Ac-SDKP对TGF-β1诱导的大鼠肾小管上皮细胞转分化的影响

The role of Ac-SDKP in TGF-β1-induced transdifferentiation of rat renal tubular epithelial cells

目的:观察氮乙酰丝氨酰天冬氨酰赖氨酰脯氨酸(Acetyl-N-ser-asp-lys-pro,Ac-SDKP)对肾小管上皮细胞表达胸腺肽β4(Thymosinβ4,Tβ4)及上皮间充质转分化(Epithelial-mesenehymal transition,EMT)的影响,探讨Ac-SDKP与肾小管上皮细胞EMT及Ac-SDKP之间的关系。方法:用转化生长因子β1(Transforming growth factor-β1,TGF-β1)诱导大鼠肾小管上皮细胞发生EMT后,再予外源性不同浓度的Ac-SDKP干预,通过细胞免疫组化染色的方法检测肾小管上皮细胞Tβ4和α-平滑肌肌动蛋白(α-Smooth muscle actin,α-SMA)表达。结果:1.TGF-β1(5 ng/mL)刺激48 h后肾小管上皮细胞形态明显拉长,呈梭长形,似成纤维细胞;予Ac-SDKP(10 nmol/L)干预后,细胞形态似有恢复类圆形,但未恢复至正常的细胞形态;2.在空白组,Tβ4在肾小管上皮细胞胞浆及胞核内表达,平均光密度(OD)值为(0.203±0.009);α-SMA仅在肾小管上皮细胞胞浆内微量表达,OD值为(0.184±0.007);TGF-β1刺激组Tβ4和α-SMA表达显著升高,OD值分别为(0.346±0.016)和(0.345±0.008),与空白组比,差异均有统计学意义(P<0.05);3.TGF-β1+卡托普利组Tβ4及α-SMA表达减少,OD值分别为(0.337±0.021)和(0.334±0.023),与TGF-β1刺激组比,差异均无统计学意义(P>0.05);4.Ac-SDKP为0.1、1、10nmol/L时,Tβ4和α-SMA表达均下降,且与TGF-β1刺激组比较差异均有统计学意义(P<0.05),其中10 nmol/L下降最显著,OD值分别为(0.237±0.008)和(0.225±0.018);5.肾小管上皮细胞中Tβ4和α-SMA的表达量之间存在直线正相关关系(r=0.995,P<0.01)。结论:TGF-β1诱导肾小管上皮细胞EMT后,Tβ4在肾小管上皮细胞内表达明显上调,予不同浓度的Ac-SDKP刺激后,Tβ4表达下调,在10 nmol/L Ac-SDKP时下降最为显著。

Objective:To investigate the effects of Acetyl-N-ser-asp-lys-pro(Ac-SDKP)on thymosinβ4(Tβ4)expression and epithelial-mesenehymal transition(EMT)in renal tubular epithelial cells,and the relationship between Ac-SDKP,renal tubular epithelial cells EMT and Tβ4.Methods:EMT was induced in rat renal tubular epithelial cells by transformed growth factor-β1(TGF-β1),and then exogenously treated with different concentrations of Ac-SDKP.Cell immunohistochemical staining was used to detect the expression of Tβ4 andα-Smooth muscle actin(α-SMA)in renal tubular epithelial cells.Results:1.After TGF-β1(5 ng/mL)stimulation for 48 h,the morphology of renal tubular epithelial cells was obviously elongated,appearing spindle-shaped and resembling fibroblasts.After intervention with Ac-SDKP(10 nM),the cell morphology seemed to resume a round-shape but did not really return to normal cell morphology.2.In the blank group,Tβ4 was expressed in the cytoplasm and nucleus of renal tubular epithelial cells,with a mean optical density(OD)value of(0.203±0.009);α-SMA was expressed in trace amounts in the cytoplasm only,with a mean OD of(0.184±0.007).In cells stimulated by TGF-β1,the Tβ4 andα-SMA expressions were significantly increased,with OD values of(0.346±0.016)and(0.345±0.008),respectively.Compared with the blank group,the differences were statistically significant(P<0.05).3.The expression of Tβ4 andα-SMA was decreased in the TGF-β1+captopril group,with OD values of(0.337±0.021)and(0.334±0.023),respectively.Compared with the TGF-β1 stimulation group,these differences were not statistically significant(P>0.05).4.Ac-SDKP at doses of 0.1 nM,1 nM,and 10 nM caused lowered expression of Tβ4 andα-SMA with statistically significant differences compared with the TGF-β1 stimulation group(P<0.05).The decreases in Tβ4 andα-SMA expression were greatest at an Ac-SDKP dose of 10 nM,yielding OD values of(0.237±0.008)and(0.225±0.018),respectively.5.There was a linear correlation between the expression levels of Tβ4 andα-SMA in renal tubular epithelial cells(r=0.995,P<0.01).Conclusion:Under TGF-β1 induced EMT,the expression of Tβ4 in renal tubular epithelial cells is remarkably up-regulated.Down-regulation of Tβ4 expression appears after treatment with different levels of Ac-SDKP,and is most prominent with an Ac-SDKP dose of 10 nM.