本文關(guān)鍵詞： 細(xì)胞外囊泡 前列腺癌 尿液 液壓透析法 miRNA　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景與目的前列腺癌(Prostate cancer,PCa)是危害男性健康的惡性疾病,中國(guó)前列腺癌發(fā)病率呈上升趨勢(shì)。目前常用于前列腺癌輔助診斷的指標(biāo)為前列腺特異性抗體(Prostate-specific antigen,PSA),由于缺少特異性,基于PSA的前列腺癌監(jiān)測(cè)和篩查被認(rèn)為有過(guò)度診治的風(fēng)險(xiǎn)。為了更準(zhǔn)確地發(fā)現(xiàn)前列腺癌患者、減少不必要的前列腺活檢,新型的循環(huán)腫瘤標(biāo)志物亟待被發(fā)現(xiàn),其中細(xì)胞外囊泡(Extracellularvesicles,EVs)的研究受到了廣泛的關(guān)注。EVs是細(xì)胞釋放的用于細(xì)胞間信息傳遞的膜性囊泡,EVs攜帶有親代細(xì)胞特異性的核酸、蛋白質(zhì)等物質(zhì),這些生物活性物質(zhì)的含量變化可反映機(jī)體的生理病理狀態(tài),因此EVs有作為腫瘤生物標(biāo)志物的潛力。EVs可從血清/血漿、尿液、腦脊液、唾液、乳汁等體液中分離得到,尿液作為一種可無(wú)創(chuàng)、大樣本獲得的體液標(biāo)本,是泌尿系統(tǒng)疾病研究的理想標(biāo)本類型。目前對(duì)尿液細(xì)胞外囊泡(Urinary extracellular vesicles,UEVs)的分離方法尚未統(tǒng)一,因此本研究利用新型的液壓透析(Hydrostatic filtration dialysis,HFD)法分離健康人尿液中的EVs,與經(jīng)典的超高速離心法(Ultracentrifugation,UC)對(duì)比分離UEVs的能力和微小RNA(microRNA,miRNA)的檢出情況,隨后在前列腺癌、前列腺增生和健康對(duì)照者的UEVs中驗(yàn)證已報(bào)道的與前列腺癌相關(guān)的miRNA,評(píng)價(jià)UEVs中候選miRNA的診斷效能。本研究主要分為以下兩個(gè)部分:第一部分 液壓透析法和超高速離心法UEVs分離效果的對(duì)比研究一、實(shí)驗(yàn)?zāi)康淖C實(shí)新型HFD法的UEVs分離能力,探索HFD分離的UEVs(HFD-UEVs)是否適用于后續(xù)的miRNA研究,并與經(jīng)典的UC法進(jìn)行比較。二、實(shí)驗(yàn)方法分別用HFD法和UC法分離健康志愿者的UEVs,通過(guò)透射電子顯微鏡、NanoSight納米顆粒跟蹤分析儀、Bradford蛋白定量和蛋白印跡技術(shù)對(duì)比UEVs的形態(tài)、粒徑分布和特異標(biāo)志物的表達(dá),評(píng)估HFD和UC法的分離效率,采用Nnaodrop 2000、Agilent 2100生物分析儀和實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(Quantitative reverse transcription-polymerase chain reaction,qRT-PCR)檢測(cè) RNA總量、RNA組分和miRNA水平,并以配對(duì)t檢驗(yàn)作統(tǒng)計(jì)分析。三、實(shí)驗(yàn)結(jié)果HFD法和UC法分離得到的UEVs有相似的形態(tài)和粒徑分布范圍,均表達(dá)EVs特異標(biāo)志物,蛋白濃度及RNA總量差異無(wú)統(tǒng)計(jì)學(xué)意義,在RNA組分和miRNA水平方面也有一致結(jié)果。四、結(jié)論HFD法可簡(jiǎn)便地用于大樣本UEVs的分離,其分離效率與經(jīng)典的UC法相當(dāng),且HFD-UEVs可適用于后續(xù)的miRNA研究。第二部分UEVs前列腺癌相關(guān)miRNAs的檢測(cè)及評(píng)價(jià)研究一、實(shí)驗(yàn)?zāi)康臋z測(cè)已報(bào)道的與前列腺癌相關(guān)的miRNA,篩選有作為診斷標(biāo)志物潛力候選miRNA,對(duì)比血清和尿液EVs miRNA表達(dá)水平,分析EVs miRNA作為前列腺癌標(biāo)志物的能力。二、實(shí)驗(yàn)方法從文獻(xiàn)中篩選已報(bào)道的與前列腺癌相關(guān)的miRNA,分離前列腺癌、前列腺增生、健康對(duì)照者UEVs和配對(duì)血清EVs,逐步檢測(cè)miRNA的表達(dá)水平,第一組檢測(cè)標(biāo)本由21例前列腺癌和14例前列腺增生患者組成,對(duì)差異有統(tǒng)計(jì)學(xué)意義的miRNA繼續(xù)在第二組標(biāo)本中檢測(cè),根據(jù)數(shù)據(jù)類型以t檢驗(yàn)、one way ANOVA、Welch's ANOVA統(tǒng)計(jì)miRNA表達(dá)水平的差異,以LSD、Games-Howell進(jìn)行多重比較,繪制受試者工作曲線(Receiver operating characteristic curve,ROC)分析miRNA的診斷效能。三、實(shí)驗(yàn)結(jié)果初步檢測(cè)結(jié)果顯示 miR-107、miR-375、miR-21、miR-27a 和 miR-574 在各組中差異無(wú)統(tǒng)計(jì)學(xué)意義,選取miR-145、miR-1290、miR-141和miR-572在后續(xù)驗(yàn)證中檢測(cè)。在UEVs中,前列腺癌患者miR-145和miR-1290水平顯著高于前列腺增生患者和健康對(duì)照,在血清EVs中,miR-572、miR-1290、miR-141和miR-145在前列腺癌患者中均顯著高于前列腺增生患者,且在血清EVs中的表達(dá)水平顯著高于在UEVs中的表達(dá)水平。ROC曲線和曲線下面積(Areaunder ROC curve,AUC)提示血清 PSA 聯(lián)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單獨(dú)檢測(cè)(AUC:0.805)的診斷效能,且UEVsmiR-145在Gleason Score≥8的患者中表達(dá)水平顯著高于Gleason Score≤7的患者。四、結(jié)論miR-145在前列腺癌患者和前列腺增生患者的UEVs和血清EVs中均有差異表達(dá),血清或尿液EVs中的miR-145聯(lián)合血清PSA均可提高血清PSA的診斷效能,UEVs miR-145是前列腺癌的潛在診斷標(biāo)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

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