NF—κB通路在单核细胞诱导肾小管上皮细胞转分化中的作用

Role of NF-κB in monocytes-induced HK-2 cells transdifferentiation

目的观察单核细胞（U937细胞）对人近端肾小管上皮细胞（HK-2细胞）转分化的影响及其分子机制。方法将HK-2细胞与人单核细胞系U937细胞共培养；倒置相差显微镜观察HK-2细胞形态；Western印迹、实时荧光定量PCR法检测仅平滑肌肌动蛋白（α-SMA）、纤连蛋白（FN）、E钙黏蛋白（E—cadherin）和胞间黏附分子1（ICAM-1）表达；BCECF—AM荧光染色法测定单核细胞黏附；流式细胞仪法检测HK-2细胞表面分子ICAM-1表达；基因芯片筛选HK-2细胞基因变化；利用信号阻断剂阻断基因芯片筛选出的信号通路，进一步验证单核细胞诱导肾小管上皮细胞转分化的分子机制。结果单核细胞可直接诱导HK-2细胞发生形态变化，减少HK-2细胞E-cadherin表达（均P〈0．05），并上调α-SMA、FN表达（均P〈0．05）。应用CD18抗体阻断CD18-ICAM-1可抑制单核细胞黏附及其诱导的HK-2细胞形态变化。基因芯片结果显示，NF—κB信号通路分子CC亚族趋化因子配体20（CCL20）、白细胞介素（IL）2、IL-8、脂磷壁酸（LTA）及血小板内皮细胞黏附分子1（PECAMl）表达明显增加（均P〈0．05）。NF—KB信号阻断剂吡咯烷二硫氨基甲酸（PDTC）能显著抑制HK-2细胞形态变化及表面ICAM-1表达，抑制单核细胞诱导的肾小管上皮细胞转分化。结论单核细胞通过CD18分子与HK-2细胞表面ICAM-1结合，从而启动NF—κB信号通路介导的特定基因转录，最终导致肾小管上皮细胞发生转分化。

Objective To investigate the effects of monocytes on phenotypic changes of human proximal tubular HK-2 cells and the mechanism. Methods Monocytes were co-cuhured with HK-2 cells. Morphological changes of HK-2 cells were detected by inverted phase contrast microscope. Expressions of E-cadherin, α-SMA and fibronectin were assessed by RT-PCR, Western blotting and immunocytochemical staining. Flow cytometry techniques was applied to evaluate intercellular cell adhesion molecule-1 （ICAM-1） expression on HK-2 cells. The intracellular signal was investigated by gene microarray. Results The typical epithelial cell morphology of HK-2 cells disappeared after co-culture with monocytes, accompanied by decreased E-cadherin expression and increased α-SMA and fibronectin expression （all P〈0.05）. The expression of ICAM-1 on HK-2 cells was increased by monocytes stimulation. Interestingly, administration of CD18 antibody directly inhibited the phenotypic change of HK-2 cells. Furthermore, NF-κB signaling might be critical in mediating this process, and blockade of this signaling pathway could inhibit ICAM-1 expression and epithelial mesenchymal transition （EMT） formation. Conclusion Monocytes can directly induce EMT of HK-2 cells via up-regulating ICAM-1 through NF-κB signaling pathway.