Neuritin与HSP60表达水平与肝细胞凋亡调节的相关性

Expressions of Neuritin and HSP60 in hepatocytes and the regulation effect on apoptosis of hepatocytes

目的探讨Neuritin与HSP60在肝细胞中的表达水平及对细胞凋亡调节的分子机制。方法将含有肝细胞的培养皿,随机分为4大组,分别为Neuritin低表达组、Neuritin高表达组、HSP60低表达组、HSP60高表达组。每个大组分为5个小组,对照组(n=4)和实验组(n=16),对照组不做任何处理,Neuritin低表达组分别给予0.1、0.3、0.5、0.7μg/ml的Neuritin抗体;Neuritin高表达组分别给予0.5、1.0、1.5、2.0μg/ml的Neuritin蛋白;HSP60低表达组分别给予0.6、0.8、1.0、1.2μg/ml的HSP60抗体;HSP60高表达组分别给予10×10^-3、50×10^-3、100×10^-3、200×10^-3μg/ml的HSP60蛋白,BRI肝细胞培养48 h后使用Western印迹测定Neuritin、HSP60及Caspase-3在肝细胞中的表达水平。结果与对照组相比,Neuritin低表达、高表达各剂量组Neuritin相对表达程度渐渐减低、增高,差异均有统计学意义(P<0.05);HSP60相对表达水平差异无统计学意义(P>0.05);Caspase-3相对表达水平逐渐增高、减低,差异有统计学意义(P<0.05)。Neuritin表达量与Caspase-3表达量呈明显负相关性(r=-0.993,P<0.05)。与对照组相比,HSP60低表达、高表达各剂量组HSP60相对表达程度渐渐减低和增高,差异有统计学意义(P<0.05);Neuritin表达水平基本一致,差异无统计学意义(P>0.05);Caspase-3相对表达量渐渐增高和减低,差异有统计学意义(P<0.05)。HSP60表达量与Caspase-3表达量呈明显负相关性(r=-0.996,P<0.05)。结论Neuritin蛋白和HSP60蛋白的表达变化与凋亡蛋白Caspase-3呈现相反的变化趋势,提示Neuritin和HSP60可能通过某种协同作用共同参与肝细胞的抗凋亡调控机制。

Objective To investigate the expressions of Neuritin and HSP60 in hepatocytes and their molecular mechanism on apoptosis regulation.Methods Petri dishes containing hepatocytes were randomly divided into Neuritin low,high expression,HSP60 low and high expression groups.Each group was divided into 5 subgroups,the control(n=1)and experimental subgroups(n=4).Control subgroup had not any treatment.Experimental subgroup of Neuritin low expression group was given 0.1,0.3,0.5 and 0.7μg/ml Neuritin antibody respectively;experimental subgroup of Neuritin high expression group was given 0.5,1.0,1.5,2.0μg/ml Neuritin protein respectively;experimental subgroup of HSP60 low expression group was given 0.6,0.8,1.0,1.2μg/ml of HSP60 antibody respectively;experimental subgroup of HSP60 high expression group was given 10×10^-3,50×10^-3,100×10^-3 and 200×10^-3μg/ml HSP60 protein respectively.After 48 h,the expressions of Neuritin,HSP60 and Caspase-3 in hepatocytes were determined by Western blot.The location and colocalization of Neuritin and HSP60 in hepatocytes were detected by immunofluorescence.Results(1)Compared with control subgroup,expression of Neuritin in experimental subgroup of Neuritin low or high expression group was decreased or increased gradually,the difference was statistically significant(P<0.05);compared with control subgroup,expression of HSP60 in experimental subgroup of HSP60 low or high expression group was basically the same,there was no significant difference(P>0.05);expression of Caspase-3 gradually increased and decreased,the difference was statistically significant(P<0.05).There was a significant negative correlation between the trend of Neuritin relative expression and the change of Caspase-3 relative expression(r=-0.993,P<0.05).(2)The low expression and over-expression of HSP60 in hepatocytes showed that compared with the control subgroup,the relative expression of the experimental subgroup gradually decreased and increased,the difference was statistically significant(P<0.05);compared with control subgroup,the Neuritin relative expressions were basically the same,and the difference was not statistically significant(P>0.05).The relative expression of Caspase-3 was gradually increased and decreased,and the difference was statistically significant(P<0.05).There was also a significant negative correlation between the change in the relative expression of HSP60 and the change in the relative expression of Caspase-3(r=-0.996,P<0.05).(3)Neuritin and HSP60 were located in the cell membrane after merge in cellular immunofluorescence.Conclusions The expression changes of Neuritin and HSP60 proteins are opposite to those of the apoptotic protein Caspase-3,which suggest that Neuritin and HSP60 might participate in the anti-apoptotic regulation mechanism of hepatocytes through some synergistic effects.