, and down regulated the collagen Ⅰexpression in response to TGF β1 ,but sense and missense oligonucleotides not.Conclusion Direct ASON transfection is effctive and has less cytotoxity. CTGF ASON can abrogate the influence of TGF β1 on collagen type Ⅰsecretion in tubular epithelial cells. CTGF may be a potential therapeutic target to renal tubulointerstitial fibrosis.更多还原