本文關(guān)鍵詞： 膽囊癌 循環(huán)腫瘤細(xì)胞 診斷 預(yù)后 膽囊癌 慢性膽囊炎 外泌體microRNA 測序 診斷　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分膽囊癌循環(huán)腫瘤細(xì)胞的檢測及其臨床意義研究背景膽囊癌是惡性程度較高的膽道腫瘤,容易出現(xiàn)臨近臟器侵犯和轉(zhuǎn)移,預(yù)后極差。外周血循環(huán)中存在循環(huán)腫瘤細(xì)胞是目前認(rèn)為與轉(zhuǎn)移密切相關(guān)的重要因素之一。因此在膽囊癌病人的外周血中檢測CTCs對患者病情的評估,治療方案的選擇和預(yù)后判斷有著重要的指導(dǎo)意義。目的研究膽囊癌患者外周血CTCs情況及其臨床應(yīng)用。方法對2014年12月至2016年12月在我院腹腔鏡科住院的80例膽囊病變患者術(shù)前及術(shù)后采集外周靜脈血,檢測CTCs情況。采血方法為采集空腹肘正中靜脈血5ml。應(yīng)用武漢友芝友公司生產(chǎn)的CTC異常細(xì)胞分離染色儀分離和富集CTCs,并運用專利技術(shù)對CTCs進行染色鑒定。同時使用CK8/CK18/CK19/CK133/CD45/DAPI免疫熒光染色結(jié)果鑒定CTCs。并回顧性分析患者的臨床資料(臨床病理特征、治療情況、生存隨訪),統(tǒng)計分析CTCs與預(yù)后生存的關(guān)系。結(jié)果共對80例膽囊病變進行外周血分離,術(shù)后病理證實63例為膽囊癌,CTCs陽性者50例,陽性率79.4%(50/63),CTCs數(shù)量分布于0~19個/5ml,中位值6個/5ml,平均值6.43±0.65個。CTM陽性組11例(均合并CTC陽性),陽性率17.5%(11/63)。17例為膽囊良性病變,CTC陽性0例,陽性率0%(0/17)。63例膽囊癌患者43例得到根治,TNM分期II期2例,III期27例,IV期34例。對患者進行定期隨訪,隨訪日期截止至2017年03月01日,隨訪率達(dá)到100%,隨訪時間1~26個月。累計0.5,1,2年生存分別為77.8%,49.5%,36.1%,中位生存期11.9月。CTCs和患者TNM分期、肝侵犯及CA199水平有相關(guān)性。CTCs也是膽囊癌預(yù)后多因素分析的獨立危險因素,并可用于病情治療效果的判斷和監(jiān)測病情進展。合并CTM陽性的CTCs陽性組患者比單純CTCs陽性預(yù)后更差,中位生存期分別為10.2月VS 3.4月。結(jié)論應(yīng)用CTC異常細(xì)胞分離染色儀結(jié)合免疫熒光的方法捕獲和鑒定膽囊癌外周血CTCs是可行的。CTCs是膽囊癌診斷、病情監(jiān)測和預(yù)后預(yù)測的重要指標(biāo),在膽囊癌的臨床診治中具有較大的應(yīng)用價值。第二部分基于外泌體micro RNA測序應(yīng)用于膽囊癌早期診斷研究背景膽囊癌是膽道系統(tǒng)最常見的惡性腫瘤,近年來發(fā)病率上升較快。我國膽囊癌相對高發(fā),其日益成為嚴(yán)重威脅廣大人民群眾生命的惡性疾病。由于缺乏有效的早期診斷策略,多數(shù)患者確診時已為晚期,失去了根治性切除的手術(shù)機會,術(shù)后的5年生存率不到5%,膽囊癌被認(rèn)為是侵襲性和致死性最高的惡性腫瘤之一。因此,膽囊癌的早期診斷和早期治療就顯得尤其重要,尋找早期膽囊癌特異性的診斷標(biāo)志物具有重要的臨床意義。目的尋找膽囊癌早期診斷的特異性標(biāo)志物及臨床應(yīng)用。方法通過體液活檢方法,收集并分析膽囊癌(Tis N0M0或T1N0M0期)與慢性膽囊炎患者各10例,深度測序比較循環(huán)血中外泌體(exosomes)中micro RNAs的改變,以期發(fā)現(xiàn)膽囊癌早期診斷的特異性標(biāo)志物。結(jié)果20例患者均能成功提取出Exosomes micro RNAs,順利建庫測序12例,成功率60%,其中6例為膽囊癌組,6例為慢性膽囊炎組。在當(dāng)前讀取深度,共檢測出615種外泌體micro RNA,其中186種差異表達(dá)有統(tǒng)計學(xué)意義。相比于慢性膽囊炎組,膽囊癌組患者外泌體內(nèi)容物上調(diào)的差異表達(dá)micro RNA共56種,下調(diào)的差異表達(dá)micro RNA共130種。成功對差異表達(dá)micro RNA進行靶基因預(yù)測及GO、KEGG功能分析。結(jié)論我們的研究發(fā)現(xiàn)外泌體中含有豐富的micro RNA,應(yīng)用測序的方法可從外泌體中篩選出膽囊癌早期診斷的特異性標(biāo)志物,發(fā)現(xiàn)上調(diào)的差異micro RNA共56種,下調(diào)的差異micro RNA共130種,并對上調(diào)最顯著的mi R-552-3p進行靶基因預(yù)測及GO、KEGG功能分析。該方法無創(chuàng),對病人損傷小,應(yīng)用方便,具有較為廣闊的用于膽囊癌早期診斷的潛在應(yīng)用價值。
[Abstract]:The first part of the detection of circulating tumor cells of gallbladder carcinoma and its clinical significance in the research background of gallbladder carcinoma is highly malignant tumor of biliary tract, prone to adjacent organ invasion and metastasis and poor prognosis. There are circulating tumor cells in peripheral blood is currently considered one of the most important factors associated with metastasis. Therefore the detection of peripheral blood CTCs in gallbladder cancer patients in assessment of patients with the disease, has an important guiding significance and prognosis of treatment. CTCs of peripheral blood of patients with gallbladder cancer and clinical application. Methods from December 2014 to December 2016 hospitalized in our hospital 80 cases of laparoscopic cholecystectomy in patients with preoperative and postoperative peripheral vein blood, detection of CTCs. CTC method for separation of abnormal cells blood fasting elbow venous blood 5ml. application of Wuhan youzhiyou company production separation and dyeing apparatus The enrichment of CTCs, and use the patent technology of CTCs staining. At the same time using CK8/CK18/CK19/CK133/CD45/DAPI immunofluorescence staining and identification of CTCs. were analyzed retrospectively (clinical and pathological features, treatment, follow-up, survival) between the survival statistic analysis of CTCs and pre. Results a total of 80 cases of gallbladder lesions were isolated from peripheral blood, postoperative pathology confirmed 63 cases of gallbladder carcinoma, 50 cases were CTCs positive, the positive rate was 79.4% (50/63), the number of CTCs located on 0~19 /5ml, the median value of 6 /5ml, the average value of 6.43 + 0.65.CTM positive group (11 cases with CTC positive), the positive rate was 17.5% (11/63).17 cases of gallbladder benign lesions, 0 cases were CTC positive, the positive rate was 0% (0/17).63 cases 43 cases were cure, 2 cases of TNM stage II, 27 stage III cases, 34 cases of stage IV. Regular follow-up of patients, follow-up date up to 03 2017 01, follow-up rate reached 100%, 闅忚鏃墮棿1~26涓湀.绱0.5,1,2騫寸敓瀛樺垎鍒負(fù)77.8%,49.5%,36.1%,涓綅鐢熷瓨鏈，

本文編號：1502374