胆囊癌外周血循环肿瘤细胞和外泌体的检测及其临床应用

发布时间：2018-02-11 06:07

  本文关键词： 胆囊癌 循环肿瘤细胞 诊断 预后 胆囊癌 慢性胆囊炎 外泌体microRNA 测序 诊断　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：第一部分胆囊癌循环肿瘤细胞的检测及其临床意义研究背景胆囊癌是恶性程度较高的胆道肿瘤,容易出现临近脏器侵犯和转移,预后极差。外周血循环中存在循环肿瘤细胞是目前认为与转移密切相关的重要因素之一。因此在胆囊癌病人的外周血中检测CTCs对患者病情的评估,治疗方案的选择和预后判断有着重要的指导意义。目的研究胆囊癌患者外周血CTCs情况及其临床应用。方法对2014年12月至2016年12月在我院腹腔镜科住院的80例胆囊病变患者术前及术后采集外周静脉血,检测CTCs情况。采血方法为采集空腹肘正中静脉血5ml。应用武汉友芝友公司生产的CTC异常细胞分离染色仪分离和富集CTCs,并运用专利技术对CTCs进行染色鉴定。同时使用CK8/CK18/CK19/CK133/CD45/DAPI免疫荧光染色结果鉴定CTCs。并回顾性分析患者的临床资料(临床病理特征、治疗情况、生存随访),统计分析CTCs与预后生存的关系。结果共对80例胆囊病变进行外周血分离,术后病理证实63例为胆囊癌,CTCs阳性者50例,阳性率79.4%(50/63),CTCs数量分布于0~19个/5ml,中位值6个/5ml,平均值6.43±0.65个。CTM阳性组11例(均合并CTC阳性),阳性率17.5%(11/63)。17例为胆囊良性病变,CTC阳性0例,阳性率0%(0/17)。63例胆囊癌患者43例得到根治,TNM分期II期2例,III期27例,IV期34例。对患者进行定期随访,随访日期截止至2017年03月01日,随访率达到100%,随访时间1~26个月。累计0.5,1,2年生存分别为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水平有相关性。CTCs也是胆囊癌预后多因素分析的独立危险因素,并可用于病情治疗效果的判断和监测病情进展。合并CTM阳性的CTCs阳性组患者比单纯CTCs阳性预后更差,中位生存期分别为10.2月VS 3.4月。结论应用CTC异常细胞分离染色仪结合免疫荧光的方法捕获和鉴定胆囊癌外周血CTCs是可行的。CTCs是胆囊癌诊断、病情监测和预后预测的重要指标,在胆囊癌的临床诊治中具有较大的应用价值。第二部分基于外泌体micro RNA测序应用于胆囊癌早期诊断研究背景胆囊癌是胆道系统最常见的恶性肿瘤,近年来发病率上升较快。我国胆囊癌相对高发,其日益成为严重威胁广大人民群众生命的恶性疾病。由于缺乏有效的早期诊断策略,多数患者确诊时已为晚期,失去了根治性切除的手术机会,术后的5年生存率不到5%,胆囊癌被认为是侵袭性和致死性最高的恶性肿瘤之一。因此,胆囊癌的早期诊断和早期治疗就显得尤其重要,寻找早期胆囊癌特异性的诊断标志物具有重要的临床意义。目的寻找胆囊癌早期诊断的特异性标志物及临床应用。方法通过体液活检方法,收集并分析胆囊癌(Tis N0M0或T1N0M0期)与慢性胆囊炎患者各10例,深度测序比较循环血中外泌体(exosomes)中micro RNAs的改变,以期发现胆囊癌早期诊断的特异性标志物。结果20例患者均能成功提取出Exosomes micro RNAs,顺利建库测序12例,成功率60%,其中6例为胆囊癌组,6例为慢性胆囊炎组。在当前读取深度,共检测出615种外泌体micro RNA,其中186种差异表达有统计学意义。相比于慢性胆囊炎组,胆囊癌组患者外泌体内容物上调的差异表达micro RNA共56种,下调的差异表达micro RNA共130种。成功对差异表达micro RNA进行靶基因预测及GO、KEGG功能分析。结论我们的研究发现外泌体中含有丰富的micro RNA,应用测序的方法可从外泌体中筛选出胆囊癌早期诊断的特异性标志物,发现上调的差异micro RNA共56种,下调的差异micro RNA共130种,并对上调最显著的mi R-552-3p进行靶基因预测及GO、KEGG功能分析。该方法无创,对病人损伤小,应用方便,具有较为广阔的用于胆囊癌早期诊断的潜在应用价值。
[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�挎椂闂�1~26涓�鏈�.绱�璁�0.5,1,2骞寸敓瀛樺垎鍒�涓�77.8%,49.5%,36.1%,涓�浣嶇敓瀛樻湥�

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