中文摘要：

研究转运膜蛋白21（TMP21）在小鼠成纤维细胞中超表达状态下。对γ分泌酶活性的影响．利用TMP21eDNA转染敲除淀粉样前体蛋白表达基因的小鼠胚胎成纤维细胞系（MEF（KO）），以梯度浓度G418筛选TMP21表达量最高的细胞．处理超表达组（T）和对照组（C）细胞，并收集纯化含γ分泌酶复合物的样本．用Western blotting检测TMP21蛋白表达量，选用饱和浓度的γ分泌酶底物C99，运用ELISA检测Aβ-peptide40和AIβ-peptide42的生成总量．结果表明，样本T1、T2及IPT2所含TMP21的蛋白表达量较相应对照组样本C1、C2和IPC2分别上升11．5％（P=0．08），28．1％（P〈0．01），69．1％（P〈0．001），提示经TMP21eDNA转染后MEF（K0）细胞中TMP21蛋白表达明显提高．同时，对照样本C1、C2及IPC2，样本T1、T2及IPT2中所含γ分泌酶活性分别降低33．4%（P=0．01），71．9％（P〈0．001），96．5％（P〈0．001）．转染组样本中，样本IPT2所含γ分泌酶活性最低．提示经处理γ分泌酶得到明显纯化分离，与Western blotting所得结果一致．因此，高表达状态下的TMP21蛋白，对MEF（KO）细胞中γ分泌酶的活性具有明显的降低作用．表明TMP21为γ分泌酶的负调控因子之一．

英文摘要：

Objective γ-secretase is one of the crucial enzymes which involved in the pathogenic mechanism of Alzheimer＇s disease. The impacts of the transmembrane protein 21 （TMP21） on γ-secretase activity were studied in APP knockout mouse embryonic fibroblasts（MEF （KO））. Methods MEF（KO） was transfectted with TMP21 cDNA to induce protein expression. During the purification of γ-secretase, samples comprising γ- secretase complex were collected in transfection（T） or control（C） group at three phases. The samples were respectively marked as follow： T1 and C1 which included the vchole cell membrane proteins; T2 and C2 which were the lysates by CHAPSO; IPT2 and IPC2 which were prepared after coimmunoprecipitation by presenilin-1 antibody. The protein levels of TMP21 were detected by Western blotting. The γ-secretase activity against artificial substrate C99 was measured by ELISA. Results The protein levels of TMP21 in T1,T2 and IPT2 were, compared with C1,C2 and IPC2, increased by 11.5%（P=0.08）,28. 1%（P〈0.01） and 69. 1% （P〈0. 001） respectively. The amounts of Aβ-peptide40 and Aft-peptide42 from C99 catalyzed by γ-secretase in T1, T2 and IPTx were reduced by 33.4%（P=0.01）,71.9% （P〈0. 001）, 96. 5% （P〈0, 001） when respectively compared with C1 ,C2 and IPC2. Conclusions TMP21 can be one of the negative regulators of γ-secretase as the component of γ-secretase complex. The activity of γ-secretase was reduced in MEF（KO） by the induction of TMP21 protein expression via eDNA transfeetion.