[γ-干扰素对体外肾小管上皮细胞-肌成纤维细胞转分化的影响]

[Effects of interferon-gamma on tubular epithelial-myofibroblast transdifferentiation in vitro].

作者信息

Li Qin, Fan Jun-ming, Pu Lei, Li Zi, Qin Wei, Su Bai-hai

机构信息

Department of Nephrology, West China Hospital, Sichuan University, Chengdu 610041, China.

出版信息

Sichuan Da Xue Xue Bao Yi Xue Ban. 2008 Nov;39(6):895-9, 943.

PMID:19253820

Abstract

OBJECTIVE

To investigate the effects of interferon-gamma (IFN-gamma) on tubular epithelial-myofibroblast transdifferentiation (TEMT) induced by transforming growth factor (TGF-beta1).

METHODS

The normal rat kidney tubular epithelial cells (NRK52E) were cultured and divided into blank (NRK52E cells only) control group, TGF-beta1 (3 ng/mL) treated group, IFN-gamma (1000 IU/mL) treated group, and IFN-gamma inhibition group (TGF-beta1 3 ng/mL + IFN-gamma 200, 400, 600, 1000, 2000, 3000 IU/mL). After 72 hours of treatment, the morphology of cells was observed under phase-contrast microscopy and scanning electron microscopy. The expressions of a-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were detected by immunocytochemistry. Flowcytometry was employed to measure the percentage of alpha-SMA+ cells and the mean channel fluorescence (MCF). The expressions of alpha-SMA mRNA and CTGF mRNA were examined by reverse transcription-polymerase chain reaction analyses (RT-PCR). The level of collagen in the culture supernatant was measured by Enzyme-linked immunoadsordent assay (ELISA).

RESULTS

NRK52E cells cultured in the control group showed a classic cobblestone morphology. TGF-beta1 induced NRK52E cells to transdifferentiate into myofibroblast-like cells, which showed strong alpha-SMA immunostaining. The TGF-beta1 treated cells had higher percentage of a-SMA+ cells, MCF and alpha-SMA mRNA, increased CTGF mRNA expression, and ascended collagen III than the blank controls (P<0.05). IFN-gamma treated alone did not make any changes to the cell morphology, the expressions of alpha-SMA mRNA and CTGF mRNA and the level of collagen III (P>0.05). IFN-gamma exerted a strong inhibitory effect on the TEMT induced by TGF-beta1. With the increase of IFN-gamma, the percentage of alpha-SMA+ cells, the level of collagen III, and the expressions of alpha-SMA mRNA and CTGF mRNA decreased (P<0.05).

CONCLUSION

IFN-gamma inhibits the TEMT induced by TGF-beta1 and reduces the level of collagen III.

摘要

目的

探讨γ干扰素（IFN-γ）对转化生长因子（TGF-β1）诱导的肾小管上皮-肌成纤维细胞转分化（TEMT）的影响。

方法

培养正常大鼠肾小管上皮细胞（NRK52E），分为空白对照组（仅NRK52E细胞）、TGF-β1（3 ng/mL）处理组、IFN-γ（1000 IU/mL）处理组和IFN-γ抑制组（TGF-β1 3 ng/mL + IFN-γ 200、400、600、1000、2000、3000 IU/mL）。处理72小时后，在相差显微镜和扫描电子显微镜下观察细胞形态。采用免疫细胞化学法检测α-平滑肌肌动蛋白（α-SMA）和结缔组织生长因子（CTGF）的表达。用流式细胞术检测α-SMA+细胞百分比和平均通道荧光（MCF）。通过逆转录-聚合酶链反应分析（RT-PCR）检测α-SMA mRNA和CTGF mRNA的表达。采用酶联免疫吸附测定（ELISA）法检测培养上清液中的胶原蛋白水平。

结果

对照组培养的NRK52E细胞呈现典型的鹅卵石样形态。TGF-β1诱导NRK52E细胞转分化为肌成纤维细胞样细胞，α-SMA免疫染色强阳性。与空白对照组相比，TGF-β1处理的细胞中α-SMA+细胞百分比、MCF和α-SMA mRNA更高，CTGF mRNA表达增加，Ⅲ型胶原蛋白升高（P<0.05）。单独用IFN-γ处理对细胞形态、α-SMA mRNA和CTGF mRNA表达及Ⅲ型胶原蛋白水平无影响（P>0.05）。IFN-γ对TGF-β1诱导的TEMT有强烈抑制作用。随着IFN-γ浓度增加，α-SMA+细胞百分比、Ⅲ型胶原蛋白水平以及α-SMA mRNA和CTGF mRNA表达降低（P<0.05）。