【作者】 何芙蓉；

【导师】 黄高昇； 闫庆国；

【作者基本信息】 第四军医大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 【研究背景】PBK/TOPK是新发现的一种丝苏氨酸激酶,最初发现能够与PDZ结构域结合,文献报道它能与抑癌基因hDlg、P53蛋白、raf蛋白等多个分子结合相互作用,能激活p38MAPK、Erk、JNK等多个下游分子,作为蛋白激酶分子参与信号转导通路。既往研究表明在PBK/TOPK在除睾丸组织外的正常组织中表达量均较低,而在几种肿瘤中表达升高,从而引起大家的关注。然而文献从mRNA上分析了PBK/TOPK的组织分布,所涉及的肿瘤类型也很有限,况且在人胆管细胞癌组织的表达、临床意义及其生物学功能也未见报道。目前为止蛋白水平上PBK/TOPK在正常组织及常见肿瘤的组织分布尚未见报道。【目的】1.研究PBK/TOPK在人体正常组织和常见恶性肿瘤组织的表达,探讨其作为肿瘤标志物的可能性;2.组织芯片结果显示,肝脏中PBK/TOPK仅在正常胆管细胞和胆管癌细胞中呈特异表达,因此探讨在胆管癌手术标本的表达和与患者预后的关系;3.PBK/TOPK在胆管癌细胞系中的表达及其功能初步研究。【方法】1.应用免疫组织化学EnVision法,分别在含有人体19种正常组织和含有19种恶性肿瘤组织的组织芯片上进行PBK/ TOPK染色,检测PBK/TOPK蛋白的表达;2.应用EnVision法研究PBK/TOPK在74例胆管癌(包括57例手术标本和17例肝脏穿刺标本),33例肝细胞肝癌和10例正常肝脏标本的表达,并与胆管细胞标志物CK7和CK19进行比较,并用Ki67染色分析PBK/TOPK与细胞增殖的关系;3.对57例胆管癌手术患者进行生存期随访,其中24例获得随访结果,用Kaplan–Meier方法做生存曲线,并分析PBK/TOPK的表达与患者生存期之间的关系; 4.采用RT-PCR及Western Blot方法检测胆管细胞癌细胞系QBC939细胞中的PBK/TOPK分子的表达,并测序构建PBK/TOPK真核表达载体;5.用EGF刺激胆管癌细胞,从蛋白和mRNA水平检测PBK/TOPK表达的变化;并观察其过表达对细胞周期的影响;6.用siRNA抑制胆管癌细胞PBK/TOPK的表达,利用流式细胞仪检测细胞周期的变化;7.利用免疫组织化学方法,检测CD133在胆管癌中的表达,并用胆管癌细胞系研究其功能。【结果】结果1.在19种正常组织中,只有头皮的汗腺、睾丸的精原细胞、肝脏的胆管上皮、食管黏膜下腺体、胰腺外分泌部的导管上皮、前列腺的基底细胞和肾脏的远曲小管8种组织有不同数量细胞明确阳性表达;在19种人类常见恶性肿瘤组织中,乳腺癌、子宫颈癌、甲状腺癌等多种肿瘤组织中存在PBK/TOPK的大量、强阳性表达;2. PBK/TOPK在正常胆管中明确表达,但肝细胞为阴性,肝硬化组织中的反应性增生小胆管的表达明显增强,阳性部位在胆管细胞的胞浆中;在74例胆管癌中,68例中可见PBK/TOPK的阳性表达。而33例肝细胞肝癌均为阴性,统计学分析,两者间具有非常显著的差异(P<0.001)。PBK/TOPK染色和Ki67染色无明确的相关关系。此外,PBK/TOPK与CK7或CK19相比在鉴别肝细胞肝癌上更加特异。3.在57例手术切除的胆管癌患者中,有24例获得随访资料,利用Kaplan-Meier曲线分析表明,13例PBK/TOPK低表达患者的中位生存期为8个月,而高表达患者的中位生存期为14个月,两组中位生存时间有明显差异(P=0.013)。同时发现,高分化胆管癌的中位生存时间和低分化胆管癌也有统计学差异(P=0.011)。提示PBK/TOPK可以作为评价胆管细胞癌患者预后的一个指标。4.在胆管癌细胞系QBC939细胞中,检测到了PBK/TOPK的mRNA和蛋白;经测序比对发现mRNA与GenBank中PBK/TOPK基因符合率为100%,并成功构建真核表达载体;5. RNAi抑制PBK/TOPK表达后,经流式细胞仪分析表明,对细胞的周期无明显影响;6.经EGF刺激后,发现胆管癌细胞系中PBK/TOPKmRNA和蛋白水平均增加,不影响处于G1期细胞数,却使S期细胞增加, G2/M期细胞总数减少。【结论】1、首次系统研究了PBK/TOPK蛋白在人类正常组织和常见恶性肿瘤组织的表达,发现8种正常组织表达,在乳腺癌、甲状腺癌及子宫颈癌等多种恶性肿瘤组织中表达增加;2、发现PBK/TOPK在所有标本正常胆管上皮细胞和大部分胆管癌标本中表达,而正常肝细胞和肝细胞肝癌不表达,提示PBK/TOPK有助于肝内胆管癌和肝细胞肝癌的鉴别诊断;3、PBK/TOPK的表达与胆管癌患者的预后密切相关;4、在胆管癌细胞系中,EGF刺激可使PBK/TOPK的表达上调,并影响细胞周期中从S期到G2/M的进程,但是抑制PBK/TOPK表达不影响细胞周期;5、成功构建了PBK/TOPK的真核表达载体,为下一步研究PBK/TOPK的功能奠定了基础。更多还原

【Abstract】 BackgroundThe increased expression of PBK/TOPK is associated with some malignant tumors, but PBK/TOPK expression and its function in primary liver cancer have not been studied. In our early study, it was found that PBK/TOPK was expressed only in normal bile duct but not in hepatocyte. In this study, we analyzed the expression of PBK/TOPK in hepatic primary cancer and explored its role in cholangiocarcinoma biology.Objectives1. To study the distribution of PBK/TOPK Protein in human normal and tumor tissues by immunohistochemsitry, and to know if it could be as a tumor marker; 2. To study PBK/TOPK expression in liver tissue, to reveal if PBK/TOPK expression was related to cholangiocarcinoma grade and to the patient prognosis; 3. To study PBK/TOPK physiological function in cholangiocarcinoma cell line. Methods1. The expression of PBK/TOPK was detected in normal tissues, including 19 types of normal human tissue and tumor tissues including, 19 types of tumors by using tissue chip assay and EnVision immunohistochemical staining. 2. 117 (74 cholangiocarcinomas, 33 hepatocellular carcinomas and 10 normal liver tissues) samples were prepared from paraffin-embedded surgical specimen. PBK/TOPK protein was detected by immunohistochemical staining and the relationship between PBK/TOPK and Ki67 labeling index and the tumor grade were assessed. The normal bile duct staining was used to set the score intensity and Image-Pro Plus 4.5 software was used to count and to analyze the immunostaining of PBK/TOPK and Ki67. 3. Among the 57 patients with surgical specimens of cholangiocarcinoma, clinical data for 24 patients were attained. The patients’survival time with cholangiocarcinoma and the relationship between PBK/TOPK and survival time were analyzed with the Kaplan–Meier method. 4. The mRNA and protein levels of PBK/TOPK in cholangiocarcinoma (QBC939) cells and other cell lines were examined by RT-PCR and Western Blot, and eukaryotic expression vector for PBK/TOPK was constructed. 5. The suppressive effect of PBK/TOPK with small interfere RNA in cholangiocarcinoma cell line was studied by detecting cell cycle. 6. QBC939 cells were stimulated with EGF and the alteration of mRNA and protein were detected; and cell cycle of the cells was examined by FCM analysis.Results1. PBK/TOPK was expressed in 8 normal tissues and special cells, such as the sweat glands of scalp, renal distal tubular epithelial cells, esophageal mucous gland, pancreatic ductal epithelial cells and biliary epithelium. High level expression of PBK/TOPK protein was observed especially in breast carcinoma, thyroid carcinoma, and cervical cancer.2. We found PBK/TOPK was usually expressed in normal bile duct epithelial cells, and much more expressed in cholangiocarcinomas (68/74) but never expressed in hepatocytes and hepatocellular carcinomas.Statistically, PBK/TOPK expression was significantly different between cholangiocarcinoma and hepatocellular carcinoma(P<0.001). There was no positive correlation between the expression of Ki67 and PBK/TOPK (P=0.286). A correlation between the expression of PBK/TOPK and the histopathological grading of the tumor was also not found(P=0.67).3. The median survival time was 8 months for13 patients with low expression of PBK/TOPK and 14 months for 11 patients with high expression of PBK/TOPK. Low expression of PBK/TOPK was predictive of poor survival of the patients with cholangiocarcinoma (P=0.013). It was also found that the median survival for the patients was related with the differentiation of cholangiocarcinoma (P=0.011) and the gender (P=0.009).4. We detected PBK/TOPK mRNA and Protein for by RT-PCR and Western blotting in cholangiocarcinoma cell line. PBK/TOPK gene was cloned from the total RNA of QBC939 cells by RT-PCR, and their sequences were identical to the report in GenBank. The full length eukaryotic expression vector pcDNA3.1+PBK/TOPK was constructed.5. After the stimulation with EGF (20ng/ml) for about 2h, the mRNA of PBK/TOPK in QBC939 cells began to increase and followed by the increased protein. Then we analyzed the cell cell cycle of QBC939 cells after the stimulation with EGF and found that the cell number of G1 phase remained stable but the cell number of G2/M period reduced with that of S phase increased.6. After the transfection of the siRNA into QBC939 cells, PBK/TOPK expression was decreased compared with the negative control. No significant difference was observed in the cell cycle profile between PBK/TOPK the transfected cells and the negative control.ConclusionsPBK/TOPK is expressed in several normal tissues and some special cells,and certain carcinoma tissues strongly expressed PBK/TOPK. As PBK/TOPK was usually expressed in normal bile duct cell and most of human cholangiocarcinomas, but never in hepatocyte and hepatocelllular carcinomas, it indicates that PBK/TOPK protein could serve as a potential diagnostic marker to make the differential diagnosis of CC from HCC. Furthermore, the low expression of PBK/TOPK is predicative of poor survival in CC patients. EGF stimulation can enhance the expression of PBK/TOPK in cholangiocarcinoma cells but suppression of PBK/TOPK in cholangiocarcinoma cells with siRNA did not affect its cell cycle. The data indicate that PBK/TOPK may not be a key protein in cell cycle control for cholangiocarcinoma cells. However, the exact biologic function of PBK/TOPK in both normal bile duct epithelial cells and in cholangiocarcinoma cells needs to be further investigated in our future research work..更多还原