PS1基因突变转染细胞中α-分泌酶活性的实验研究

Experimental study on activity ofα-secretase in PS1 mutational transfected cells

目的通过观察两种细胞系3个不同位点早老素基因1(PS1)突变转染细胞中α-分泌酶活性的变化,探讨PS1基因突变对α-分泌酶活性的影响,及两者在家族性阿尔茨海默病病理机制中可能存在的联系。方法利用荧光酶标反应法和Western blot免疫印迹方法分别检测突变型PS1M146L和野生型APP_(751)双转染稳定表达CHO细胞系,及突变型PS1V97L、PS1A136G单转染稳定表达SH-SY5Y细胞系,3种类型的PS1突变细胞α-分泌酶活性及其作用产物分泌型α淀粉样蛋白(sAPPα)表达。结果PS1M146L/APP_(751),双转染CHO细胞系与野生型PS1/APP_(751)双转染CHO细胞系相比α-分泌酶活性有降低,统计学检验差异显著(P＜<0．05)。PS1V97L和PS1A136G突变单转染稳定表达SH-SY5Y细胞系较野生型PS1组均有降低,其中PS1V97L与野生型PS1组α-分泌酶活性比较有显著性差异(P＜0．05),PS1A136G组较野生型PS1组有降低,但无统计学意义。Western blot方法检测α-分泌酶活性作用产物可溶性α淀粉样蛋白结果显示,未转染CHO、SH- SY5Y细胞系与其对应的转染野生型PS1基因的细胞系sAPPα蛋白表达量基本相同,突变型PS1V97L和PS1A136G单转染SH-SY5Y细胞系较野生型PS1组蛋白表达量少,突变型PS1M146L/ APP双转染CHO细胞系中sAPPα蛋白表达量也少于野生型PS1 M146L/APP双转染CHO细胞系。结论PS1基因突变促使α-分泌酶活性降低可能是PS1基因突变导致家族性阿尔茨海默病病理机制之一,PS1致病性与α-分泌酶活性的降低程度可能存在正相关,PS1M146L/APP双转染CHO细胞系与PS1V97L单转染细胞系均可适用于作为探讨家族性阿尔茨海默病中PS1突变与α-分泌酶功能关系研究的细胞模型。

Objective To investigate the influence of presenilin genel （ PSI ） mutation on the activity of α-secretase and the relationship between PSI mutation and the pathological pathogenesis of familial Alzheimer disease （AD） through the observation on the changes of α-secretase activity in PSI mutational transfected cells of 3 sites of 2 cell lines. Methods Fluorometric reaction and Western blot test were used respectively to examine the double transfected stable expressions of CHO cell lines in mutant PS1 M146L and wild-type amyloid precursor protein 751 （APP751 ） , the single transfected stable expression of SH-SY5Y cell line in mutant PS1V97L and PS1A136G, and the α-secretase activities and expression of sAPPPα in 3 types of PS1 mutation cells. Results The α-secretase activity was decreased with a significant difference statistically （P 〈 0.05 ） after the comparison between the double transfected stable expressions of CHO cell lines in PS1M146L/APP751 and wild -type PS1/APP751. The single transfected stable expression of SH-SY5Y cell line in mutant PS1V97L and PS1A136G were decreased compared with that of wild-type PS1 group. There was a significant difference in the α-secretase activity between PS1V97L and wild - type PS1 groups （ P 〈 0.05 ）, and the α-secretase activity was decreased in PS1A136G group than that of wild-type PS1 group, but there was no statistical significance. The result of Western blot test in the examination of sAPPer showed that the sAPPer expressions of non-transfected CHO and SH-SYSY cell lines were as the same as that of transfected wild-type PS1. The protein expressions of mutant PS1V97L and single transfected SH-SY5Y cell line in PS1A136G were decreased compared with that of wild-type PS1 group. The sAPPer expression of double transfected stable expressions of CHO cell lines in mutant PS1M146L/APP was decreased compared with that in wild-type PS1M146L/APP. Conclusion It may be one of the pathological mechanisms of familial AD that the α - secretase activity is decreased induced by PS1 mutation. The pathogenic effect of PS1 mutation may be correlated to the reducing degree of the α-secretase activity. The double transfected CHO cell lines in PS1M146L/APP and single transfected cell line in PS1V97L can established as the cell model for the study on the relationship between PS1 mutation of familial AD and the α-secretase function.