尿液细胞外囊泡液压透析分离方法建立用于前列腺癌miRNAs检测和评价研究

发布时间：2018-01-29 22:12

  本文关键词： 细胞外囊泡 前列腺癌 尿液 液压透析法 miRNA　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景与目的前列腺癌(Prostate cancer,PCa)是危害男性健康的恶性疾病,中国前列腺癌发病率呈上升趋势。目前常用于前列腺癌辅助诊断的指标为前列腺特异性抗体(Prostate-specific antigen,PSA),由于缺少特异性,基于PSA的前列腺癌监测和筛查被认为有过度诊治的风险。为了更准确地发现前列腺癌患者、减少不必要的前列腺活检,新型的循环肿瘤标志物亟待被发现,其中细胞外囊泡(Extracellularvesicles,EVs)的研究受到了广泛的关注。EVs是细胞释放的用于细胞间信息传递的膜性囊泡,EVs携带有亲代细胞特异性的核酸、蛋白质等物质,这些生物活性物质的含量变化可反映机体的生理病理状态,因此EVs有作为肿瘤生物标志物的潜力。EVs可从血清/血浆、尿液、脑脊液、唾液、乳汁等体液中分离得到,尿液作为一种可无创、大样本获得的体液标本,是泌尿系统疾病研究的理想标本类型。目前对尿液细胞外囊泡(Urinary extracellular vesicles,UEVs)的分离方法尚未统一,因此本研究利用新型的液压透析(Hydrostatic filtration dialysis,HFD)法分离健康人尿液中的EVs,与经典的超高速离心法(Ultracentrifugation,UC)对比分离UEVs的能力和微小RNA(microRNA,miRNA)的检出情况,随后在前列腺癌、前列腺增生和健康对照者的UEVs中验证已报道的与前列腺癌相关的miRNA,评价UEVs中候选miRNA的诊断效能。本研究主要分为以下两个部分:第一部分 液压透析法和超高速离心法UEVs分离效果的对比研究一、实验目的证实新型HFD法的UEVs分离能力,探索HFD分离的UEVs(HFD-UEVs)是否适用于后续的miRNA研究,并与经典的UC法进行比较。二、实验方法分别用HFD法和UC法分离健康志愿者的UEVs,通过透射电子显微镜、NanoSight纳米颗粒跟踪分析仪、Bradford蛋白定量和蛋白印迹技术对比UEVs的形态、粒径分布和特异标志物的表达,评估HFD和UC法的分离效率,采用Nnaodrop 2000、Agilent 2100生物分析仪和实时荧光定量聚合酶链反应(Quantitative reverse transcription-polymerase chain reaction,qRT-PCR)检测 RNA总量、RNA组分和miRNA水平,并以配对t检验作统计分析。三、实验结果HFD法和UC法分离得到的UEVs有相似的形态和粒径分布范围,均表达EVs特异标志物,蛋白浓度及RNA总量差异无统计学意义,在RNA组分和miRNA水平方面也有一致结果。四、结论HFD法可简便地用于大样本UEVs的分离,其分离效率与经典的UC法相当,且HFD-UEVs可适用于后续的miRNA研究。第二部分UEVs前列腺癌相关miRNAs的检测及评价研究一、实验目的检测已报道的与前列腺癌相关的miRNA,筛选有作为诊断标志物潜力候选miRNA,对比血清和尿液EVs miRNA表达水平,分析EVs miRNA作为前列腺癌标志物的能力。二、实验方法从文献中筛选已报道的与前列腺癌相关的miRNA,分离前列腺癌、前列腺增生、健康对照者UEVs和配对血清EVs,逐步检测miRNA的表达水平,第一组检测标本由21例前列腺癌和14例前列腺增生患者组成,对差异有统计学意义的miRNA继续在第二组标本中检测,根据数据类型以t检验、one way ANOVA、Welch's ANOVA统计miRNA表达水平的差异,以LSD、Games-Howell进行多重比较,绘制受试者工作曲线(Receiver operating characteristic curve,ROC)分析miRNA的诊断效能。三、实验结果初步检测结果显示 miR-107、miR-375、miR-21、miR-27a 和 miR-574 在各组中差异无统计学意义,选取miR-145、miR-1290、miR-141和miR-572在后续验证中检测。在UEVs中,前列腺癌患者miR-145和miR-1290水平显著高于前列腺增生患者和健康对照,在血清EVs中,miR-572、miR-1290、miR-141和miR-145在前列腺癌患者中均显著高于前列腺增生患者,且在血清EVs中的表达水平显著高于在UEVs中的表达水平。ROC曲线和曲线下面积(Areaunder ROC curve,AUC)提示血清 PSA 联合尿液 EVs miR-145(AUC:0.863)或血清PSA血清EVsmiR-145(AUC:0.895)均可提高血清PSA单独检测(AUC:0.805)的诊断效能,且UEVsmiR-145在Gleason Score≥8的患者中表达水平显著高于Gleason Score≤7的患者。四、结论miR-145在前列腺癌患者和前列腺增生患者的UEVs和血清EVs中均有差异表达,血清或尿液EVs中的miR-145联合血清PSA均可提高血清PSA的诊断效能,UEVs miR-145是前列腺癌的潜在诊断标志物。
[Abstract]:Background and purpose: prostate cancer (Prostate, cancer, PCa) is a malignant disease that endangers male health, Chinese prostate cancer incidence is rising. The current commonly used in prostate cancer diagnosis index for prostate specific antibody (Prostate-specific, antigen, PSA), due to lack of specificity, monitoring and screening for prostate cancer is considered PSA there is a risk of over diagnosis and treatment based on. In order to more accurately identify patients with prostate cancer, reduce unnecessary prostate biopsies, circulating tumor markers were found to be novel, including extracellular vesicles (Extracellularvesicles, EVs) have been widely concerned.EVs is released from cells for cell membrane cystic information transmission global, EVs carrying parental cell specific nucleic acids, proteins and other substances, the content changes of these bioactive substances can reflect the physiological and pathological state of the body, so EVs as a tumor biomarker potential.EVs from serum / plasma, urine, saliva, cerebrospinal fluid, isolated body fluids such as milk, urine as a noninvasive, body fluid specimens obtained from large samples, is a disease of the urinary system of ideal type. The urine samples of extracellular vesicles (Urinary extracellular vesicles, UEVs) separation method is not unified, so this research uses hydraulic dialysis model (Hydrostatic filtration dialysis, HFD) were isolated in human urine EVs, and ultra high speed centrifugation classic (Ultracentrifugation, UC) UEVs and the separation capability of micro RNA (microRNA, miRNA) detection. Then in prostate cancer, validation has been reported associated with prostate cancer miRNA prostate hyperplasia and healthy controls UEVs, diagnostic efficacy evaluation candidate miRNA UEVs. This research is mainly divided into the following two parts Divided into: a comparative study of the first part of hydraulic dialysis and ultra high speed centrifugation UEVs separation effect, the purpose of the experiment confirmed that the new HFD method UEVs separation ability, exploring the HFD separation of UEVs (HFD-UEVs) is suitable for the follow-up study on miRNA, and compared with the classical UC method. Two experimental methods were used HFD method and UC were isolated from healthy volunteers by UEVs, transmission electron microscopy, NanoSight nano particle tracking analyzer, Bradford protein assay and Western blotting contrast UEVs morphology, particle size distribution and expression of specific markers, separation efficiency evaluation of HFD and UC method, using Nnaodrop 2000, Agilent 2100 Bioanalyzer and real-time fluorescence quantitative polymerase chain reaction (Quantitative reverse transcription-polymerase chain reaction, qRT-PCR) detection of total RNA, RNA components and miRNA level, and the paired t test for statistical analysis. Three, experiment The results of HFD method and UC method, the isolated UEVs has a range of similar shape and particle size distribution, both the expression of EVs specific markers, there was no significant difference in total protein concentration and RNA, RNA components and miRNA levels also have consistent results. Four. Conclusion HFD method can be conveniently used in the separation of the sample UEVs rather, the separation efficiency and the classical UC method, and HFD-UEVs can be applied to the follow-up study on miRNA. The second part is the research of UEVs detection and evaluation of prostate cancer related miRNAs, miRNA experiment to detect associated with prostate cancer have been reported, as a diagnostic marker for screening potential candidates for miRNA, the expression level of serum and contrast EVs miRNA EVs miRNA analysis of urine, as a marker for prostate cancer. The ability of two experimental screening methods have been reported associated with prostate cancer miRNA from the literature, the separation of prostate cancer, benign prostatic hyperplasia, healthy controls UEVs And paired serum EVs, the expression level gradually to detect miRNA, the first group of specimens from 21 cases of prostate cancer and 14 cases of benign prostatic hyperplasia patients, there were significant differences in the miRNA continue to test the second groups of specimens, according to the data type in the t test, one way ANOVA, Welch's ANOVA miRNA statistical differential expression in LSD, Games-Howell multiple comparison, receiveroperating curve (Receiver operating characteristic curve, ROC) diagnostic efficacy analysis of miRNA. Three. Preliminary experimental results showed that the miR-107, miR-375, miR-21, miR-27a and miR-574 in each group no significant difference in the selection of miR-145, miR-1290, miR-141, and miR-572 in the subsequent verification in the detection of prostate cancer. In UEVs, miR-145 and miR-1290 were significantly higher than that of patients with benign prostatic hyperplasia patients and healthy controls, the serum EVs, miR-572, miR-1290, miR- 141 and miR-145 in patients with prostate cancer were significantly higher than in patients with benign prostatic hyperplasia, and the expression level of EVs in serum was significantly higher than that in the expression level of.ROC curve and the area under the curve in UEVs (Areaunder ROC curve, AUC) suggested that serum PSA and urine EVs miR-145 (AUC:0.863) or serum PSA serum EVsmiR-145 (AUC:0.895) can be improved serum PSA detection (AUC:0.805) diagnostic performance, and the expression level of UEVsmiR-145 was significantly higher than that of Gleason Score is less than or equal to 7 of the patients in Gleason Score more than 8 of patients. Four. Conclusion the expression of miR-145 in both prostate cancer and prostate hyperplasia in patients with UEVs and serum EVs in different serum or urine EVs in miR-145 serum PSA can improve the diagnostic efficiency of serum PSA, UEVs, miR-145 is a potential diagnostic marker for prostate cancer.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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