【作者】 熊力；

【导师】 杨竹林；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 第一部分TGF-β1诱导胆囊癌EMT发生中差异表达基因分析目的为研究胆囊癌侵袭转移的可能机制,我们利用TGF-β1诱导胆囊癌GBC-SD细胞发生上皮-间质转化(EMT),然后应用基因芯片技术检测GBC-SD细胞与发生EMT的GBC-SD细胞的基因,分析其差异表达的基因。方法使用5ng/ml的TGF-β1诱导胆囊癌GBC-SD细胞株,分别提取该组和未经诱导的胆囊癌GBC-SD细胞株组的总RNA,纯化后再进行荧光标记、对寡核苷酸芯片进行杂交,激光扫描仪对其进行扫描,根据荧光强度来筛选出两者之间的差异基因,然后进行在线生物信息学分析。结果基因芯片实验共筛选出264个在胆囊癌GBC-SD细胞和经TGF-β1诱导后胆囊癌GBC-SD细胞间表达差异的基因。166个基因在经TGF-β1诱导后胆囊癌GBC-SD细胞组中表达上调,98个基因表达下调,经基因功能分类注释系统查询(GO分类),他们涉及到细胞粘附、细胞周期等；信号通路分析发现他们涉及到细胞周期蛋白调节、磷酸肌醇介导的信号通路等。结论我们利用基因芯片技术检测胆囊癌GBC-SD细胞及TGF-β1诱导发生EMT后的胆囊癌GBC-SD细胞差异表达的基因。结合文献检索分析我们认为NT5E和FcGBP可能涉及到胆囊癌的侵袭转移过程,因此将这两个基因作为下一步深入研究的目标。第二部分NT5E和FcGBP基因及其蛋白在胆囊癌组织中的表达及意义目的通过检测胆囊腺癌及慢性胆囊炎组织中NT5E和FcGBP的mRNA和蛋白质的表达,验证第一部分基因芯片的结果,并探讨这两个蛋白与胆囊癌侵袭转移的关系。方法随机收取了15例未行放化疗患者的胆囊腺癌标本和15例慢性结石性胆囊炎患者的慢性胆囊炎标本,获取其总RNA和总蛋白。NT5E和：FcGBP的mRNA表达水平采用定量RT-PCR检测；NT5E和FcGBP的蛋白质表达水平采用Western-blot检测。结果1.通过定量RT-PCR分析显示,胆囊腺癌NT5EmRNA的表达明显上调,与慢性胆囊炎比较有高度显著差异(1.012+0.019vs0.225±0.021,P<0.01)；而FcGBP mRNA在胆囊腺癌中的表达则与此相反,为明显下调且有高度显著差异(0.078±0.020vs0.321+0.017,P<0.01)。2.通过Western-blot分析发现NT5E蛋白在胆囊腺癌中的表达有明显上调,与慢性胆囊炎比较有高度显著差异(0.826±0.011vs0.412±0.017,P<0.01)；而FcGBP蛋白在胆囊腺癌中的表达有明显下调,与慢性胆囊炎比较有高度显著差异(0.084±0.017vs0.187±0.025,P<0.01)。结论胆囊腺癌中的NT5E mRNA以及其蛋白较慢性胆囊炎中的明显高表达,而FcGBP mRNA则相反,为低表达,这正与前述基因芯片结果相符合：NT5E、FcGBP基因可能从两个相反的方面涉及胆囊癌EMT调节,进而与胆囊癌侵袭转移相关。第三部分胆囊良恶性病变组织中NT5E和FcGBP的表达及其临床病理意义目的检测胆囊腺癌及其癌旁组织、腺瘤、息肉和慢性胆囊炎标本中NT5E和FcGBP的表达,探讨其在胆囊良恶性病变中的临床病理意义以及与胆囊腺癌侵袭转移的关系。方法共收集到了108例胆囊腺癌、46例癌旁组织、30例腺瘤、15例息肉和35例慢性胆囊炎组织标本切片,采用En Vision免疫组化二步法对NT5E和FcGBP进行染色,整理数据后利用SPSS软件分析。结果(1)108例胆囊腺癌中NT5E阳性表达为59例(54.6%),46例癌旁组织中14例阳性(30.4%),30例腺瘤中5例阳性(16.7%),15例息肉中2例阳性(13.3%),35例慢性胆囊炎胆囊上皮中4例阳性(11.4%)：胆囊腺癌中NT5E表达阳性率显著高于癌旁组织、腺瘤、息肉和慢性胆囊炎胆囊上皮,差异均有高度显著性(P<0.01)；另一方面108例胆囊腺癌中FcGBP阳性表达为52例(48.1%),46例癌旁组织中中阳性为35例(76.1%),30例腺瘤中阳性为24例(80.0%),15例息肉中阳性为13例(86.7%),35例慢性胆囊炎胆囊上皮中阳性为30例(85.7%),FcGBP在胆囊腺癌中的阳性表达率明显低于其在癌旁组织、腺瘤、息肉和慢性胆囊炎胆囊上皮中的阳性表达率,并有显著性差异(P<0.05或P<0.01)。此外,良性病变的胆囊上皮不论NT5E阳性表达、FcGBP阴性表达,光镜下均可见呈不同程度不典型增生。(2)NT5E在高分化的腺癌、最大直径<2cm的肿块以及无淋巴结转移、无周围组织侵犯的病例中,其阳性表达率明显低于其在低分化的腺癌、最大直径≥2cm肿块以及发现淋巴结转移和周围组织浸润的病例的阳性表达率(P<0.05或P<0.01)；但FcGBP在上述组织中的表达阳性率结果与NT5E相反(P<0.05或P<0.01)；(3)采用Kaplan-Meier单因素生存分析,发现胆囊癌患者术后的平均生存期与其病理类型、有无淋巴结转移、肿块最大直径、周围组织侵犯的状况等均有密切关系(P<0.05或P<0.01)；NT5E表达阳性的患者术后生存期明显低于阴性表达的患者(P<0.05),而FcGBP阳性表达的患者则生存期明显长于其阴性表达的患者(P<0.05)。NT5E阳性表达、FcGBP阴性表达患者的生存时间显著少于NT5E阴性表达、FcGBP阳性表达患者(P<0.01)；Cox回归多因素分析也表明NT5E表达阳性(P=0.019)或FcGBP表达阴性(P=0.004)均是胆囊腺癌预后不良的评价指标。结论NT5E及FcGBP表达水平均为反映胆囊癌发生、进展以及临床生物学行为的重要指标；NT5E阳性表达者和(或)FcGBP阴性表达者预后较差。更多还原

【Abstract】 Part I Differential analysis of the genes in gallbladder cancer with TGF-β1induced epithelial-mesenchymal transitionObjectiveTo explore the mechanism of invasion and metastasis of gallbladder cancer, gene chips were applied to investigate the differential expression of genes between gallbladder cancer cells with TGF-β1induced epithelial mesenchymal transition (EMT) and gallbladder cancer cells without TGF-β1induced EMT, which might provide potential therapeutic intervention of gallbladder cancer.MethodsThe preliminary research in our lab had optimazied5ng/ml as the concentration of TGF-β1for EMT induction. GBC-SD cells with and without TGF-β1induced EMT were cultured, then the total RNA were extracted and labeled with fluorescent probe. The hybridazition was performed by using the human genome oligonucleotide microarray containing21,522transcripts. The image data were initially collected by LuxScan10KA dual Channel laser scanner. Final analysis was conducted by MAS2.0.Results264differentially expressed genes were definited between GBC-SD cells with and without TGF-β1induced EMT, among which166genes were up-regulated and98genes were down-regulated in gallbladder cancer cells after TGF-β1induced EMT. According to GO classification, the differentially expressed genes belong to the groups such as reduction, oxidation, protein binding, and cell adhesion. Most interestingly, revealed by the pathway analysis, these genes were associated with the cyclin dependent kinase pathway regulation, intrinsic prothrombin activation pathway, cell cycle regulation, phosphoinositide-mediated signaling, etc.ConclusionsThrough the anlysis and comparison of the gene expression profile, quite a few differentially expressed genes were newly identified. These genes were involved in a variety of signaling pathways. After reference review, we thought NT5E and FcGBP might play important roles in the invasion and metastasis of gallbladder cancer with TGF-β1induced EMT. So these two genes were choosed for further research. Part Ⅱ The significance of NT5E and FcGBP expression in gallbladder adenocarcinomaObjectiveThe in vivo study of gallbladder adenocarcinoma and chronic cholecystitis was performed to verify the results obtained in microarray study and further investigate the role of NT5E and FcGBP in invasion and metastasis of gallbladder adenocarcinoma.MethodsThe total RNA and protein were extracted from specimens both of15patients diagnosed as gallbladder adenocarcinoma and15patients diagnosed as chronic cholecystitis. The mRNA transcription and protein expression levels of NT5E and FcGBP were evaluated by RT-PCR and Western-blot respectively. Statistic analysis was performed afte data collectionResults1. NT5E mRNA was significantly lower in chronic cholecystitis than that in gallbladder adenocarcinoma,0.225±0.021vsl.012±0.019, P<0.01,while FcGBP mRNA was significantly higher in chronic cholecystitis than that in gallbladder adenocarcinoma (0.321±0.017vs0.078±0.020, P<0.01)2. Western blot demonstrated that NT5E expression was significantly higher in gallbladder adenocarcinoma than that in chronic cholecystitis (0.826±0.011vs0.412±0.017, P<0.01),while the expression of FcGBP protein was significantly lower in gallbladder adenocarcinoma than that in chronic cholecystitis (0.084±0.017vs0.187±0.025, P<0.01)ConclusionsNT5E expression was upregulated and FcGBP expression was downregulated in gallbladder adenocarcinoma compared to chronic cholecystitis in vitro that was constistent with in vitro study in part one.It was suggested that NT5E and FcGBP might play an important role in the process of gallbladder epithelial malignant transformationwhich is associated with gallbladder adenocarcinoma invasion and metastasis.Part Ⅲ Differential expression of NT5E and FcGBPand their clinicopathological significance in benign and malignant lesions of the gallbladderObjectiveThe differential expression of NT5E and FcGBP in benign and malignant lesions of gallbladder is investigated immunochemically and the clinicopathological significance between them is evaluated.MethodsSpecimens of gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenoma (n=30), polypus (n=15) and chronic cholecystitis (n=35) were paraffin-embedded to sections which detected by En VisionTM immunohistochemistry to evaluate the expression level of NT5E and FcGBP.Results(1) The NT5E expression was significantly up-regulated in gallbladder adenocarcinoma (54.6%) than that in peritumoral tissues (30.4%) and other benign lesions including adenoma (16.7%), polypus (13.3%) and chronic cholecystitis (11.4%)(P<0.01). The ratio of positive FcGBP expression was significantly down-regulated in gallbladder adenocarcinoma (48.1%) than that in peritumoral tissues (76.1%), adenoma (80.0%), polypus (86.7%) and chromic cholecystitis (85.7%)(P<0.05). Atypical hyperplasia in gallbladder epithelium was observed in the benign lesions with NT5E and/or FcGBP expression.(2) The NT5E expression in well-differentiated adenocarcinoma, small tumor size (diameter<2cm), no lymph node metastasis and no peripheral tissue invasion was significantly down-regulated than that in poor-differentiated adenocarcinoma, larger tumor size (diameter≥2cm), lymph node metastasis and peripheral tissue invasion (P<0.5or P<0.01). However, the expression of FcGBP showed a negative correlation in those cases (P<0.05or P<0.01).(3) The monovariable Kaplan-Meier survival analysis showed that the pathological type, tumor maxium diameter, lymph node metastasis, peripheral tissue invasion, NT5E and FcGBP expression were significantlyrelated to the average survival time of patients with the gallbladder adenocarcinoma (P<0.05or P<0.01). Furthermore, the survival time of patients with NT5E expression was significantly shorter than those without NT5E expression (P<0.05). And FcGBP showed a negative correlation in those cases (P<0.05). Interestingly, the overall survival time of cases withNT5E(-)FcGBP(+) was significantly longer than those with NT5E(+)FcGBP(-). Multivariable Cox analysis revealed that NT5E expression (P=0.019) or FcGBP expression (P=0.004) was negatively associated with survival period and positively associated with mortality after surgery.ConclusionsThe expression of NT5E and FcGBP in gallbladder adenocarcinoma is an independent marker for evaluation of the disease progression, clinical biological behaviors and prognosis. Patients with FcGBP expression had a better prognosis while patients with NT5E expression got a worse prognosis.更多还原