肿瘤坏死因子-α在慢性移植肾间质纤维化形成中的作用机制研究

The role and mechanism of tumor necrosis factor-α in the formation of chronic transplant renal interstitial fibrosis

目的观察肿瘤坏死因子-α（TNF-α）在慢性移植肾间质纤维化形成中的作用，及smad泛素化调节因子2（Smurf2）在此过程中的表达变化及意义。方法通过对26例正常肾组织样本和26例慢性移植肾功能丧失（CAD）患者移植肾组织样本进行HE染色、马松三色（Masson）染色及免疫组织化学染色，检测两组肾组织中B钙粘蛋白（E-cad）和α-平滑肌肌动蛋白（α-SMA）及TNF-α，Smurf2等的表达和分布，初步探讨TNF-α在CAD形成中的作用。并进一步以人肾小管上皮细胞（HK2）为体外研究对象，以不同浓度（0、10、20、50、100ng／ml）TNF-α分别刺激HK2细胞48h后，应用蛋白质印迹法检测每组细胞中E-cad、α-SMA及Smurf2等指标的表达变化。结果HE及Masson染色结果显示，与正常组相比，CAD患者移植肾组织中明显出现间质纤维化表现。免疫组织化学结果显示，与正常组相比，CAD患者移植肾组织中E-cad阳性表达率减少（P〈0．01），而α-SMA、TNF-α、Smurf2阳性表达率增加（P〈0．01）。体外实验也显示，随着TNF-α浓度增加，HK2细胞中E-cad表达下调（P〈0．05），而α-SMA和Smurf2表达上调（P〈0．05）。结论TNF-α可能通过上调Smurf2表达诱导肾小管上皮细胞表型向间质细胞转分化，进而促进移植肾间质纤维化的形成。

Objective To explore the role and mechanism of tumor necrosis factor-α （TNF-α） in the formation of chronic transplant renal interstitial fibrosis and to study Smurf2 expression change and role played in this process. Methods We collected 26 cases of normal renal tissues and 26 cases of renal allograft specimens from chronic allograft dysfunction patients to observe the degree of renal interstitial fibrosis by Hematoxylin and eosin （ HE ） staining and Masson staining. Immunohistochemical staining was applied to detect the expression and distribution of E-cadherin, α-smooth muscle actin （a-SMA） and TNF-α, Smad ubiquitination regulatory factor 2 （Smurf2）. Furthermore, The HK2 ceils were divided into five groups depending on different concentrations of TNF-α （0,10,20, 50, and 100 ng/mL）. After 48 h, we collected the cells to analyze the expression changes of Smurf2, E-cad, α-SMA by Western blotting. Results As compared with normal group, the degree of renal interstitial fibrosis in CAD group was aggravated according to the results of HE and Masson staining （P〈0. 01）. Immunohistochemical staining results showed that the positive expression of E-cadherin was reduced, and that of α-SMA, TNF-α and Smurf2 increased greatly in CAD group as compared with normal group （P〈 0. 01）. The results of Western blotting also revealed that after treatment with different concentrations of TNF-α for 48 h, the expression of E-cadherin was down- regulated,and that of α-SMA and smurf2 was up-regulated in HK2 cells （P〈0. 05）. Conelnsion TNF-α may promote the formation of allograft renal interstitial fibrosis via transdifferentiation of human tubular epithelial cells to mesenchymal cells by up-regulating the expression of Smurf2.