论文部分内容阅读
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 investigate the expression of peroxisome proliferator activated receptor B (PPARB) during the apoptosis of HaCaT cells induced by tumor necrosis factor α (TNF-α), and to explore the protective mechanism of epidermal growth factor (EGF) on HaCaT cells. Methods The cultured HaCaT cells were divided into normal control group (without any treatment), low dose TNF-α group (10 ng / ml TNF-α treatment), high dose TNF- , EGF + TNF-α low dose group and EGF + TNF-α high dose group. The latter two groups were treated with 20 ng / ml EGF for 4 h and then treated with 10, 20 ng / ml TNF-α. The apoptosis of cells was detected by flow cytometry. The activity of caspase-3 was detected by caspase-3 fluorescence detection kit. Cell viability was assayed by thiazolyl blue colorimetry. HaCaT cells were treated with 5, 10, 20 and 40 ng / ml of EGF, and the expression of PPARβ mRNA and its protein was detected by reverse transcription-PCR and Western blot. Results Compared with the low dose group of TNF-α [(32 ± 6)%] and the high dose group of TNF-α (57 ± 6%), the high dose group of EGF + TNF- The apoptotic rate was significantly decreased (P <0.01), the activity of caspase-3 was decreased and the survival rate was increased (P <0.01). At 20 ng / ml EGF, the expression of PPARβ mRNA and its protein was the highest. Conclusion EGF inhibits the apoptosis of HaCaT cells induced by TNF-α and enhances the expression of PPARβ.