發(fā)布時(shí)間：2018-04-03 18:33

  本文選題：阿爾茨海默病　切入點(diǎn)：β-分泌酶　出處：《遼寧醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的 本文旨在研究當(dāng)細(xì)胞內(nèi)環(huán)境呈酸化改變時(shí)，，細(xì)胞內(nèi)β-分泌酶mRNA表達(dá)、蛋白含量及活性的變化，進(jìn)而說(shuō)明細(xì)胞內(nèi)pH值變化與β-分泌酶的關(guān)系。 方法 培養(yǎng)人神經(jīng)母細(xì)胞瘤細(xì)胞（Human neuroblastoma cell line, SH-SY5Y），將構(gòu)建好的靶向人NHE-1基因的siRNA腺病毒及腺病毒空載體感染SH-SY5Y細(xì)胞，建立NHE-1基因敲低細(xì)胞模型及負(fù)對(duì)照組；用配制好的無(wú)血清的培養(yǎng)基培養(yǎng)SH-SY5Y細(xì)胞，收集前5天培養(yǎng)的細(xì)胞，建立無(wú)血清培養(yǎng)組。實(shí)驗(yàn)分成4組，即正常對(duì)照組、NHE-1基因敲低組、負(fù)對(duì)照組和無(wú)血清培養(yǎng)組。運(yùn)用熒光分光光度計(jì)測(cè)定各組細(xì)胞內(nèi)pH值，采用Real-time PCR技術(shù)檢測(cè)各組細(xì)胞內(nèi)β-分泌酶mRNA表達(dá)，通過(guò)Western Blotting檢測(cè)β-分泌酶蛋白表達(dá)水平，通過(guò)ELISA檢測(cè)β-分泌酶的活性變化。利用單因素方差分析及LSD-t檢驗(yàn)統(tǒng)計(jì)分析各組細(xì)胞的檢測(cè)指標(biāo)差異情況。 結(jié)果 無(wú)血清條件下培養(yǎng)細(xì)胞，當(dāng)培養(yǎng)4天時(shí)細(xì)胞狀態(tài)最佳。熒光探針?lè)y(cè)得NHE-1基因敲低組細(xì)胞內(nèi)pH值為6.91±0.055，無(wú)血清培養(yǎng)組細(xì)胞內(nèi)pH值為7.03±0.062，負(fù)對(duì)照組胞內(nèi)pH值為7.22±0.026，正常對(duì)照組細(xì)胞內(nèi)pH值為7.19±0.066，與正常對(duì)照組和負(fù)對(duì)照組相比，NHE-1基因敲低組和無(wú)血清培養(yǎng)組細(xì)胞內(nèi)pH值明顯降低，差異具有統(tǒng)計(jì)學(xué)意義（p0.05），而正常對(duì)照組和負(fù)對(duì)照組相比細(xì)胞內(nèi)pH值無(wú)明顯變化，差異無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。Real-time PCR及Western Blotting結(jié)果顯示：NHE-1基因敲低組細(xì)胞內(nèi)β-分泌酶mRNA表達(dá)和蛋白含量分別為5.65±0.33和0.37±0.030，無(wú)血清培養(yǎng)組細(xì)胞內(nèi)β-分泌酶mRNA表達(dá)和蛋白含量分別為2.40±0.55和0.32±0.031，與正常對(duì)照組（0.92±0.34；0.12±0.020）和負(fù)對(duì)照組（1.00±0.00；0.14±0.021）相比，NHE-1基因敲低組和無(wú)血清培養(yǎng)組細(xì)胞內(nèi)β-分泌酶mRNA表達(dá)和蛋白含量明顯增多，差異具有統(tǒng)計(jì)學(xué)意義（p0.05），而正常對(duì)照組和負(fù)對(duì)照組相比細(xì)胞內(nèi)β-分泌酶mRNA表達(dá)和蛋白含量無(wú)明顯變化，差異無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。ELISA結(jié)果顯示：NHE-1基因敲低組細(xì)胞內(nèi)β-分泌酶的蛋白水平為449.45±65.95ng/L，無(wú)血清培養(yǎng)組細(xì)胞內(nèi)β-分泌酶的蛋白水平為468.60±58.59ng/L，與正常對(duì)照組（290.65±82.33ng/L）和負(fù)對(duì)照組（327.11±82.32ng/L）相比，NHE-1基因敲低組和無(wú)血清培養(yǎng)組細(xì)胞內(nèi)β-分泌酶的活性明顯增強(qiáng)，差異具有統(tǒng)計(jì)學(xué)意義（p0.05），而正常對(duì)照組和負(fù)對(duì)照組相比細(xì)胞內(nèi)β-分泌酶的活性無(wú)明顯變化，差異無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論 通過(guò)成功構(gòu)建NHE-1基因敲低組及無(wú)血清培養(yǎng)組，使細(xì)胞內(nèi)環(huán)境呈酸化改變，證實(shí)細(xì)胞內(nèi)pH降低能夠使β-分泌酶的表達(dá)上調(diào)并增強(qiáng)其活性，進(jìn)而可能影響APP水解剪切，促進(jìn)Aβ的生成積聚。
[Abstract]:objective
The aim of this study is to investigate the expression of mRNA, the protein content and activity of beta secretase in cells when the intracellular environment is acidified, and to further illustrate the relationship between pH value and beta secretase.
Method
Cultured human neuroblastoma cells (Human neuroblastoma cell line, SH-SY5Y siRNA), adenovirus and adenovirus constructed targeting human NHE-1 gene in SH-SY5Y cells infected with NHE-1 gene knockdown cell model is established and the negative control group; SH-SY5Y cells cultured with the prepared serum-free culture medium 5 days before the collection of cells, establish the serum-free group. The experiment was divided into 4 groups: normal control group, NHE-1 knockdown group, negative control group and serum-free group. Spectrophotometric determination of the intracellular pH value by fluorescence, using Real-time PCR technology to detect the intracellular beta secretase mRNA the expression level of Western Blotting expression by detecting beta secretase protein, the activity of ELISA in the detection of beta secretase. Using single factor analysis of variance and LSD-t test statistical analysis indexes of cells were difference.
Result
Cells cultured in serum-free conditions, when cells cultured for 4 days. The best knockdown group of intracellular pH 6.91 + 0.055 NHE-1 gene measured by fluorescence probe, no group of intracellular pH 7.03 + 0.062 serum culture negative group, the intracellular pH value was 7.22 + 0.026 group, normal control group cells the pH value is 7.19 + 0.066, compared with the normal control group and negative control group, NHE-1 knockdown group and serum-free group of intracellular pH levels decreased significantly, the difference was statistically significant (P0.05), and the normal control group the intracellular pH values had no significant changes compared with the negative control group, the difference was not statistically significant (P0.05).Real-time PCR and Western Blotting showed that NHE-1 mutant enzyme mRNA expression and protein content were low in beta cell secretion group were 5.65 + 0.33 and 0.37 + 0.030, serum-free group of intracellular beta secretase mRNA expression and protein content were 2.40 + 0.55 and 0.32 + 0 .031, and the normal control group (0.92 + 0.34; 0.12 + 0.020) and negative control group (1 + 0; 0.14 + 0.021) compared with NHE-1 gene knockdown group and serum-free group of intracellular beta secretase mRNA expression and protein content increased, the difference was statistically significant (P0.05), and normal the control group of intracellular beta secretase mRNA expression and protein content had no significant change compared with the negative control group, the difference was not statistically significant (P0.05).ELISA showed that NHE-1 mutant protein level low intracellular beta secretase is 449.45 + 65.95ng/L, no intracellular beta secretase serum protein level the 468.60 + 58.59ng/L, and normal control group (290.65 + 82.33ng/L) and negative control group (327.11 + 82.32ng/L) compared with NHE-1 gene knockdown group and serum-free group of intracellular beta secretase activity increased significantly, the difference was statistically significant (P0.05), and normal control There was no significant difference in the activity of intracellular beta secretase in the group and the negative control group, and there was no significant difference between the two groups (P0.05).
conclusion
Through successful construction of NHE-1 knockdown group and serum-free culture group, the intracellular environment is acidified. It is confirmed that the decrease of intracellular pH can increase the expression of beta secretase and enhance its activity, which may affect APP hydrolysis and shear, and promote the accumulation of A beta.

【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R749.16

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本文編號(hào)：1706415