尿液外泌体内Lnc-RNA作为肾癌早期诊断标记物相关研究

发布时间：2018-04-06 07:19

  本文选题：肾癌　切入点：早期诊断　出处：《第二军医大学》2017年硕士论文

【摘要】：研究背景及目的肾癌是少数几个全球范围内发病率逐年上升的肿瘤之一,早期没有显著的临床表现,目前只能通过超声、CT等影像学检查发现,而缺少能直接通过体液检测的肿瘤标记物。大约有30-40%的肾癌患者在确诊的时候就已经发生转移,并且早期诊断的肾癌患者相较于晚期转移的患者其5年生存率相差甚远。所以,实现肾癌的早期诊断和筛查具有极其重要的临床意义。本研究旨在通过对尿液外泌体中的Lnc-RNA进行新一代测序(next-generation sequencing,NGS)分析,寻找能够提示早期肾癌的Lnc-RNA作为其早期诊断标记物,从而结合影像学检查,更早地诊断肾癌。外泌体(exosome)是由细胞内多泡小体(multivesicular body,MVB)与细胞膜融合后,释放到细胞外微环境中的一种直径约30-120nm的膜性囊泡。其内含有蛋白质、各种RNA等多种成分。包括肿瘤细胞在内的多种细胞都会分泌外泌体,并在血液、尿液、唾液、乳汁等多种体液中可分离、检测。长链非编码RNA(Long non-coding RNA,LncRNA)是指长度大于200个核苷酸(bp)的非编码RNA。现有研究认为,Lnc-RNA具有良好的生物标记开发潜力。研究方法1)选取肾透明细胞癌患者及年龄、性别相匹配的正常对照者各10人,留取晨尿。2)应用超速离心法分离出外泌体,并对所获得的外泌体进行透射电镜检查、粒径分布、蛋白标记物(CD63、ALIX、TSG101)等验证。3)利用新一代测序技术分析肾透明细胞癌患者和正常人尿液外泌体内差异表达的LncRNA,并跟据差异倍数、P值,筛选出差异表达显著的LncRNA。4)将所筛选出的差异最明显的LncRNA在成熟的肾癌细胞系中进一步验证其表达量。5)加大尿液外泌体的样本量,分析目标LncRNA的表达水平。7)绘制目标LncRNA的ROC曲线,评价其敏感性和特异性等临床诊断应用价值。研究结果1)通过超速离心法,可以成功分离富集尿液外泌体;2)成功筛选出可以提示肾癌早期诊断的LncRNA NR_040448和NR_033390,发现其在成熟的肾癌细胞系、扩大的肾癌尿液外泌体样本中均高表达。结论尿液外泌体内的LncRNA NR_040448和NR_033390有希望成为肾癌早期诊断的潜在标记物,其敏感性和特异性还需要更大样本的临床资料进行验证。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11
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本文编号：1718536