摘要
目的观察结缔组织生长因子(CTGF)对体外培养的人肾小管上皮细胞转分化的直接影响,并探讨阻断内源性CTGF表达对转化生长因子-β1(TGF-β1)诱导人肾小管上皮细胞转分化的干预效果,以阐明CTGF在肾小管上皮细胞转分化中的作用。方法体外培养人近曲小管上皮细胞(HKC)。在观察CTGF对HKC的直接作用时,将HKC细胞分为三组:(1)对照组;(2)小剂量CTGF组:培养液中加入重组人CTGF(rhCTGF),终浓度为2·5μg/L;(3)大剂量CTGF组:rhCTGF终浓度为5·0μg/L。在探讨CTGF在TGF-β1诱导HKC转分化中的作用时,将细胞分为4组:(1)正常对照组;(2)TGF-β1刺激组(培养液加入重组人TGF-β1蛋白,浓度为10·0μg/L);(3)TGF-β1+CTGF正义寡核苷酸(ODN)组(先以CTGF正义ODN转染HKC,再加TGF-β1刺激);(4)TGF-β1+CTGF反义ODN组(先以CTGF反义ODN转染HKC,再加TGF-β1刺激)。用逆转录-聚合酶链反应(RT-PCR)方法测定HKCα-平滑肌肌动蛋白(α-SMA)和Ⅳ型胶原(colⅣ)mRNA水平的变化;间接免疫荧光方法检测HKC胞浆内α-SMA的表达;流式细胞仪检测α-SMA阳性细胞百分率;ELISA方法测定培养液上清中colⅣ的浓度。结果不同浓度rhCTGF作用于HKC24h后,α-SMA mRNA水平显著升高(P<0·01),而colⅣmRNA表达水平明显下降(均P<0·01);刺激48h后,胞浆α-SMA表达明显增强,流式细胞仪测得三组细胞α-SMA阳性百分率依次为:2·4%、38·9%、65·5%(P<0·01);ELISA结果表明,rhCTGF抑制了colⅣ的分泌(P<0·01)。TGF-β1可诱导HKC高表达CTGF和α-SMA。转染后6h,CTGF反义ODN可显著抑制HKC表达CTGF和α-SMA mRNA(P<0·01)。转染后48h,CTGF反义ODN可明显抑制胞内α-SMA蛋白合成。结论CTGF在体外能刺激肾小管上皮细胞向肌成纤维细胞(MyoF)转分化,而CTGF反义ODN的导入可有效抑制TGF-β1诱导的转分化过程。因此,CTGF可能是调节肾小管上皮细胞转分化的重要因子。
Objective To observe the effect of connective tissue growth factor （CTGF） on the transdifferentiation of human renal tubular epithelial cells and to explore the influence of CTGF antisense oligodeoxynucleotide （ASODN） transfection on the transdifferentiation process induced by transforming growth factor-β1 （TGF-β1）. Methods Human renal tubular epithelial cells of the strain HKC were cultured and divided into 3 groups ： （ 1 ） negative control group, （ 2 ） low dose CTGF group, treated with recombinant human CTGF （ rhCTGF ） with the terminal concentration of 2.5 μg/L, and （ 3 ） high dose CTGF group, treated with rhCTGF with the terminal concentration of 5. 0 μg/L）. To evaluate the contribution of CTGF to the transdifferentiation induced by TGF-1, Another HKC cells were divided into 4 groups： （ 1 ） untreated control group （ Group C） , （2） Group T, stimulated by TGF-β1 （ 10.0 μg,/L） , （3） Group S, stimulated by sense ODN transfection ＋ TGF-β1 （ 10.0 μg,/L） , and （4） Group A, stimulated by antisense ODN transfection ＋ TGF-β1 （ 10.0 μg,/L）. RT-PCR was used to detect the mRNA expression of a-smooth muscle actin （ct-SMA） and collagen type Ⅳ （col Ⅳ ） mRNA. Indirect immunofluorescence assay and flow cytometry were used to assess the level of intracellular α-SMA protein. ELISA was used to determine the concentration of col Ⅳ in the media. Results The normal HKC cells were round and the HKC cells stimulated with rhCTGF became elongated. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA mRNA increased markedly （both P 〈 0.01 ） , while the mRNA expression of collagen type IV gene was down-regulated significantly （ both P 〈 0. 01 ）. The percentage of α-SMA positive cells was significantly higher in the stimulated groups than that in negative control with significant difference among any 2 groups （38.9% , 65.5% vs. 2.4% respectively, all P 〈 0.01 ）. Under this condition, collagen type IV secreted into the culture medium was lowered markedly upon the induction of CTGF （P 〈 0.01 ）. RT-PCR analysis showed that the CTGF gene expression was upregulated by TGF-β1 stimulation and peaked in 3 hours, and the α-SMA expression was upregulated by TGF-β1 stimulation, however, peaked in 6 hours. The CTGF mRNA expression of the HKC cells transfected with CTGF ASODN that was stimulated by TGF-β1 10 μg/L was significantly suppressed （ P 〈 0.01 ） and the α-SMA mRNA expression induced by TGF-β1 10 μg/L was significantly inhibited by CTGF ASODN transfection （ P 〈 0.01 ）. Indirect immunofluorescence assay showed that normal HKC cells did not express α-SMA, 48 hours after stimulation of TGF-β1 10 μg/L the HKC cells showed expression of α-SMA in the cytoplasm, and the intracytoplasmic α-SMA expression was significantly down0regulated by the transfection of CTGF ASODN （ P 〈0.01）. Conclusion CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast （MyoF） in vitro, and CTGF blockade results in a dramatic inhibition of TGF-β-induced transdifferentiation of renal tubular cells. So CTGF may be a crucial factor in promoting tubularepithelial myofibroblast transdifferentiation.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第41期2920-2925,共6页
National Medical Journal of China
基金
湖北省科技攻关计划资助项目(2003AA301C14)
关键词
生长物质
肾小管
上皮细胞
肌动蛋白类
胶原
Growth substances
Kidney tubules
Epithelial cells
Actins
Collagen Growth substances
Kidney tubules
Epithelial cells
Actins
Collagen
