【作者】 余春开；

【导师】 陈杰；

【作者基本信息】 北京协和医学院 ， 病理学与病理生理学， 2012， 博士

【摘要】 胰腺导管腺癌(pancreatic ductal adenocarcinoma, PDAC)是一种高度侵袭性的肿瘤,临床症状出现晚,难以早期发现,多数病例发现时已属晚期,是实性肿瘤中预后最差的。如果在胰腺非浸润性癌前病变时期进行有效的干预,将大大提高胰腺癌的生存率。胰腺导管上皮内瘤变(pancreatic intraepithelial neoplasia, PanIN)是胰腺导管腺癌最常见最重要的癌前病变。miRNA是一种内源性非编码单链小分子RNA,长度19-24个核苷酸,成熟miRNA发挥癌基因或抑癌基因的作用。本课题组之前的研究中采用芯片技术及定量PCR均证实hsa-miR-96及hsa-miR-217在胰腺导管腺癌组织中呈低表达,功能研究提示hsa-miR-96及hsa-miR-217发挥抑癌基因的作用。因此我们提出了这样一个问题：hsa-miR-96及hsa-miR-217在胰腺导管腺癌最重要的癌前病变(胰腺导管上皮内瘤变)中是否就出现了表达的异常?hsa-miR-96及hsa-miR-217在胰腺导管腺癌形成过程中是否起到一定的作用?对于这些问题目前还没有文献报道。本研究在课题组之前研究结果的基础上用核酸锁定原位杂交(Locked Nucleic Acid In situ Hybridization LNA-ISH)的方法检测了hsa-miR-96及hsa-miR-217在胰腺导管腺癌及胰腺导管上皮内瘤变的定位表达情况,以此来了解hsa-miR-96及hsa-miR-217在胰腺导管腺癌癌变过程中的作用,以及在胰腺导管腺癌早期诊断中的潜在作用。目的：研究hsa-miR-96及hsa-miR-217在胰腺癌的癌变过程中的表达以及在胰腺导管腺癌诊断的潜在作用。方法：(1)收集2008年-2010年北京协和医院手术切除的胰腺石蜡标本,其中病理切片完整的191例,包括胰腺导管腺癌119例、慢性胰腺炎22例及其他的胰腺良恶性肿瘤47例,并进行随访,随访时间为17-50个月,失访2例。。对所有病例的临床资料及病理切片进行重新回顾整理,对PanIN病变进行分级,统计不同类型胰腺病变PanIN的发生率。(2)选取伴有胰腺导管上皮内瘤变的胰腺石蜡标本,包括45例胰腺导管腺癌、10例慢性胰腺炎及5例其他类型胰腺肿瘤。用核酸锁定原位杂交的方法检测miRNA (hsa-miR-96及hsa-miR-217)的表达。所有结果应用SPSS Statistics统计软件进行数据分析。结果：(1)PanIN总发生率在其他类型胰腺肿瘤周边组织、慢性胰腺炎及胰腺导管腺癌的癌旁胰腺组织逐渐升高(分别为24.0%,12/50；54.5%,12/22；70.6%,84/119),三者之间存在显著性差异。胰腺导管腺癌的癌旁胰腺组织中高级另PanIN的发生率(31.1%,37/119)明显高于慢性胰腺炎(0%,0/22)或其他类型良恶性肿瘤周边组织(2%,1/50))(P<0.05)。PanIN的发生率与年龄有相关性,经统计学分析不同年龄组的PanIN发生率有显著性差异(P<0.05)。PanIN随着年龄增加发生率升高。男女性别间的PanIN发生率没有显著性差异。(2)hsa-miR-96和hsa-miR-217的表达随着PanIN级别的升高而降低。hsa-miR-96在PanIN-1、PanIN-2、PanIN-3及导管腺癌中的表达分别为91.3%(22/23)、70.6%(12/17)、22.2%(4/18)及31.1%(14/45)。hsa-miR-217在PanIN--1、PanIN-2、PanIN-3及导管腺癌中的表达分别为95.7%(22/23)、70.6%(12/17)、27.8%(5/18)及42.2%(19/45)。hsa-miR-96及hsa-miR-217在PanIN-2/3中的表达与正常胰腺组织及PanIN-1的表达有统计学差异(P<0.01)。(3)hsa-miR-96及hsa-miR-217在胰腺导管腺癌肿瘤细胞的阳性率分别为31.1%及42.2%,而在对应癌旁胰腺组织及慢性胰腺炎中全部有阳性或弱阳性表达,差异具有显著性(P<0.05)。(4) hsa-miR-96及hsa-miR-217在胰腺导管腺癌间质纤维母细胞的表达率分别为22.2%和24.4%,较慢性胰腺炎中的77.8%和66.7%亦明显降低(P=0.003)。(5)hsa-miR-96及hsa-miR-217阴性表达与阳性表达之间Kaplan-Meier生存分析,阴性组与阳性组之间生存均无统计学差异。结论：(1)胰腺导管腺癌癌旁组织中发现PanIN特别是高级另IJPanIN的检出率明显高于慢性胰腺炎或其他类型良恶性肿瘤周边组织,支持PanIN是胰腺导管腺癌最常见的癌前病变。(2)在胰腺导管腺癌的癌前病变(PanIN-2/PanIN-3)发现hsa-miR-96及hsa-miR-217的异常表达,提示hsa-miR-96及hsa-miR-217可能在胰腺癌的形成过程中起到重要的作用、可能是胰腺癌早期诊断的潜在生物学标记物。(3) hsa-miR-96及hsa-miR-217在胰腺导管腺癌与慢性胰腺炎存在差异表达。(4)miRNA在胰腺导管腺癌及慢性胰腺炎中的表达差异不仅存在于上皮细胞,间质纤维母细胞间也存在显著差异,这可能为胰腺导管腺癌的微环境研究提供新思路。更多还原

【Abstract】 BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly invasive tumor which is difficult to be early detected because the clinical symptoms usually appear too late. Most cases are diagnosed at an advanced stage, and the prognosis is the worst among all solid tumors. If effective interventions could be provided during non-invasive precancerous period, the survival rate of pancreatic cancer would be greatly improved. Pancreatic intraepithelial neoplasia (PanIN) is the most common non-invasive precursor lesion of PDAC.MicroRNAs (miRNAs) is an endogenous non-coding single-stranded small RNA of19to24nucleotides(nt) in length that function as a post-transcriptional regulating molecule. Mature miRNAs can function as oncogenes or tumor suppressor genes. In recent years, many studies have confirmed that many miRNAs are up-regulated or down-regulated in PDAC. Previous studies in this group have both indicated that hsa-miR-96and hsa-miR-217are down-regulated in PDAC using microarray technology and quantitative PCR. hsa-miR-96and hsa-miR-217may function as tumor suppressor genes. Whether hsa-miR-96and hsa-miR-217have been down-regulated in the most important non-invasive precursor lesion(PanIN) of PDAC, and play a potential role in carcinogenesis of PDAC, which has not yet been reported in the literatur. Based on the previouse study in our group, in this study the expression of hsa-miR-96and hsa-miR-217in PanIN and PDAC were analyzed using the method of locked nucleic acid in situ hybridization (LNA-ISH), with chronic pancreatitis and normal pancreatic tissue as reference control tissues.Objective:To study the role of hsa-miR-96and hsa-miR-217in pancreatic ductal adenocarcinoma development and potential role in diagnosis of PDAC.Methods:(1)191cases of paraffin-embedded pancreatic specimens, including119cases of PDAC,22cases of chronic pancreatitis and47cases of other pancreatic tumors were collected from the archives of the Department of Pathology, Peking Union Medical College Hospital from the year2008-2010. Follow up was made, and the time of follow-up was17-50months. All the clinical data were reviewed. And all available slides were evaluated for the presence, grade of PanIN lesions using the four-tier classification.(2)Paraffin-embedded pancreatic specimens contain with PanIN, including45cases of PDAC,10cases of chronic pancreatitis and cases of other pancreatic tumors were selected in this study. The miRNAs hsa-miR-96and hsa-miR-217were analyzed using locked nucleic acid in situ hybridization (LNA-ISH). The differences of miRNA expression among sample sets were analyzed with the Chi-squared test.Results:(1) There was a progressive increase from other types of pancreatic tumors to pancreatitis and to ductal adenocarcinoma in the frequency of overall pancreatic intraepithelial neoplasia lesions (24.0%,12/50;54.5%,12/22; and70.6%,84/119; respectively). The frequency of higher grade pancreatic intraepithelial neoplasia lesions (2and3) in ductal adenocarcinoma patients was31.1%(37/119), which was significantly higher than the one in chronic pancreatitis(0%,0/22) or other types pancreatic tumors(2%,1/50)(P<0.05). The frequency of PanIN was significant difference among the different age groups (P<0.05), and was increased with the age. There was no significant difference of the frequency of the PanIN between male and female gender.(2) There was a progressive reduce from PanIN-1, PanIN-2, to PanIN-3in the expression of hsa-miR-96(91.3%,22/23;70.6%,12/17; and22.2%,4/18; respectively) and hsa-miR-217(95.7%,22/23;70.6%,12/17;27.8%,5/18; respectively). The expressions of hsa-miR-96and hsa-miR-217in PanIN-2/3lesions were significantly different from normal pancreatic tissue and PanIN-1(P<0.01).(3) In PDAC,31.1%(14/45) and42.2%(19/45) of samples showed positive expression of hsa-miR-96and hsa-miR-217in tumor cells, respectively, whereas all corresponding adjacent normal pancreatic tissue and chronic pancreatitis samples were positive or weakly positive (P<0.05).(4)Positive expression of hsa-miR-96and hsa-miR-217was observed in the fibroblasts of22.2%(10/45) and24.4%(11/45) of PDAC, respectively, which was significantly lower than chronic pancreatitis (70%,7/10and60%,6/10). These differences were statistically significant (P=0.003).(5)There was no statistical difference in survival between the negative group and positive group of hsa-miR-96or hsa-miR-217.Conclusion:(1) The frequency of overall PanIN lesions and higher grade PanIN lesions (2and3) in PDAC were significantly higher than the one in chronic pancreatitisor other types pancreatic tumors.(2)hsa-miR-96and of hsa-miR-217abnormal expression was observed in early stage (PanIN-2/3) of PDAC.(2)These microRNAs(hsa-miR-96and hsa-miR-217) may play an important role in the development of PDAC and may serve as potential biomarker.(3)There was different expression of hsa-miR-96and hsa-miR-217between PDAC and chronic pancreatitis.(4) Down-regulation of hsa-miR-96and hsa-miR-217in PDAC exists not only in cancer cells, but also in the stromal fibroblasts, which maybe provide new ideas about the microenvironment of PDAC.更多还原