表皮生长因子对肿瘤坏死因子α致HaCaT细胞凋亡的抑制作用

Inhibitory effect of epidermal growth factor on apoptosis in HaCaT keratinocytes induced by TNF-α

目的了解肿瘤坏死因子α(TNF-α)导致HaCaT细胞凋亡过程中过氧化物酶体增殖物激活受体B(PPARB)的表达情况,探讨表皮生长因子(EGF)对HaCaT细胞的保护机制。方法将培养的HaCaT细胞分为正常对照组(不作任何处理)、TNF-α小剂量组(10 ng/ml TNF-α处理)、TNF-α大剂量组(20 ng/ml TNF-α处理)、EGF+TNF-α小剂量组、EGF+TNF-α大剂量组,后2组先用20 ng/ml EGF培养4 h后,再分别予以10、20 ng/ml TNF-α处理。采用流式细胞仪检测细胞凋亡情况,半胱氨酸天冬氨酸蛋白酶3(caspase-3)荧光检测试剂盒测定caspase-3的活性,噻唑蓝比色法检测细胞存活率。以5、10、20、40 ng/ml的EGF处理HaCaT细胞,采用反转录-PCR和蛋白质印迹法检测PPARβmRNA及其蛋白的表达。结果与TNF-α小剂量组[(32±6)%]、TNF-α大剂量组[(57±6)%]比较,EGF+TNF-α小剂量组、EGF+TNF-α大剂量组细胞凋亡率[(20±3)%、(28±4)%]明显下降(P<0.01),caspase-3活性降低、存活率上升(P<0.01)。20 ng/ml EGF处理细胞时,PPARβmRNA及其蛋白表达最强。结论EGF在抑制TNF-α导致的HaCaT细胞凋亡的同时,亦增强细胞中PPARβ的表达。

Objective To explore the effect of epidermal growth factor （EGF） on apoptosis induced by TNF-α and the expression of PPAR[3 in HaCaT keratinocytes. Methods HaCaT keratinocytes were cultured and randomly divided into A （normal control） , B（ with treatment of 10 ng/ml TNF-α for 24 hours） , C （with treatment of 20 ng/ml TNF-α for 24 hours） , D（ with treatment of 10 ng/ml TNF-α after 20 ng/ml EGF treatment for 4 hours） , E（ with treatment of 20 ng/ml TNF-α after 20 ng/ml EGF treatment for 4 hours） groups. The apoptosis of HaCaT keratinocytes was observed by flow cytometry. The proliferative activity of HaCaT keratinocytes was evaluated by MTT method. The activity of caspase-3 was analyzed with caspase colorimetric assay Kit. The changes in the mRNA and protein expression of PPARβ in HaCaT keratinocytes were observed by RT-PCR and western-blotting after treatment with different concentrations （5, 10, 20, 40 ng/ml） of EGF for 4 or 24 hrs. Results Compared with A and B groups[（32±6）%,（57± 6） % ] , the apoptosis of HaCaT keratinocytes in D and E groups were significantly increased [ （ 20 ±3 ） % , （28± 4 ）% , respectively, P 〈 0.01 ] , while the survival rate of HaCaT keratinocytes in D and E groups increased, and the caspase-3 activity were decreased （ P 〈0.01 ）. The expression of PPARβ mRNA and protein in HaCaT keratinocytes reached the peak with the treatment of 20 ng/ml EGF. Conclusion EGF can inhibit the apoptosis of HaCaT keratinocytes induced by TNF-α, and it can also increase the expression of PPARβ.