本文選題：腎癌　切入點：早期診斷　出處：《第二軍醫(yī)大學》2017年碩士論文

【摘要】：研究背景及目的腎癌是少數(shù)幾個全球范圍內(nèi)發(fā)病率逐年上升的腫瘤之一,早期沒有顯著的臨床表現(xiàn),目前只能通過超聲、CT等影像學檢查發(fā)現(xiàn),而缺少能直接通過體液檢測的腫瘤標記物。大約有30-40%的腎癌患者在確診的時候就已經(jīng)發(fā)生轉(zhuǎn)移,并且早期診斷的腎癌患者相較于晚期轉(zhuǎn)移的患者其5年生存率相差甚遠。所以,實現(xiàn)腎癌的早期診斷和篩查具有極其重要的臨床意義。本研究旨在通過對尿液外泌體中的Lnc-RNA進行新一代測序(next-generation sequencing,NGS)分析,尋找能夠提示早期腎癌的Lnc-RNA作為其早期診斷標記物,從而結(jié)合影像學檢查,更早地診斷腎癌。外泌體(exosome)是由細胞內(nèi)多泡小體(multivesicular body,MVB)與細胞膜融合后,釋放到細胞外微環(huán)境中的一種直徑約30-120nm的膜性囊泡。其內(nèi)含有蛋白質(zhì)、各種RNA等多種成分。包括腫瘤細胞在內(nèi)的多種細胞都會分泌外泌體,并在血液、尿液、唾液、乳汁等多種體液中可分離、檢測。長鏈非編碼RNA(Long non-coding RNA,LncRNA)是指長度大于200個核苷酸(bp)的非編碼RNA�，F(xiàn)有研究認為,Lnc-RNA具有良好的生物標記開發(fā)潛力。研究方法1)選取腎透明細胞癌患者及年齡、性別相匹配的正常對照者各10人,留取晨尿。2)應用超速離心法分離出外泌體,并對所獲得的外泌體進行透射電鏡檢查、粒徑分布、蛋白標記物(CD63、ALIX、TSG101)等驗證。3)利用新一代測序技術(shù)分析腎透明細胞癌患者和正常人尿液外泌體內(nèi)差異表達的LncRNA,并跟據(jù)差異倍數(shù)、P值,篩選出差異表達顯著的LncRNA。4)將所篩選出的差異最明顯的LncRNA在成熟的腎癌細胞系中進一步驗證其表達量。5)加大尿液外泌體的樣本量,分析目標LncRNA的表達水平。7)繪制目標LncRNA的ROC曲線,評價其敏感性和特異性等臨床診斷應用價值。研究結(jié)果1)通過超速離心法,可以成功分離富集尿液外泌體;2)成功篩選出可以提示腎癌早期診斷的LncRNA NR_040448和NR_033390,發(fā)現(xiàn)其在成熟的腎癌細胞系、擴大的腎癌尿液外泌體樣本中均高表達。結(jié)論尿液外泌體內(nèi)的LncRNA NR_040448和NR_033390有希望成為腎癌早期診斷的潛在標記物,其敏感性和特異性還需要更大樣本的臨床資料進行驗證。
[Abstract]:Background and objective Renal cell carcinoma (RCC) is one of the few tumors with increasing incidence in the world. There is no significant clinical manifestation in the early stage.There is a lack of tumor markers that can be detected directly by body fluids.About 30-40% of RCC patients had metastases at the time of diagnosis, and the 5-year survival rate of early diagnosed RCC patients was significantly different from that of advanced RCC patients.Therefore, the realization of early diagnosis and screening of renal cell carcinoma has an extremely important clinical significance.The purpose of this study was to analyze the Lnc-RNA in urine exocrine by a next-generation sequencing assay, and to search for Lnc-RNA as an early diagnostic marker for early renal cell carcinoma (RCC), so as to diagnose RCC earlier with imaging examination.Exosome is a membranous vesicle about 30-120nm diameter which is released into the extracellular microenvironment by the fusion of multivesical body (MVB) with the cell membrane.It contains protein, various RNA and other components.Many kinds of cells, including tumor cells, secrete exocrine, and can be separated and detected in blood, urine, saliva, milk and other body fluids.LncRNAs with long chain noncoding RNA(Long non-coding (LncRNAs) are non-coding RNAs with a length of more than 200 nucleotides (BP).Current studies suggest that Lnc-RNA has good biomarker potential.Methods 1) 10 patients with renal clear cell carcinoma (RCC) and 10 normal controls matched with age and sex were selected to retain morning urine. The exocrine was isolated by ultracentrifugation, and the obtained exocrine was examined by transmission electron microscopy (TEM) and particle size distribution.Using a new generation of sequencing techniques to analyze the differential expression of LncRNAs in urine secreted from patients with clear cell carcinoma of kidney and normal people, and according to the difference multiple P value, the protein marker CD63, ALIXT TSG101, and so on, was used to analyze the differential expression of LncRNAs in the urine of patients with renal clear cell carcinoma and normal people.LncRNA.4) to further verify the expression of LncRNA.4 in mature renal cell carcinoma cell lines, to increase the amount of urine exocrine, and to analyze the expression level of target LncRNA. 7) to draw the ROC curve of target LncRNA.To evaluate its sensitivity and specificity in clinical diagnosis.Results 1) LncRNA NR_040448 and NR033390, which can be successfully isolated and enriched by ultracentrifugation and enriched in urine, were successfully screened and found in mature renal carcinoma cell lines.Overexpression was found in urine exocrine samples of enlarged renal cell carcinoma.Conclusion LncRNA NR_040448 and NR_033390 in urine exocrine may be potential markers for early diagnosis of renal cell carcinoma. Its sensitivity and specificity need to be verified by larger clinical data.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.11
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本文編號：1718536