本文選題：電化學(xué)信號(hào)放大檢測(cè) + miRNA�。� 參考：《中南大學(xué)》2012年碩士論文

【摘要】：人體內(nèi),生物分子miRNA和BACE1與膠質(zhì)瘤和阿爾茨海默病存在密切的關(guān)系。1niRNA-182的表達(dá)水平可作為臨床上膠質(zhì)瘤早期診斷的依據(jù),抑制BACE1的活性可在一定程度上減少Ap的產(chǎn)生,因此,研究miRNA和BACE1對(duì)產(chǎn)生Aβ的前體蛋白的剪切具有重要的生物學(xué)意義。鑒于miRNA在體內(nèi)的低含量和以及BACE1對(duì)產(chǎn)生Ap的前體蛋白的切割缺乏有效的檢測(cè)手段,本論文中,我們建立了基于一種巰基二茂鐵修飾的納米金/抗生物素蛋白復(fù)合物電化學(xué)信號(hào)放大方法來(lái)檢測(cè)miRNA和BACE1對(duì)多肽的切割。 miRNA(MicroRNA)是一種廣泛存在于真核生物中的小RNA,成熟體的miRNA長(zhǎng)度大約為17-25nt。機(jī)體內(nèi)miRNA-182的表達(dá)水平在一定程度上可反映膠質(zhì)瘤的產(chǎn)生及發(fā)展過(guò)程。本論文中,我們采用電化學(xué)信號(hào)放大的方法,檢測(cè)了膠質(zhì)瘤中miRNA的表達(dá)水平。首先在金電極的表面修飾DNA單分子層,然后利用生物素標(biāo)記的miRNA和靶點(diǎn)miRNA與表面固定的DNA發(fā)生競(jìng)爭(zhēng)反應(yīng),隨后巰基二茂鐵修飾的納米金/抗生物素蛋白復(fù)合物衍生到電極表面。二茂鐵的電化學(xué)信號(hào)與靶點(diǎn)miRNA濃度成反比例。該方法成功應(yīng)用于膠質(zhì)瘤患者血清中miRNA-182的測(cè)定。結(jié)果表明,癌癥患者中miRNA-182的含量是健康人的3倍左右,該結(jié)果和實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(RT-qPCR)結(jié)果一致。此外,電化學(xué)方法操作簡(jiǎn)單、重現(xiàn)性好(RSD5%)、檢測(cè)限低至10fM。 BACE1是一種與阿爾茨海默病相關(guān)的重要的蛋白水解酶。Ap是β-淀粉樣蛋白前體(APP)的水解產(chǎn)物之一,APP依次由p-分泌酶(BACE1)、γ-分泌酶特異性酶切產(chǎn)生Ap。目前大量的學(xué)者認(rèn)為Aβ的異常表達(dá)與阿爾茨海默病存在密切的關(guān)系,因此抑制BACE1的活性成為治療AD的重要途徑之一。本論文中模擬BACE1酶切APP的生物過(guò)程,設(shè)計(jì)一條含BACE1酶切位點(diǎn)的一段生物素標(biāo)記的多肽序列并修飾于金電極表面,經(jīng)過(guò)BACE1剪切,隨后用巰基二茂鐵修飾的納米金/抗生物素蛋白復(fù)合物衍生到電極表面,通過(guò)BACE1酶切前后電化學(xué)信號(hào)的變化來(lái)檢測(cè)BACE1切割多肽的過(guò)程。該方法可初步用于BACE1抑制劑的篩選。當(dāng)BACE1抑制劑存在時(shí),電化學(xué)信號(hào)不降低或者降低程度較小。
[Abstract]:In human body, the expression of biomolecular miRNA and BACE1 is closely related to glioma and Alzheimer's disease. The expression level of .1niRNA-182 may serve as a basis for early diagnosis of glioma, and inhibition of BACE1 activity can reduce the production of AP to some extent. The study of miRNA and BACE1 has important biological significance for the shearing of precursor protein producing A 尾. In view of the low content of miRNA in vivo and the lack of effective detection of BACE1 for the cleavage of precursor proteins that produce AP, in this paper, We have established a novel electrochemical signal amplification method based on thioglycol-ferrocene modified nano-gold / biotin protein complex to detect the cleavage of polypeptides by miRNA and BACE1. MiRNA-MicroRNAs are a kind of small RNAs widely present in eukaryotes and mature. The length of miRNA is about 17-25 NT. The expression level of miRNA-182 can reflect the formation and development of glioma to some extent. In this paper, we detected the expression of miRNA in gliomas by electrochemical signal amplification. First, DNA monolayers were modified on the surface of gold electrode, then biotin-labeled miRNA and target miRNA were used to compete with the surface immobilized DNA, and then thioglycol-ferrocene modified nano-gold / biotin protein complex was derived to the electrode surface. The electrochemical signal of ferrocene is inversely proportional to the concentration of miRNA. The method has been successfully applied to the determination of miRNA-182 in serum of glioma patients. The results showed that the content of miRNA-182 in cancer patients was about three times as high as that in healthy persons, which was consistent with the results of real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). In addition, the electrochemical method is simple to operate, The detection limit is as low as 10fM.BACE1 is an important proteolytic enzyme associated with Alzheimer's disease. AP is one of the hydrolysates of 尾 -amyloid protein precursor APP. At present, a large number of scholars believe that the abnormal expression of A 尾 is closely related to Alzheimer's disease, so inhibiting the activity of BACE1 is one of the important ways to treat AD. In this paper, we simulated the biological process of BACE1 digesting app, designed a biotin labeled polypeptide sequence containing BACE1 restriction site and modified it on the surface of gold electrode, and then cut it by BACE1. Then the nano-gold / biotin protein complex modified by thioglycol-ferrocene was derived to the electrode surface. The process of peptide cleavage of BACE1 was detected by the change of electrochemical signal before and after BACE1 enzyme digestion. The method can be used to screen BACE1 inhibitors. When BACE1 inhibitor exists, the electrochemical signal does not decrease or decrease to a lesser extent.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16

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