海生素靶向ULK1蛋白诱导HL-60细胞分化机制的研究

Mechanism Inducing the HL-60 Cell Differentiation by Haishengsu Tegillarca Granosa Extract via Targeting ULK1 Protein

目的:探讨泥蚶提取物海生素通过靶向调控ULK1蛋白诱导HL-60细胞增殖和分化潜能,以及自噬机制。方法:实验分为海生素10、100和1000 mg/L处理组、全反式维甲酸(ATRA)5μmol/L处理组和对照组,共5组,分别培养HL-60细胞12、24、48和72 h,采用MTT法检测各组细胞增殖率,采用流式细胞术检测细胞分化成熟标志分子CD11b和CD15的表达,采用Western blot法检测自噬分子ULK1、LC-3蛋白的表达。结果:海生素100和1000 mg/L处理HL-60细胞48和72 h,HL-60细胞增殖率均显著低于ATRA组(P<0.05)和海生素10 mg/L组和对照组;处理72h后,HL-60细胞CD11b、CD15表达百分比和ULK1、LC-3蛋白相对表达量均显著高于ATRA组和海生素10 mg/L组和对照组(P<0.05)。海生素10、100和1000 mg/L组同时给予ULK1蛋白抑制剂培养HL-60细胞72 h,各组细胞增长率均明显增加,CD11b、CD15表达百分比和ULK1、LC-3蛋白水平均明显降低(P<0.05)。结论:泥蚶提取物海生素通过靶向调控ULK1蛋白诱导HL-60细胞增殖和分化潜能,且具有浓度和时间依赖性;通过介导细胞自噬活性促进细胞分化成熟。

Objective:To investigate the proliferation,differentiation potential and autophagic mechanism HL-60 cells via ULK1 protein targeted by Haishengsu extracted from Tegillarca granosa.Methods:The HL-60 cells w ere divided into five groups:Haishengsu of 10,100,1000 mg/L-treated group,all-trans retinoic acid(ATRA)5μmol/L-treated group and control group.The cell proliferation rates w ere detected by M TT after culture 12,24,48 h and 72 h.The expression of differentiation and maturation markers CD11 b and CD15 w ere detected by flow cytometry,and the expression of autophagy molecules ULK1 and LC-3 proteins w ere measured by Western blot.Results:After cultured for 48 and 72 h the cell proliferation rates of HL-60 in Haishengsu 100 and 1000 mg/L groups w ere significantly low er than those of ATRA 5μmol/L group and Haishengsu 10 mg/L group and control group(P<0.05).After 72 h,the levels of CD11 b,CD15,ULK1 and LC-3 expression of HL-60 cell in Haishengsu 100 and 1000 mg/L groups w ere higher than those in ATRA 5μmol/L group,Haishengsu 10 mg/L group and control group(P<0.05).While the HL-60 cells in Haishengsu 10,100 and 1000 mg/L group w ere treated w ith ULK1 protein inhibitor and after cultured for 72 h,the proliferation rate of HL-60 cell increased,levels of CD11 b and CD15 expression decreased at the same time,and the levels of ULK1 and LC-3 proteins in HL-60 cells also decreased(P<0.05).Conclusion:Haishengsu,w hich extracted from Tegillarca granosa,can induce HL-60 cell proliferation and differentiation by targeting ULK1 protein in a concentration and time dependent manner,its effective mochanism may be mediating autophagic activity,promoting cell differentiation and maturation.