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目的:探讨肾小管上皮细胞G2-M期阻滞在缺氧诱导的肾间质纤维化中的作用。方法:体外实验,人肾小管上皮细胞(HK2)分别置于常氧21%和缺氧1%的孵箱里,培养24,48 hrs。采用流式细胞术检测缺氧48 hrs后上皮细胞的周期分布;采用Western blot法检测Collagen4A1(COL4A1)及α-平滑肌肌动蛋白(α-SMA)的蛋白表达水平。单侧输尿管梗阻(UUO)14 d小鼠模型,HE及Masson染色观察肾组织病理改变及纤维化程度,免疫组化染色观察α-SMA的表达及部位;Western blot检测低氧标记分子HIF-1α、细胞周期蛋白cyclinB1和cyclinD1的蛋白水平。结果:与常氧组比较,缺氧组肾小管上皮细胞G2/M期比例增高(P<0.05),同时α-SMA和COL4A1的蛋白表达水平增高(P<0.05);与假手术组比较,HE及Masson染色显示模型组肾组织纤维化程度增高,α-SMA的表达增高且主要分布在间质中;Western blot结果显示:HIF-1α及G2/M期标记物cyclinB1/cyclinD1比值增加。结论:肾小管上皮细胞G2-M期阻滞参与缺氧诱导的肾间质纤维化。
Objective: To investigate the role of G2-M phase arrest in renal tubular epithelial cells induced by hypoxia in renal interstitial fibrosis. Methods: In vitro experiments, human renal tubular epithelial cells (HK2) were placed in normoxia 21% and hypoxia 1% incubator, cultured for 24,48 hrs. The cell cycle distribution of epithelial cells was detected by flow cytometry (FCM) 48 hrs after hypoxia. The protein expression of Collagen4A1 (COL4A1) and α-smooth muscle actin (α-SMA) was detected by Western blot. Unilateral ureteral obstruction (UUO) 14 d mouse model, HE and Masson staining of renal tissue pathological changes and fibrosis, immunohistochemical staining α-SMA expression and location; Western blot detection of hypoxia marker HIF-1α , CyclinB1 and cyclinD1 protein levels. Results: Compared with the normoxia group, the proportion of G2 / M phase in hypoxia group was increased (P <0.05), and the protein expression of α-SMA and COL4A1 increased (P <0.05). Compared with the sham-operated group, HE and Masson staining showed that the degree of renal fibrosis was increased and the expression of α-SMA was increased in the model group and mainly distributed in the interstitium. Western blot results showed that the ratio of cyclinB1 / cyclinD1 in HIF-1α and G2 / M increased. Conclusion: G2-M phase arrest of renal tubular epithelial cells is involved in hypoxia-induced renal interstitial fibrosis.