人近端肾小管上皮细胞诱导表达吲哚胺2,3-加双氧酶的实验研究

Induced expression of indoleamine 2,3-dioxygenase in epithelial cells of human proximal renal tubule

目的了解培养的人近端肾小管上皮HK2细胞株吲哚胺2,3-加双氧酶(IDO)的诱导表达情况及其影响因素。方法实验分为不同浓度(0、500、1000、2000U/ml)γ-干扰素(IFN-γ)刺激培养组和IFN-γ2000U/ml刺激12、24、48、72h不同时点培养组,以10%胎牛血清DMEM培养基作为正常对照组。RT-PCR检测不同培养条件下HK2细胞IDOmRNA的表达,免疫细胞化学染色检测24h后HK2细胞IDO的表达,高效液相色谱法检测培养基中犬尿氨酸、色氨酸浓度。结果正常HK2细胞不表达IDOmRNA和IDO蛋白。不同浓度IFN-γ刺激后,HK2细胞IDO mRNA的表达呈剂量依赖性升高,与正常对照组相比差异显著(P<0.05),同时IDOmRNA表达还呈时间依赖性地增高,以48h为最佳刺激时间。免疫组化染色显示,经IFN-γ刺激24h后,HK2细胞IDO蛋白表达增强,高效液相色谱检测显示刺激组培养基上清中犬尿氨酸特异性代谢率升高,表明表达的IDO具有生物活性,并且其活性呈IFN-γ剂量依赖性地升高。结论IFN-γ刺激培养的HK2细胞IDO表达升高并具有活性,推测病理状态下人肾小管上皮细胞诱导表达的IDO有可能参与了肾小管-间质的病理损害过程。

Objective To investigate the induced expression of indoleamine 2,3-dioxygenase in epithelial cells of cultured human proximal renal tubule under the stimulation of interferon-γ （IFN-γ） in vitro. Methods The expression of IDOmRNA in epithelial cells of human proximal renal tubule under normal and different dosage or time of IFN-γ stimulation were assayed by semi-quantitative RT-PCR, the expression of IDO protein under normal and 2000U/ml IFN-γ stimulation for 24h were assayed by immunocytochemistry, and the activity of IDO under different dosage of IFN-γ stimulation was assessed by the special kynurenine metabolizable ratio in culture medium detected by high performance liquid chromatography （HPLC）. Results No expression of IDO mRNA or IDO was found in the epithelial cells of human proximal renal tubule under normal culture conditions. IDO mRNA expression increased significantly after IFN-γ stimulation in a dose dependent manner compared with that of control group （P〈0. 05）, meanwhile its expression increased significantly at 12h after 2000U/ml IFN-γ stimulation, and peaked 48h later. The increase in IDO protein was confirmed by immunohistochemical staining 24h after 2000U/ml IFN-γ stimulation. The activity of IDO increased significantly in a dose dependent manner by estimated special kynurenine metabolizable ratio in culture medium. Conclusion The expressions of IDO mRNA and IDO protein in epithelial cells of human proximal renal tubule are up-regulated markedly following the stimulation of IFN-γ in a dose and time dependent manner. IDO may play an important role in the process of tubulo-interstitial lesion.