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-东南大学学报医学版

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

作者：刘婷¹ 党杨杰² 刘利敏¹ 张磊¹ 付爱萍¹ 张玉明¹ 吴笛³ 杜锐⁴ 孙世仁¹

单位：1. 第四军医大学西京医院肾内科, 国家肿瘤重点实验室陕西西安 710032;
2. 军事口腔医学国家重点实验室, 口腔疾病国家临床医学研究中心, 陕西省口腔生物工程技术研究中心, 第四军医大学口腔医学院麻醉科陕西西安 710032;
3. 第四军医大学学员一旅陕西西安 710032

关键词：缺氧 G2-M阻滞肾小管上皮细胞肾间质纤维化

分类号：R692.2

出版年·卷·期（页码）：2017·36·第二期（240-245）

摘要：

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 effect of G2/M phase arrest in renal tubular epithelial cells on hypoxia-induced renal interstitial fibrosis.Methods: In vitro, HK2 cells were subjected to hypoxia(1%O₂) or normoxia(21%O₂) for 24 and 48 hrs. We examined the cell cycle distribution of HK2 cells by flow cytometry, the expression of COL4A1 and α-SMA was detected by Western blot 48 hrs after hypoxia;We observed the pathological changes and renal tissue fibrosis degree by HE and Masson staining. The expression of α-SMA was detected by immunohistochemical staining in UUO model. Then we tested the protein level of HIF-1α, cyclinB1and cyclinD1 by Western blot in UUO model. Results: Compared with normoxia group, hypoxia group showed a significant increase in the percentage of G2/M stage in HK2 cells(P<0.05). Hypoxia induced a significant increase of α-SMA and COL4A1 in protein level(P<0.05); Compared with the sham group, HE and Masson staining revealed the increased degree of renal tissue fibrosis and increased expression of α-SMA that was mainly expressed in interstitial; HIF-1α and G2/M phase marker cyclinB1/cyclinD1 was increased in UUO model by Western blot. Conclusion: Chronic hypoxia induced renal interstitial fibrosis is associated with G2/M arrest in renal tubular epithelial cells.

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