本文選題：DNA納米結(jié)構(gòu) + 癌細胞　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：癌癥是危害全人類健康和生命安全的重大疾病。目前,研究表明早期診斷和治療能夠提高惡性腫瘤的臨床療效、降低其死亡率。因此,腫瘤發(fā)生早期的靈敏和準確檢測具有十分重要的現(xiàn)實意義。傳統(tǒng)檢測癌癥的方法在特異性、靈敏度等方面仍存在不足,無法滿足腫瘤早期檢測的要求。近年來,一系列針對腫瘤標志物(如mRNA、膜蛋白等)的新型檢測分子不斷被開發(fā)出來,為腫瘤早期診斷提供了新的機遇。DNA納米技術(shù)自提出以來,因具有結(jié)構(gòu)可編程性、良好的生物相容性和生物穩(wěn)定性、無明顯的細胞毒性和免疫刺激性、能夠自主入胞等特點,在生物醫(yī)藥領(lǐng)域引起了廣泛的關(guān)注,已經(jīng)用于腫瘤的成像與檢測。基于以上論述,本文利用DNA納米技術(shù)構(gòu)建了新型腫瘤檢測系統(tǒng),用于癌細胞中mRNA的邏輯檢測以及膜蛋白免標記、信號放大型檢測,實現(xiàn)了腫瘤細胞的靈敏、準確檢測,以期為臨床惡性腫瘤的早期檢測提供依據(jù)。本論文構(gòu)建了雙識別探針共軛的DNA四面體通過mRNA的原位邏輯檢測實現(xiàn)HepG2細胞的準確檢測。該結(jié)構(gòu)包括:(1)邏輯識別兩種mRNA的探針。只有兩種特定的mRNA——GalNAc-T mRNA和TK1 mRNA同時存在的基礎(chǔ)上才能輸出一種熒光信號,一種或者沒有指定mRNA存在的情況下就沒有熒光信號,準確區(qū)分肝癌細胞HepG2(存在GalNAc-TmRNA和TK1 mRNA兩種mRNA)與正常肝細胞HL7702(只存在GalNAc-T mRNA);(2)DNA四面體結(jié)構(gòu)。作為探針的載體,生物相容性好、易合成與修飾、結(jié)構(gòu)穩(wěn)定以及能夠自主入胞等,具有運載探針入胞以及保護探針的作用。最終成功實現(xiàn)兩種細胞的區(qū)分,避免了只檢測一種腫瘤相關(guān)mRNA存在的假陽性現(xiàn)象。同時采用邏輯輸出方式,結(jié)果簡單直觀,避免多色檢測方式會受到熒光光譜重疊以及檢測通道數(shù)目等的限制。本論文還構(gòu)建了共區(qū)域化適體探針膜上觸發(fā)信號放大裝置用于Hela細胞的免標記、靈敏檢測。在所設(shè)計的共區(qū)域化適體探針的基礎(chǔ)上,結(jié)合經(jīng)典的HCR反應(yīng)膜上觸發(fā)信號放大裝置——DNA納米條帶,在生成的HCR雙鏈部分插入Eve Green染料就可以輸出熒光信號了,多余未反應(yīng)完的探針可以被加入的石墨烯吸附掉,其生成的背景信號。檢測的有效細胞個數(shù)為100個。另外這種策略是在識別與信號放大過程后插入染料,具有不干擾識別與信號放大過程的優(yōu)點。
[Abstract]:Cancer is a major disease that endangers the health and safety of all mankind. At present, studies have shown that early diagnosis and treatment can improve the clinical efficacy of malignant tumors and reduce their mortality. Therefore, the sensitive and accurate detection of early tumorigenesis is of great practical significance. The traditional methods of cancer detection are still insufficient in specificity and sensitivity, and can not meet the requirements of early detection of cancer. In recent years, a series of new detection molecules for tumor markers (such as mRNAs, membrane proteins, etc.) have been developed, which provide a new opportunity for early diagnosis of tumor. Because of its good biocompatibility and stability, no obvious cytotoxicity and immune irritation, and the ability to enter the cell independently, it has attracted wide attention in the field of biomedicine and has been used in tumor imaging and detection. Based on the above discussion, a new tumor detection system was constructed by using DNA nanotechnology, which can be used for the logical detection of mRNA in cancer cells, as well as for the detection of membrane proteins without labeling and signal amplification, so as to realize the sensitive and accurate detection of tumor cells. In order to provide basis for early detection of clinical malignant tumor. In this paper, a conjugated DNA tetrahedron with double recognition probe was constructed to detect HepG2 cells accurately by in situ logical detection of mRNA. The structure consists of 1: 1) logical recognition of two mRNA probes. Only when two specific mRNA--GalNAc-T mRNA and TK1 mRNA exist at the same time can one kind of fluorescence signal be outputted, and there is no fluorescence signal in the presence of one or no specified mRNA. To distinguish HepG2 (GalNAc-TmRNA and TK1 mRNA) from HL7702 (only GalNAc-T mRNA2) tetrahedron in hepatoma cell line (HepG2) and normal hepatocyte (HL7702). As the carrier of the probe, it has good biocompatibility, easy synthesis and modification, stable structure and ability to enter the cell independently. It has the function of carrying the probe into the cell and protecting the probe. Finally, the differentiation of two kinds of cells was successfully realized, avoiding the false positive phenomenon of detecting only one tumor associated mRNA. At the same time, the method of logical output is used, the result is simple and intuitionistic, and the multi-color detection method is avoided by the limitation of fluorescence spectrum overlap and the number of detection channels. In this paper, we also constructed a co-regionalized aptamer probe on the membrane trigger signal amplification device for Hela cell labeling, sensitive detection. On the basis of the designed co-regionalized aptamer probe and the classic trigger signal amplification device on the HCR reaction film, the fluorescent signal can be output by inserting the Eve Green dye into the generated HCR double strand. The superfluous unreacted probe can be adsorbed by the added graphene and the generated background signal. The number of effective cells detected was 100. In addition, this strategy is to insert dyes after the recognition and signal amplification process, which has the advantage of not interfering with the process of recognition and signal amplification.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R730.4

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本文編號：1889012