Zhang Chun, Zhu Zhong-hua, Liu Jian-she, Yang Xiao, Deng An-Guo
Department of Nephrology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zhonghua Yi Xue Za Zhi. 2005 Nov 2;85(41):2920-5.
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-beta1 (TGF-beta1).
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 microg/L, and (3) high dose CTGF group, treated with rhCTGF with the terminal concentration of 5.0 microg/L). To evaluate the contribution of CTGF to the transdifferentiation induced by TGF-beta1, Another HKC cells were divided into 4 groups: (1) untreated control group (Group C), (2) Group T, stimulated by TGF-beta1 (10.0 microg/L), (3) Group S, stimulated by sense ODN transfection + TGF-beta1 (10.0 microg/L), and (4) Group A, stimulated by antisense ODN transfection + TGF-beta1 (10.0 microg/L). RT-PCR was used to detect the mRNA expression of alpha-smooth muscle actin (alpha-SMA) and collagen type IV (col IV) mRNA. Indirect immunofluorescence assay and flow cytometry were used to assess the level of intracellular alpha-SMA protein. ELISA was used to determine the concentration of col IV in the media.
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 alpha-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 alpha-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-beta1 stimulation and peaked in 3 hours, and the alpha-SMA expression was upregulated by TGF-beta1 stimulation, however, peaked in 6 hours. The CTGF mRNA expression of the HKC cells transfected with CTGF ASODN that was stimulated by TGF-beta1 10 microg/L was significantly suppressed (P < 0.01) and the alpha-SMA mRNA expression induced by TGF-beta1 10 microg/L was significantly inhibited by CTGF ASODN transfection (P < 0.01). Indirect immunofluorescence assay showed that normal HKC cells did not express alpha-SMA, 48 hours after stimulation of TGF-beta1 10 microg/L the HKC cells showed expression of alpha-SMA in the cytoplasm, and the intracytoplasmic alpha-SMA expression was significantly down-regulated by the transfection of CTGF ASODN (P < 0.01).
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-beta-induced transdifferentiation of renal tubular cells. So CTGF may be a crucial factor in promoting tubular-epithelial myofibroblast transdifferentiation.
观察结缔组织生长因子（CTGF）对人肾小管上皮细胞转分化的影响，探讨CTGF反义寡脱氧核苷酸（ASODN）转染对转化生长因子-β1（TGF-β1）诱导的转分化过程的影响。
培养人HKC肾小管上皮细胞并分为3组：（1）阴性对照组；（2）低剂量CTGF组，用终浓度为2.5μg/L的重组人CTGF（rhCTGF）处理；（3）高剂量CTGF组，用终浓度为5.0μg/L的rhCTGF处理。为评估CTGF对TGF-β1诱导的转分化的作用，将另一组HKC细胞分为4组：（1）未处理对照组（C组）；（2）T组，用TGF-β1（10.0μg/L）刺激；（3）S组，用正义寡脱氧核苷酸转染+TGF-β1（10.0μg/L）刺激；（4）A组，用反义寡脱氧核苷酸转染+TGF-β1（10.0μg/L）刺激。采用逆转录-聚合酶链反应（RT-PCR）检测α-平滑肌肌动蛋白（α-SMA）和IV型胶原（col IV）mRNA的表达。采用间接免疫荧光法和流式细胞术评估细胞内α-SMA蛋白水平。采用酶联免疫吸附测定（ELISA）法测定培养基中col IV的浓度。
正常HKC细胞呈圆形，经rhCTGF刺激的HKC细胞变长。不同浓度rhCTGF刺激后，α-SMA mRNA表达明显增加（均P<0.01），而IV型胶原基因的mRNA表达明显下调（均P<0.01）。刺激组中α-SMA阳性细胞百分比明显高于阴性对照组，任意两组间差异均有统计学意义（分别为38.9%、65.5%和2.4%，均P<0.01）。在此条件下，CTGF诱导后分泌到培养基中的IV型胶原明显降低（P<0.01）。RT-PCR分析显示，TGF-β1刺激可上调CTGF基因表达，并在3小时达到峰值，TGF-β1刺激可上调α-SMA表达，但在6小时达到峰值。用10μg/L TGF-β1刺激并用CTGF ASODN转染的HKC细胞的CTGF mRNA表达明显受到抑制（P<0.01），CTGF ASODN转染可明显抑制10μg/L TGF-β1诱导的α-SMA mRNA表达（P<0.01）。间接免疫荧光法显示，正常HKC细胞不表达α-SMA，10μg/L TGF-β1刺激48小时后HKC细胞在细胞质中显示α-SMA表达，CTGF ASODN转染可明显下调细胞质内α-SMA表达（P<0.01）。
CTGF在体外可促进人肾小管上皮细胞向肌成纤维细胞转分化，阻断CTGF可显著抑制TGF-β诱导的肾小管细胞转分化。因此，CTGF可能是促进肾小管上皮-肌成纤维细胞转分化的关键因素。
