肾小管上皮细胞微环境对后肾间充质细胞增殖、迁移与分化的影响

doi:10.3877/cma.j.issn.2095-3216.2025.04.002

中华肾病研究电子杂志 ›› 2025, Vol. 14 ›› Issue (04) : 188 -195. doi: 10.3877/cma.j.issn.2095-3216.2025.04.002

论著

肾小管上皮细胞微环境对后肾间充质细胞增殖、迁移与分化的影响

纪鹏程¹^,², 国文凯²^,³, 毕靖茹²^,³, 韩冰⁴, 傅博², 谢院生¹^,²^,³^,^†(

)

¹100853　北京，解放军医学院
²100853　北京，解放军总医院第一医学中心肾脏病医学部、肾脏疾病全国重点实验室、国家慢性肾病临床医学研究中心、重症肾脏疾病器械与中西医药物研发北京市重点实验室、数智中医泛血管疾病防治北京市重点实验室、国家中医药管理局高水平中医药重点学科(zyyzdxk-2023310)
³300071　天津，南开大学医学院
⁴300131　天津市人民医院

收稿日期:2025-04-25 出版日期:2025-08-28
通信作者: 谢院生
基金资助:
国家自然科学基金面上项目(82174115，81774027)

Effects of renal tubular epithelial cell microenvironments on proliferation, migration, and differentiation of metanephric mesenchymal cells

Pengcheng Ji¹^,², Wenkai Guo²^,³, Jingru Bi²^,³, Bing Han⁴, Bo Fu², Yuansheng Xie¹^,²^,³^,^†()

¹Chinese PLA Medical School, Beijing 100853
²Department of Nephrology, First Medical Center of Chinese PLA General Hospital, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Medical Devices and Integrated Traditional Chinese and Western Drug Development for Severe Kidney Diseases, Beijing Key Laboratory of Digital Intelligent TCM for the Prevention and Treatment of Pan-vascular Diseases, Key Disciplines of National Administration of Traditional Chinese Medicine (zyyzdxk-2023310), Beijing 100853
³College of Medicine, Nankai University, Tianjin 300071
⁴Tianjin People′s Hospital Tianjin 300131; China

Received:2025-04-25 Published:2025-08-28
Corresponding author: Yuansheng Xie

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引用本文：

纪鹏程, 国文凯, 毕靖茹, 韩冰, 傅博, 谢院生. 肾小管上皮细胞微环境对后肾间充质细胞增殖、迁移与分化的影响[J/OL]. 中华肾病研究电子杂志, 2025, 14(04): 188-195.

Pengcheng Ji, Wenkai Guo, Jingru Bi, Bing Han, Bo Fu, Yuansheng Xie. Effects of renal tubular epithelial cell microenvironments on proliferation, migration, and differentiation of metanephric mesenchymal cells[J/OL]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2025, 14(04): 188-195.

目的

探讨肾小管上皮细胞系TCMK-1细胞正常微环境和损伤微环境对后肾间充质细胞增殖、迁移与分化能力的影响，并探讨相关的信号通路。

方法

采用肾小管上皮细胞系TCMK-1细胞培养，分别制备正常微环境上清液和损伤微环境上清液(5 μg/ml顺铂处理)。采用50 ng/ml碱性成纤维细胞生长因子和10 ng/ml转化生长因子α制备上皮诱导培养基。在增殖实验中，将后肾间充质细胞分为对照组(常规培养基)、正常微环境组和损伤微环境组，应用细胞计数试剂盒-8法检测细胞增殖。在迁移实验中，将后肾间充质细胞分为对照组、顺铂组(常规培养基＋5 μg/ml顺铂)、正常微环境组和损伤微环境组，采用Transwell小室法检测细胞迁移能力。在分化实验中，后肾间充质细胞分为对照组、上皮诱导组(上皮诱导培养基)、上皮诱导＋正常微环境组和上皮诱导＋损伤微环境组，培养10 d后采用Western印迹法检测E-钙粘蛋白、间充质标志物α-平滑肌肌动蛋白和肾小管上皮特异性标志物水通道蛋白2表达。在通路验证实验中，利用Western印迹法检测经典无翅/整合基因(wingless/integrated, Wnt)通路蛋白Wnt3A、β-连环蛋白和T细胞因子4的表达情况，加入经典Wnt通路小分子抑制剂MSAB检测其对上皮分化的影响。

结果

与对照组相比，正常微环境组与损伤微环境组的后肾间充质细胞增殖活性均被抑制(P均<0.01)，但两组的迁移能力增加(P均<0.05)。相较对照组和上皮诱导组，正常微环境组和损伤微环境组的后肾间充质细胞E-钙粘蛋白表达均增加，而α-平滑肌肌动蛋白表达减少，损伤微环境组的水通道蛋白2表达也有所增加。损伤微环境组的经典Wnt通路相关蛋白Wnt3A、β-连环蛋白和T细胞因子4表达较多(P均<0.05)；加入MSAB能够抑制损伤微环境促进后肾间充质细胞向上皮分化的作用(P<0.05)。

结论

肾小管上皮细胞的正常微环境与损伤微环境均轻度抑制后肾间充质细胞增殖，但可促进其迁移；肾小管上皮细胞的损伤微环境能激活经典Wnt通路，并促进后肾间充质细胞向肾小管上皮细胞分化。

关键词: 微环境, 后肾间充质细胞, 经典Wnt通路, 上皮分化

Objective

To investigate the effects of normal and injured microenvironments derived from renal tubular epithelial cell line TCMK-1 cells on the proliferation, migration, and differentiation of metanephric mesenchymal cells (MMCs), and to identify the signaling pathways involved.

Methods

The normal microenvironment supernatant was prepared using the TCMK-1 cells, while the injured microenvironment supernatant was generated by treating TCMK-1 cells with 5 μg/mL cisplatin. The epithelial induction medium was prepared by using 50 ng/ml basic fibroblast growth factor and 10 ng/ml transforming growth factor α. In the proliferation experiment, the MMCs were divided into control group (regular culture medium), normal microenvironment group, and injured microenvironment group. Cell proliferation was detected by the cell counting kit-8 method. In the migration experiment, the MMCs were divided into control group, cisplatin group (regular culture medium+ 5 μg/ml cisplatin), normal microenvironment group, and injured microenvironment group. The cell migration ability was assessed by means of the transwell chamber assay. In the differentiation experiment, the MMCs were divided into control group, epithelial induction group (epithelial induction medium), epithelial induction plus normal microenvironment group, and epithelial induction plus injured microenvironment group. After 10 days of culture, Western blotting was used to detect the protein expression of E-cadherin, mesenchymal marker α-smooth muscle actin, and renal tubular epithelial-specific marker aquaporin 2. In the pathway verification experiment, Western blotting was used to detect the expression levels of canonical wingless/integrated (Wnt) pathway proteins, including Wnt3A, β-catenin, and T-cell factor 4. The small molecule inhibitor MSAB of the canonical Wnt pathway was added to assess its effect on epithelial differentiation.

Results

Compared with the control group, the proliferative activity of MMCs was inhibited in both the normal microenvironment group and the injured microenvironment group (both P<0.05), while the migration ability of the two groups increased (both P<0.01). Compared with the control group and the epithelial induction group, the expression of E-cadherin in the MMCs of both the normal microenvironment group and the injured microenvironment group increased, while the expression of α-smooth muscle actin decreased. Additionally, the expression of aquaporin 2 in the injured microenvironment group also increased. The expression of canonical Wnt pathway-related proteins as Wnt3A, β-catenin, and T-cell factor 4 was higher in the injured microenvironment group (all P<0.05), and the addition of MSAB could inhibit the effect of the injured microenvironment in promoting the epithelial differentiation of MMCs (P<0.05).

Conclusion

Both the normal and injured microenvironments of the renal tubular epithelial cells slightly inhibited the proliferation of the MMCs, but promoted their migration. The injured microenvironment of the renal tubular epithelial cells activated the canonical Wnt pathway and promoted the differentiation of MMCs into renal tubular epithelial cells.

Key words: Microenvironment, Metanephric mesenchymal cells, Canonical Wnt pathway, Epithelial differentiation

图1 细胞计数试剂盒-8测定正常微环境与损伤微环境后肾间充质细胞增殖活性注：与CON组相比，^aP<0.01；与TCMK1-con组相比，^bP<0.01

图2 后肾间充质细胞Transwell小室迁移细胞结晶紫染色(×40倍)及比较注：采用10%氯化十六烷基吡啶溶液洗涤Transwell小室迁移细胞的结晶紫染液490 nm波长下检测吸光度；与CON组相比，^aP<0.05；与CP组相比，^bP<0.05；与TCMK1-con组相比，^cP<0.05

图3 Western印迹法检测后肾间充质各组上皮标志物与间充质标志物相关蛋白的表达水平与半定量分析注：E-cadherin：E-钙粘蛋白；α-SMA：α-平滑肌肌动蛋白；AQP2：水通道蛋白2；GAPDH：甘油醛-3-磷酸脱氢酶；与CON组相比，^aP<0.05；与EIM组相比，^bP<0.05；与EIM＋TCMK1-con组相比，^cP<0.05

图4 Western印迹法检测各组后肾间充质细胞的经典Wnt通路相关蛋白表达水平与半定量分析注：Wnt3A：wingless/integrated 3A，无翅/整合基因家族蛋白3A；β-catenin：β-连环蛋白；TCF4：T细胞因子4；GAPDH：甘油醛-3-磷酸脱氢酶；与CON组相比，^aP<0.05；与EIM组相比，^bP<0.05；与EIM＋TCMK1-con组相比，^cP<0.05

图5 Western印迹法检测后各组肾间充质细胞的上皮标志物、间充质标志物与Wnt经典通路相关蛋白表达水平与半定量分析注：E-cadherin：E-钙粘蛋白；α-SMA：α-平滑肌肌动蛋白；AQP2：水通道蛋白2；Wnt3A：wingless/integrated 3A，无翅/整合基因家族蛋白3A；β-catenin：β-连环蛋白；TCF4：T细胞因子4；GAPDH：甘油醛-3-磷酸脱氢酶；与EIM组相比，^aP<0.05；与EIM＋TCMK1-cis组相比，^bP<0.05

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