本文選題：原發(fā)性肝癌 + 肝良性病��； 參考：《南方醫(yī)科大學》2017年博士論文

【摘要】：研究背景和目的原發(fā)性肝癌是世界上最常見的具有高發(fā)病率和死亡率的惡性腫瘤之一,手術(shù)切除是大多數(shù)原發(fā)性肝癌(primary liver cancer,PLC)的首選治療方式,然而術(shù)后復(fù)發(fā)是影響PLC預(yù)后的主要原因。目前對于PLC的復(fù)發(fā)監(jiān)測只要依靠B超、CT及血清AFP,這些傳統(tǒng)監(jiān)測手段是無法做到早期診斷復(fù)發(fā)。因此,發(fā)現(xiàn)具有早期診斷PLC復(fù)發(fā)的敏感性生物標志物至關(guān)重要。在過去的幾十年中,腫瘤患者外周血中循環(huán)腫瘤細胞的檢測已經(jīng)獲得越來越多的關(guān)注。因為外周血循環(huán)腫瘤細胞檢測是一種簡單、可重復(fù)和微創(chuàng)的操作,其可作為理想的取樣檢測手段。循環(huán)腫瘤細胞(circulating tumor cells,CTCs)是指存在于實體瘤外的腫瘤細胞。事實上,源自原發(fā)性腫瘤灶的具有高度侵襲性的腫瘤細胞不斷增殖,它們在離開原發(fā)腫瘤灶并進入外周血后成為具有侵襲和轉(zhuǎn)移潛能的循環(huán)腫瘤細胞。因此,近年來CTCs在腫瘤診斷,復(fù)發(fā)和轉(zhuǎn)移中的作用得到越來越多的研究。已有研究證實,上皮-間質(zhì)轉(zhuǎn)化是一種細胞形態(tài)轉(zhuǎn)化過程:細胞失去其上皮特征并獲得間充質(zhì)性質(zhì)。同時,間充質(zhì)細胞可能轉(zhuǎn)化為上皮表型細胞,從而促使腫瘤細胞轉(zhuǎn)移定值。許多研究已經(jīng)建立了上皮-間質(zhì)轉(zhuǎn)化和各型CTCs形成之間的聯(lián)系。然而,目前基于CTCs的檢測技術(shù)有數(shù)十種,其每種檢測技術(shù)的方法及原理各有不同,因而選取并深入研究肝癌CTCs檢測技術(shù)對于實施本項目起著關(guān)鍵作用。在本研究中,我們首先選取并確立了CanPatrolrM肝癌CTCs檢測技術(shù),明確其檢測技術(shù)的基本方法和原理,研究其檢測技術(shù)的靈敏度、特異度及可重復(fù)性;在此檢測技術(shù)的基礎(chǔ)上分析了 EMT表型CTCs在PLC和非惡性肝臟疾病(nonmalignant liver diseases,NLD)之間的差異,試圖提高PLC診斷的準確性。眾所周知,腫瘤復(fù)發(fā)和轉(zhuǎn)移是一個非常復(fù)雜的過程,而其血型轉(zhuǎn)移過程通常需要CTCs從原發(fā)性腫瘤灶進入外周血.并且由于外周血CTCs檢測是一種簡單,可重復(fù)和微創(chuàng)過程,因此在過去幾十年中,CTCs在腫瘤診斷、復(fù)發(fā)和轉(zhuǎn)移中的功能一直在積極研究。但是對于各亞型循環(huán)腫瘤細胞與腫瘤復(fù)發(fā)的研究目前卻甚少,本研究通過檢測PLC患者外周血中的各亞型CTCs,分析其與PLC復(fù)發(fā)的關(guān)系,初步探討各亞型CTCs檢測在早期預(yù)測PLC復(fù)發(fā)中的臨床價值。方法:采集PLC患者外周血,利用多重原位RNA雜交檢測技術(shù)分離檢測外周血中的CTCs,并通過細胞上皮型標志物(EpCAM、CK8,18,19)及間質(zhì)型標志物(Vimentin、Twist)對CTCs進一步分型,第一組共采集73例入院診斷為肝占位患者的5-mL外周血用來檢測CTCs,其中最后入組45位原發(fā)性肝癌患者(年齡中位數(shù):55,范圍:18至77)和13位肝良性病患者(年齡中位數(shù):50,范圍:35至74)。第二組選取2014年3月至2016年3月期間行根治性切除術(shù)的原發(fā)性肝癌患者62例(男:58例,女:4例;年齡:median:55,rang:30～79)行前瞻性研究,均抽取5-mL外周血用來檢測CTCs。我們通過采用CanPatro1TM CTCs富集技術(shù)來檢測外周血中的EMT表型CTCs。通過使用該技術(shù),判斷并計數(shù)不同表型腫瘤細胞,研究其在PLC中診斷及術(shù)后預(yù)測復(fù)發(fā)的臨床價值。統(tǒng)計學處理:所有統(tǒng)計計算用SPSS v21.0進行,P值0.05被認為具有統(tǒng)計學意義。結(jié)果:我們應(yīng)用CanPatrolrM系統(tǒng)將檢測到的外周血中的EMT表型CTCs分成以下三類:上皮型CTCs(上皮標記染色),間質(zhì)型CTCs(間質(zhì)標記染色),混合型CTCs(上皮和間質(zhì)標記染色)同時,為了檢測該技術(shù)分離鑒定CTCs的回收效率及敏感性,我們將0,10,50,100,200個培養(yǎng)的腫瘤細胞加入健康人的5mL外周血樣本中,與實際加入的腫瘤細胞數(shù)目進行對比分析,結(jié)果顯示外周血中分別加入0,10,50,100,200個HepG2細胞的平均檢出數(shù)為0,8±1.41,43.33±2.49,85±3.56,182.33±4.99,線性回歸模型分析得到檢出的CTCs與加入的腫瘤細胞數(shù)量具有明顯線性相關(guān)性(r2=0.999)。通過應(yīng)用該技術(shù)對PLC及NLD的診斷性研究,首先入組73例診斷為肝占位的患者中;3例患者未行進一步診斷,10名患者最終被診斷為其他惡性腫瘤肝轉(zhuǎn)移,45名被診斷為原發(fā)性肝癌,13名被診斷為肝良性病;最終,我們發(fā)現(xiàn)除了上皮型CTCs,其它各類型EMT表型CTCs計數(shù)在PLC中顯著高于NLD,差異具有統(tǒng)計學意義(P0.05)。在深入研究EMT表型的CTCs在PLC與復(fù)發(fā)的關(guān)系中,我們?nèi)虢M了62例經(jīng)根治性手術(shù)治療的PLC患者,通過隨訪,26例最終診斷為復(fù)發(fā),36例診斷為未復(fù)發(fā)。復(fù)發(fā)組與未復(fù)發(fā)組CTCs總數(shù)(中位數(shù):6vs2.5),上皮型CTCs(中位數(shù):0.5vs0),混合型CTCs(中位數(shù):3vs1),間質(zhì)型CTCs(中位數(shù):1vs0)。最終發(fā)現(xiàn);復(fù)發(fā)組CTCs總數(shù)、間質(zhì)型CTCs、混合型CTCs計數(shù)顯著高于未復(fù)發(fā)患者。為了確定與復(fù)發(fā)相關(guān)的各型CTCs的截點值,行ROC曲線分析定義各型CTCs的截點值:CTCs總數(shù)≥4為陽性,間質(zhì)型CTCs≥1為陽性,混合型CTCs≥3為陽性.經(jīng)多因素cox回歸發(fā)現(xiàn),門脈癌栓(HR=2.905,P = 0.023)及間質(zhì)型 CTCs(HR=3.453,P = 0.007)陽性為復(fù)發(fā)的獨立危險因素。K-M檢驗進一步研究間質(zhì)型CTCs與復(fù)發(fā)時間的關(guān)系,結(jié)果發(fā)現(xiàn)間質(zhì)型CTCs陽性患者術(shù)后無瘤生存時間顯著縮短(P0.001)。結(jié)論:1、研究證明了EMT現(xiàn)象存在于原發(fā)性肝癌患者外周血CTCs中;2、外周血中間質(zhì)型CTCs和混合型CTCs計數(shù)將有望成為診斷原發(fā)性肝癌的新型生物標記物;3、原發(fā)性肝癌患者術(shù)后外周血中間質(zhì)型CTCs陽性能增加復(fù)發(fā)的風險。間質(zhì)型CTCs可以作為監(jiān)測原發(fā)性肝癌預(yù)后的生物指標;4、門脈癌栓及間質(zhì)型CTCs陽性為復(fù)發(fā)的獨立危險因素,且間質(zhì)型CTCs對復(fù)發(fā)結(jié)局的預(yù)測能力大于門脈癌栓。
[Abstract]:Background and objective: primary hepatocellular carcinoma is one of the most common with high morbidity and mortality of malignant tumors in the world, surgical resection is the most primary liver cancer (primary liver cancer, PLC) the preferred treatment, but recurrence is the main factor influencing the prognosis of PLC patients. The recurrence monitoring as long as PLC by B-ultrasound, CT and AFP in serum, the traditional monitoring methods is unable to achieve the early diagnosis of recurrence. Therefore, it is important to find sensitive biomarkers for early diagnosis of the recurrence of PLC. In the past few decades, the tumor in the peripheral blood of patients with the detection of circulating tumor cells has received more and more attention. Because the detection of circulating tumor cells the peripheral blood is a simple, repeatable and minimally invasive operation, which can be used as sampling detection means. The ideal of circulating tumor cells (circulating tumor cells, CTCs) is present in the Solid tumors of tumor cells. In fact, highly invasive tumor cells from the primary tumor to proliferate, they leave the primary tumor and enter the peripheral blood circulating tumor cells has become invasive and metastatic potential. Therefore, in recent years CTCs in tumor diagnosis, recurrence and metastasis the role of more and more research. Studies have demonstrated that epithelial mesenchymal transition is a cell transformation process: cells lose their epithelial and mesenchymal features. At the same time, mesenchymal cells may be transformed into cells from epithelial phenotype, and promote tumor cell metastasis value. Many studies have been established between epithelial mesenchymal transition and the formation of CTCs. However, at present, based on several detection techniques of CTCs ten, the method of each kind of detection technology and principle are different, thus the selection and study of liver cancer CTCs Detection technology plays a key role in the implementation of the project. In this study, we firstly selected and established CanPatrolrM hepatoma CTCs detection technology, the detection technology of the basic method and principle, the sensitivity of detection, specificity and repeatability; based on the analysis of the EMT detection technology in CTCs phenotype PLC and non malignant liver disease (nonmalignant liver, diseases, NLD) the difference between, trying to improve the diagnostic accuracy of PLC. As everyone knows, the recurrence and metastasis of tumor is a very complicated process, and the blood transfer process usually takes CTCs from the primary tumor into peripheral blood. And the detection of CTCs in peripheral blood is a simple, minimally invasive and repeatable process, so in the past few decades, CTCs in the diagnosis of tumor recurrence and metastasis, the function has been active in research. But for each subtype of circulating tumor Study on cell and tumor recurrence is little, the study of each CTCs subtype by detection of PLC in the peripheral blood of patients and analyze its relationship with the recurrence of PLC, preliminary study of various subtypes of CTCs testing to predict the clinical value of PLC in early recurrence. Methods: collected peripheral blood of PLC patients, the use of multiple in situ RNA hybridization detection technology of separation and detection of CTCs in peripheral blood, and the epithelial cell type markers (EpCAM, CK8,18,19) and mesenchymal markers (Vimentin, Twist) of CTCs furtheranalysis, the first group were collected from 73 patients diagnosed as liver lesions in patients with peripheral blood 5-mL to detect CTCs the last group of 45 patients with primary liver cancer (median age: 55, range: 18 to 77) and 13 patients with benign liver disease (median age: 50, range: 35 to 74). The second group from March 2014 to March 2016 during radical resection of primary liver cancer patients (62 cases Male: 58 cases, female: 4 cases; age: median:55, rang:30 ~ 79) study prospectively, all were extracted from 5-mL peripheral blood to detect CTCs. by adopting CTCs concentration technique CanPatro1TM to detect the phenotype of EMT CTCs. in the peripheral blood by using this technology, determine and count the different phenotypes of tumor cells, prediction to study the clinical value of PLC in diagnosis of recurrence and postoperative. Statistical analysis: all statistical calculation was carried out with SPSS v21.0, the P value of 0.05 was considered statistically significant. Results: EMT phenotype of peripheral blood CTCs we used CanPatrolrM detected in the system will be divided into the following three categories: epithelial type CTCs (epithelial staining (CTCs), mesenchymal stromal staining), mixed CTCs (epithelial and stromal staining) at the same time, in order to detect the recovery efficiency and sensitivity of the isolation and identification of CTCs technology, we will 0,10,50100200 cultured tumor cells with health Blood samples the 5mL, compared with the number of tumor cells with actual, the results showed that the addition of 0,10,50100200 HepG2 cells in the peripheral blood of the average detection number of 0,8 + 1.41,43.33 + 2.49,85 + 3.56182.33 + 4.99, linear regression analysis was CTCs and added the number of cells with obvious linear correlation get (r2=0.999). By studying the diagnostic application of the technology of PLC and NLD, into the first group of 73 cases diagnosed as liver lesions in patients; 3 patients underwent no further diagnosis, 10 patients were diagnosed as other malignant tumors of liver metastasis, 45 were diagnosed with primary liver cancer, 13 men who were diagnosed with benign liver diseases; finally, we found that in addition to epithelial type CTCs, other types of EMT phenotype CTCs count in PLC was significantly higher than that of NLD, the difference was statistically significant (P0.05). On the research of EMT in PLC and CTCs phenotype 澶嶅彂鐨勫叧緋諱腑,鎴戜滑鍏ョ粍浜，

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