本文關(guān)鍵詞：血清microRNA在非小細胞肺癌輔助診斷和化療效果評估中的應(yīng)用研究　出處：《北京市結(jié)核病胸部腫瘤研究所》2015年博士論文　論文類型：學位論文

  更多相關(guān)文章： 非小細胞肺癌 微小RNA 實時定量聚合酶鏈反應(yīng) 腫瘤標志物 早期診斷 晚期非小細胞肺癌 微小RNA 實時定量聚合酶鏈反應(yīng) 腫瘤標志物 療效評估

【摘要】：第一部分血清micro RNA在非小細胞肺癌輔助診斷中的臨床應(yīng)用研究研究背景肺癌是當今世界最主要的癌癥之一,每年大概有130萬人死于肺癌,其死亡率居各種惡性腫瘤之首,并且75-80%的肺癌患者為非小細胞肺癌(NSCLC)。由于早期NSCLC患者無明顯的臨床癥狀,因此大約75%的患者被確診時已經(jīng)屬于晚期NSCLC,失去了手術(shù)治療的最佳時機。缺乏有效的NSCLC早期診斷手段是NSCLC患者高死亡率的主要原因,早發(fā)現(xiàn)、早治療對于NSCLC患者是非常必要的。血液腫瘤標志物在腫瘤篩查中有著重要的臨床價值,并且具有取材方便、創(chuàng)傷性小、價格相對低廉、容易復檢等優(yōu)點。理想的腫瘤標志物在臨床診斷、預(yù)后評估和療效監(jiān)測等方面有著非常好的臨床應(yīng)用價值。血清微小RNA(micro RNA,mi RNA)作為新型的腫瘤標志物是當今研究的熱點。mi RNA是一類內(nèi)源性高度保守的非編碼小RNA,長度約18-25個核苷酸,主要通過轉(zhuǎn)錄后機制調(diào)控靶基因的翻譯或表達。有大量的研究表明,mi RNA參與各種類型腫瘤基因的表達和癌癥的發(fā)生、發(fā)展,包括肺癌、乳腺癌、胃癌和直腸癌等。檢測mi RNA的表達水平在癌癥的診斷、治療和預(yù)后判斷中有非常大的潛在臨床應(yīng)用價值。已經(jīng)有研究證明,mi RNA非常穩(wěn)定地存在于人類血漿和血清中,并且不受RNA內(nèi)切酶活性的影響,非常適合作為癌癥診斷或預(yù)后判斷的血液腫瘤標志物。研究目的本研究通過檢測NSCLC患者、肺部良性疾病患者和健康人對照血清中mi RNA的表達情況,探討血清mi RNA表達水平變化在NSCLC輔助診斷中的臨床應(yīng)用價值。研究方法1.收集2012年8月至2013年10月首都醫(yī)科大學附屬北京胸科醫(yī)院120例NSCLC患者、45例肺部良性疾病患者和45名健康對照者血清5ml(所有血清樣本采血前均未進行任何治療),然后應(yīng)用micro RNA提取試劑盒提取血清總micro RNA。2.選取NSCLC患者和健康對照者血清標本各3例,采用人類血清micro RNA One Array#174;基因芯片進行mi RNA表達譜分析,篩選在NSCLC患者與對照者血清中差異表達的mi RNA分子。3.所有血清mi RNA樣本先進行逆轉(zhuǎn)錄反應(yīng),然后采用實時熒光定量聚合酶鏈反應(yīng)(q RT-PCR)技術(shù)在120例NSCLC患者、45例肺部良性疾病患者和45名健康對照者樣本中對差異表達的血清mi RNA分子進行獨立驗證。應(yīng)用mi R-103做為內(nèi)部參考基因,并對所有血清mi RNA樣本的q RT-PCR實驗數(shù)據(jù)進行標準化。4.應(yīng)用受試者工作曲線(ROC曲線)分析血清mi RNA在NSCLC中的敏感性和特異性,并與其它臨床常用腫瘤標志物,如癌胚抗原(CEA)以及細胞角質(zhì)蛋白(CK)-3A9進行比較,評價血清mi RNA在NSCLC中的臨床診斷價值。研究結(jié)果1.血清micro RNA基因芯片表達譜分析結(jié)果顯示,在NSCLC血清中上調(diào)的mi RNA有26個,下調(diào)的mi RNA有24個。應(yīng)用q RT-PCR方法對其中10個候選血清mi RNA進行了驗證,包括5個上調(diào)mi RNA(mi R-22、mi R-125b、mi R-197、mi R-19b、mi R-27b)和5個下調(diào)mi RNA(mi R-15b、mi R-16、mi R-25、mi R-205、mi R-155)。2.q RT-PCR實驗結(jié)果顯示,10個候選血清mi RNA均可檢測到有效表達。NSCLC患者血清中mi R-125b的表達水平顯著高于正常人和肺部良性疾病組,差異有統(tǒng)計學意義(Z=2.238,P=0.025);NSCLC患者血清中mi R-22的表達水平顯著高于正常人和肺部良性疾病組,差異有統(tǒng)計學意義(Z=2.129,P=0.035);NSCLC患者血清中mi R-15b的表達水平顯著低于正常人和肺部良性疾病組,差異有統(tǒng)計學意義(Z=2.590,P=0.010)。血清mi R-125b在Ⅲ期+Ⅳ期中的表達水平顯著高于Ⅰ期+Ⅱ期,差異有統(tǒng)計學意義(Z=2.891,P=0.004);血清mi R-15b在Ⅲ期+Ⅳ期中的表達水平顯著低于Ⅰ期+Ⅱ期,差異有統(tǒng)計學意義(Z=2.081,P=0.040)。3.血清mi R-22對早期NSCLC(Ⅰ期+Ⅱ期)檢測的敏感性(43.5%)顯著高于CEA(21.7%),差異有統(tǒng)計學意義(χ2=4.946,P=0.026);血清mi R-15b在Ⅰ期+Ⅱ期NSCLC中檢測的敏感性(41.3%)顯著高于CEA,差異有統(tǒng)計學意義(χ2=4.079,P=0.043)。血清mi R-125b在Ⅲ期+Ⅳ期NSCLC中檢測的敏感性(58.1%)顯著高于Ⅰ期+Ⅱ期(30.4%),差異有統(tǒng)計學意義(χ2=5.875,P=0.015)。4.ROC曲線分析顯示,血清mi R-22、mi R-125b和mi R-15b診斷NSCLC的ROC曲線下面積(AUC)分別為0.725(95%CI=0.623-0.827)、0.704(95%CI=0.601-0.808)、0.619(95%CI=0.536-0.702),這3種血清mi RNA對NSCLC的診斷價值均高于血清CEA(AUC=0.594,95%CI=0.482-0.707)。結(jié)論1.本研究通過血清micro RNA One Array基因芯片篩選出的10種差異表達的mi RNA分子,又經(jīng)過q RT-PCR方法的驗證,結(jié)果表明應(yīng)用micro RNA One Array基因芯片篩選血清mi RNA差異表達譜是一種可行、可靠的方法。2.本研究結(jié)果顯示,NSCLC患者血清中mi R-125b和mi R-22表達顯著上調(diào),mi R-15b表達顯著下調(diào),表明血清mi RNA作為篩查NSCLC的腫瘤標志物具有潛在的臨床應(yīng)用價值。3.本研究發(fā)現(xiàn)血清mi R-22和mi R-15b在早期NSCLC(Ⅰ期+Ⅱ期)中的敏感性顯著高于CEA,表明血清mi R-22和mi R-15b有可能成為NSCLC早期診斷的參考指標。4.NSCLC患者血清中mi RNA差異性表達可能與NSCLC的形成和發(fā)展有關(guān),血清mi RNA的檢測對于NSCLC的輔助診斷和早期診斷有一定的臨床應(yīng)用價值。第二部分血清micro RNA在非小細胞肺癌化療效果評估中的臨床應(yīng)用研究研究背景由于早期非小細胞肺癌(NSCLC)患者無明顯的臨床癥狀,大多數(shù)NSCLC患者在臨床確診時就已經(jīng)屬于晚期,失去了手術(shù)治療的最佳時機。目前,針對晚期NSCLC患者的治療主要有化學治療或放射治療。實踐證明,聯(lián)合化療能有效地提高晚期肺癌患者的生存率。但是,一線化療方案治療的有效率只有30%左右,NSCLC患者對化療藥物的耐藥性不僅會導致化療失敗,并且嚴重損害了患者的免疫系統(tǒng),使患者不能及時地得到其它更好的治療方案。因此,對化療患者進行療效監(jiān)測是非常必要的。目前,臨床上通常在NSCLC患者進行3-4個周期后采用實體瘤評價法對化療效果進行評估,根據(jù)腫瘤體積或直徑的改變來評估化療效果的好壞。但是,實體瘤評價法不僅評價時間長,并且需要費用較高的影像學資料支持。實體瘤評價法在實際應(yīng)用中還有其它的一些局限性,比如有胸腔積液、彌漫性結(jié)節(jié)以及腫瘤邊界不清的NSCLC患者用這種方法容易導致不正確的評價結(jié)果。因此,目前迫切需要價格低廉、操作簡便的用于評價NSCLC化療效果的工具,這對于早期評估療效、及時改變治療方案以及NSCLC的個體化治療具有非常大臨床價值。目前,血液腫瘤標志物已經(jīng)廣泛應(yīng)用于NSCLC的預(yù)后評估和療效監(jiān)測,比如癌胚抗原(CEA)、特異性神經(jīng)云烯醇化酶(NSE)、細胞角質(zhì)蛋白(Cyfra21-1)等。血清微小RNA(micro RNA,mi RNA)作為新的腫瘤標志物,在NSCLC預(yù)后評估和療效監(jiān)測中臨床應(yīng)用方面的研究剛剛起步。有大量的研究表明,mi RNA參與各種類型腫瘤基因的表達和癌癥的發(fā)生、發(fā)展,檢測mi RNA的表達水平在癌癥的治療和預(yù)后判斷中有非常大的潛在臨床應(yīng)用價值。研究目的本研究通過檢測晚期(Ⅲ期+Ⅳ期)NSCLC患者化療前和化療后血清中miRNA的表達量變化情況,分析血清mi RNA濃度變化與化療效果是否存在關(guān)聯(lián),探討血清mi RNA表達水平變化在NSCLC化療效果評估中的臨床應(yīng)用價值。研究方法1.收集2012年8月至2013年10月首都醫(yī)科大學附屬北京胸科醫(yī)院74例晚期NSCLC患者化療前和化療后2周期血清各5ml,然后應(yīng)用mi RNA提取試劑盒提取血清總mi RNA。2.總計144例血清mi RNA樣本先進行逆轉(zhuǎn)錄反應(yīng),然后采用實時熒光定量聚合酶鏈反應(yīng)(q RT-PCR)技術(shù)檢測在本課題第一部分篩選并驗證的在NSCLC患者中異常表達的血清mi RNA分子(mi R-125b、mi R-22、mi R-15b)的表達量。應(yīng)用mi R-103做為內(nèi)部參考基因,并對144例血清mi RNA樣本的q RT-PCR實驗數(shù)據(jù)進行標準化。3.化療效果評估采用世界衛(wèi)生組織(WHO)推薦的實體瘤評價法,評價結(jié)果分為:完全緩解(CR),部分緩解(PR),疾病穩(wěn)定(SD)和疾病進展(PD)。4.分析晚期NSCLC患者化療前和化療后血清中mi RNA的表達水平變化,評價血清mi RNA在晚期NSCLC化療效果評估中的臨床應(yīng)用價值。研究結(jié)果1.74例晚期NSCLC患者經(jīng)過2個周期的化療后,應(yīng)用實體瘤療效評價法(RECIST)對化療效果進行評估:完全緩解0例,部分緩解34例(占45.9%),疾病穩(wěn)定33例(占44.6%)和疾病進展7例(占9.5%)。2.與化療前比較,晚期(Ⅲ期+Ⅳ期)NSCLC患者經(jīng)過2個周期的化療后,血清中mi R-125b的表達量明顯降低,差異有統(tǒng)計學意義(Z=2.196,P=0.028);而血清中mi R-15b的表達量明顯升高,差異有統(tǒng)計學意義(Z=2.650,P=0.008);血清中mi R-22的表達量無明顯變化,差異無統(tǒng)計學意義(P0.05)。3.與化療前比較,晚期NSCLC患者經(jīng)過2個周期的化療后,化療敏感組患者(CR+PR)血清中mi R-125b的表達量明顯降低,差異有統(tǒng)計學意義(Z=2.128,P=0.033);化療敏感組患者血清中mi R-15b的表達量明顯升高,差異有統(tǒng)計學意義(Z=2.794,P=0.005);而化療不敏感組患者(SD+PD)血清中mi R-125b和mi R-15b的表達量無明顯變化,差異無統(tǒng)計學意義(P0.05)。4.研究結(jié)果顯示,晚期NSCLC患者化療效果與化療前血清中mi RNA的表達量密切相關(guān)聯(lián)。在血清mi R-125b呈高表達的NSCLC患者中,有34.9%的患者對化療敏感(CR+PR);而在血清mi R-125b呈低表達的NSCLC患者中,有51.6%的患者對化療敏感,這種差異具有統(tǒng)計學意義(χ2=5.879,P=0.015)。在血清mi R-15b呈低表達的NSCLC患者中,有37.5%的患者對化療敏感;而在血清mi R-15b呈高表達的NSCLC患者中,有52.9%的患者對化療敏感,這種差異有統(tǒng)計學意義(χ2=4.795,P=0.029)。結(jié)論1.本研究結(jié)果顯示,晚期NSCLC患者化療前和化療后血清中mi R-125b和mi R-15b的表達水平有顯著的變化,并且與化療效果評價相關(guān)聯(lián),表明血清mi RNA的檢測對于晚期NSCLC的化療療效評價具有潛在的臨床應(yīng)用價值。2.晚期NSCLC患者化療前血清mi RNA的表達水平與療效評價密切相關(guān),根據(jù)晚期NSCLC患者化療前血清mi R-125b和mi R-15b的表達水平,可以用來判斷化療患者的預(yù)后情況。
[Abstract]:The first part of the clinical application of serum micro RNA background lung cancer in non small cell lung cancer diagnosis is one of the most important cancer of the world today, there are about 1 million 300 thousand people die of lung cancer each year, the mortality rate among all malignant tumors, and 75-80% lung cancer patients for non-small cell lung cancer (NSCLC). Since early NSCLC patients have no obvious clinical symptoms, about 75% of the patients were diagnosed as late NSCLC and lost the best time for surgical treatment. The lack of effective early diagnosis of NSCLC is the main cause of high mortality in NSCLC patients. Early detection and early treatment are essential for patients with NSCLC. Blood tumor markers have important clinical value in tumor screening, and have the advantages of convenient material, small trauma, relatively low price and easy reexamination. The ideal tumor markers are of great clinical value in clinical diagnosis, prognosis evaluation and therapeutic monitoring. As a new tumor marker, the serum RNA (micro RNA, MI RNA) is a hot spot of research. Mi RNA is a class of endogenous highly conserved non coded small RNA, with a length of about 18-25 nucleotides, which mainly regulate the translation or expression of target genes through post transcriptional mechanism. A large number of studies have shown that MI RNA participates in the expression of various types of tumor genes and the occurrence and development of cancer, including lung cancer, breast cancer, gastric cancer and rectal cancer. The detection of the expression level of MI RNA has a great potential clinical value in the diagnosis, treatment and prognosis of cancer. Studies have shown that MI RNA is very stable in human plasma and serum, and is not affected by the activity of RNA endonuclease. It is very suitable for cancer diagnosis or prognosis of blood tumor markers. The purpose of this study is to detect the expression of MI RNA in serum of patients with NSCLC, pulmonary benign diseases and healthy controls, and to explore the clinical application value of serum mi RNA expression level in NSCLC aided diagnosis. Methods 1. from August 2012 to October 2013 in Beijing Thoracic Hospital of Capital Medical University from 120 patients with NSCLC, 45 patients with benign lung disease and 45 healthy controls serum 5ml (all serum samples from the blood before them were without any treatment), and then apply the micro RNA extraction kit to extract serum total micro RNA. 2. select 3 serum samples from NSCLC patients and healthy controls. We used human serum micro RNA One Array#174, gene chip to analyze mi RNA expression profiles, and screen mi RNA molecules expressed in serum of NSCLC patients and controls. All 3. serum mi RNA samples before reverse transcription reaction, then by real-time fluorescence quantitative polymerase chain reaction (Q RT-PCR) in 120 NSCLC patients, 45 patients with benign lung disease and 45 healthy controls serum mi RNA expression of differences in the sample were validated independently. Mi R-103 was used as an internal reference gene and the Q RT-PCR experimental data of all serum mi RNA samples were standardized. 4. application of receiver operating characteristic curve (ROC curve) analysis of the sensitivity and specificity of serum mi RNA in NSCLC, and markers and other clinical common tumors, such as carcinoembryonic antigen (CEA) and cytokeratin (CK) -3A9 were compared to evaluate the clinical diagnostic value of serum mi RNA in NSCLC. Results 1. serum micro RNA gene chip expression profiles showed that there were 26 mi RNA up-regulated in NSCLC serum, and 24 down-regulated mi RNA. 10 candidate serum mi RNA were verified by Q RT-PCR method, including 5 upregulated mi RNA (MI R-22, MI R-125b, MI R-125b, MI, 5). The results of 2.q RT-PCR experiment showed that 10 candidate serum mi RNA could be effectively expressed. The expression level in the serum of patients with NSCLC mi R-125b was significantly higher than that in normal and benign lung disease group, the difference was statistically significant (Z=2.238, P=0.025); the expression level in the serum of patients with NSCLC mi R-22 was significantly higher than that in normal and benign lung disease group, the difference was statistically significant (Z=2.129, P=0.035); the expression level of serum NSCLC mi R-15b was significantly lower than that in normal and benign lung disease group, the difference was statistically significant (Z=2.590, P=0.010). The expression level of serum mi R-125b in stage III + IV is significantly higher than that in stage I + phase II (Z=2.891, P=0.004). The expression level of serum mi R-15b in stage III + IV is significantly lower than that in stage I + phase II, with statistically significant difference (Z=2.081, P=0.040). 3. serum mi R-22 in early NSCLC (stage I + II) detection sensitivity (43.5%) was significantly higher than that of CEA (21.7%), the difference was statistically significant (2=4.946, P=0.026); the sensitivity of serum mi R-15b in stage I + II detection in NSCLC (41.3%) was significantly higher than that of CEA, the difference was statistically meaning (x 2=4.079, P=0.043). The sensitivity of serum mi R-125b in stage III + IV NSCLC (58.1%) was significantly higher than that in phase I + II (30.4%), and the difference was statistically significant (x 2=5.875, P=0.015). 4.ROC curve analysis showed that the area of MI R-22, the MI curve of serum ROC R-125b and MI R-15b NSCLC (AUC) at diagnosis were 0.725 (95%CI=0.623-0.827), 0.704 (95%CI=0.601-0.808), 0.619 (95%CI=0.536-0.702), the value of these 3 kinds of serum mi RNA for diagnosis of NSCLC were higher in serum CEA (AUC=0.594,95%CI=0.482-0.707). Conclusion Mi expression of RNA 10 between the 1. through the study of serum micro RNA One Array gene chips were screened, and validated by Q RT-PCR method. The results show that the application of micro RNA One Array gene chips to screen the differences of serum Mi expression profile of RNA is a feasible and reliable method. 2., the results of this study showed that the expressions of MI R-125b and MI R-22 in NSCLC patients were significantly up-regulated, and the expression of MI R-15b was significantly down regulated, indicating that serum mi RNA was used as a tumor marker for screening NSCLC.
【學位授予單位】：北京市結(jié)核病胸部腫瘤研究所
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R734.2

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