siRNA干扰跨膜转运蛋白21的表达对KO小鼠胚胎成纤维细胞γ分泌酶活性的影响

目的探讨跨膜转运蛋白21（TMP21）对γ分泌酶活性的影响。 方法将淀粉样前体蛋白基因缺陷型KO小鼠胚胎成纤维细胞MEF（KO）]分为转染组（T）和对照组（C），转染组转染载有TMP21小分子干扰RNA（siRNA）的质粒，对照组转染空质粒。每组均制备包含细胞全膜蛋白的样品（T1、C1），经CHAPSO进一步溶解后超速离心制备所得纯化全膜蛋白样品（T2、C2），用γ分泌酶组分早老蛋白1（PS-1）特异性抗体免疫沉降C2或T2后获得的γ分泌酶样品（IPT2、IPC2）。蛋白质印迹法检测各类样品中γ分泌酶重要组分TMP21、PS-1和Nicastrin（NCT）蛋白表达量；应用ELISA法检测β淀粉样肽40和β＆#61472;淀粉样肽42的生成总量。 结果蛋白质印迹法检测显示，TMP21蛋白表达量在转染组样品IPT2中为（5294±247）ng/ml，在对照组样品IPC2中为（19110±579）ng/ml，组间差异有统计学意义（P〈0.05）；各类样品中PS-1与NCT蛋白表达量无明显变化；TMP21可与PS-1共沉降。ELISA法检测显示，转染组样品IPT2与饱和浓度底物C99反应生成的β淀粉样肽40和β淀粉样肽42总量为（348±18）pg/ml，对照组样品IPC2为与饱和浓度底物C99反应生成的β淀粉样肽40和β淀粉样肽42总量为（342±18）pg/ml，组间差异有统计学意义（P〈0.01）。 结论TMP21可能为γ分泌酶的组分之一。在TMP21蛋白低表达状态下γ分泌酶活性升高，提示TMP21为γ分泌酶的负调控因子之一。

Effect of siRNA-interfered transmembrane protein 21 expression on γ-secretase activity in embryonic fibroblast cells of KO mice

Objective To investigate the effect of transmembrane protein 21 (TMP 21) on the activity of γ-secretase. Methods Embryonic fibroblast cells obtained from KO mice MEF (KO)] with disrupted app gene were divided into transfection (T) and control (C) groups. The cells in the T group were transfected with the plasmids carrying the TMP 21 interfered by siRNA, and the control groups were transfected with blank plasmids. In each group, and samples of γ-secretase cell samples were prepared as follow: T1 and C1: the whole cell membrane proteins; T2 and C2: purified samples of whole membrane protein, which were the centrifugated lysates of CHAPSO, and IPT2 and IPC2: prepared by co-immunoprecipitation of by presenilin-1 (PS-1) antibody and C2 or T2. The expression levels of TMP 21, PS-1 and Nicastrin (NCT), were detected by Western blotting. The levels of Aβ&;#61472;-peptide 40 and Aβ&;#61472;-peptide 42 were measured by ELISA. Results Western blotting showed that the level of TMP 21 was 5294 ± 247 ng/ml in IPT2 , and was 19110 ± 579 ng/ml in IPC2. The difference in the TMP 21 level among the groups was significant (P < 0.05). No significant difference was found in the levels of PS-1 and NCT among the groups. TMP 21 and PS-1 were co-sedimentated. ELISA showed that the total amounts of Aβpeptide 40 and Aβpeptide 42 from C99 catalyzed by γ-secretase was 348 ± 18 pg/ml in IPT2, and was 342 ± 18 pg/ml in IPC2. The difference was significant among the groups (P < 0.05). Conclusions TMP 21 could be one of the components of γ-secretase complex. The activity of γ-secretase increases when TMP 21 expresses at a low level, indicating that TMP 21 is one of the negative modulators of γ-secretase.