【作者】 毕罡；

【导师】 靳风烁；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 前列腺癌为中老年男性常见的恶性肿瘤和主要死亡原因之一。在美国男性的癌症相关致死原因中前列腺癌占据了第二的位置。在恶性肿瘤中,前列腺癌的自然病史较为独特,它发病隐匿,早期缺乏特征性表现,易发生早期转移,出现症状后,往往已是晚期。另一方面,目前前列腺癌多采用手术治疗,创伤较大,虽然可以应用内分泌疗法进行治疗,但治疗是姑息性的,肿瘤最终将失去对内分泌治疗的敏感性而继续发展,导致患者死亡;而且前列腺肿瘤对化疗敏感性差,治疗较为棘手。上皮来源的肿瘤占了全部恶性肿瘤的90%,他们对化疗药物的不敏感成为临床上遇到的主要问题之一。在前列腺癌诊断和治疗领域,寻找和鉴定前列腺癌差异表达基因,将其作为前列腺癌诊断及治疗靶点目前已经成为国际上研究前列腺癌的热点之一。研究表明在肿瘤发生过程中,细胞间的紧密连接、连接粘附分子和基底蛋白等膜蛋白存在异常表达。这些研究提示异常表达的膜蛋白对于作用于肿瘤细胞的药物来说可能具有重要意义。Claudin蛋白是存在于紧密连接中的分子量约为23KDa的跨膜蛋白,它在紧密连接的屏障功能当中起了非常重要的作用。包括Claudin-3和Claudin-4在内,Claudin蛋白家族有超过20个成员,并且它们表现出明显的组织特异性。有趣的是,在卵巢癌、肝细胞癌、胰腺癌、前列腺癌等上皮来源的恶性肿瘤中,常常观察到Claudin蛋白有上调表达。而且研究发现Claudin-3有可能作为前列腺癌的上调表达基因,成为前列腺癌新的候选癌标进行临床诊治的研究。随着化学、药理学、蛋白质组学、基因组学等研究领域的快速发展,目前发现并研制了很多新的抗肿瘤药物,但就临床应用来说,有必要找到一种使这些新药更有选择性发挥其作用的药物传输系统。产气荚膜梭菌肠毒素(CPE)可能成为这一系统,它是一个分子量为35KDa的多肽,能导致食物中毒并与大多数的人食源性传染病相关。该肠毒素有两个功能不同的区域组成:一个是N-末端大约为22KDa的区域,主要起细胞毒作用;另一个是C-末端大约为13KDa的区域,这一结构域介导了CPE和目标细胞膜的结合并使之部分的进入胞质内部,引发细胞对大量小分子通透性的改变,从而引起细胞的空泡变和溶解。在Claudin蛋白家族中只有Claudin-3和4是CPE的受体,可以结合CPE并引起胞溶作用。这些发现表明CPE可以用来特异性结合上皮来源癌细胞中的Claudin跨膜蛋白。目前CPE已经被成功的用于治疗高表达Claudin-3和4的卵巢癌和胰腺癌中。本研究拟通过实验探讨Claudin-3作为CPE的靶标在前列腺癌诊治中可否成为一个候选癌标。方法:采用免疫组化及Western blot方法检测Claudin-3在前列腺增生及前列腺肿瘤组织的表达状态,同时分析Claudin-3与前列腺癌病理分期的关系,评价Claudin-3作为前列腺癌诊断候选癌标的价值。培养并鉴定表达肠毒素的产气荚膜梭菌标准菌株64615,接着提取细菌的基因组DNA,PCR扩增克隆出全长cpe基因并连接入pET-28a载体质粒中,构建含全长cpe基因的重组表达质粒pET-28a-cpe,经酶切及测序鉴定正确后,转化入感受态E.coli BL21中表达,并优化表达条件,进行最适诱导剂浓度和最适诱导时间的筛选;再通过Ni-NTA柱进行亲和层析纯化。纯化后的CPE作用于体外培养的人前列腺癌细胞株22RV1以及前列腺癌荷瘤裸小鼠模型,在CPE安全剂量下观察CPE激发的对前列腺癌癌细胞的抑制作用,进一步探讨Claudin-3作为前列腺癌治疗候选靶点的可行性及安全性,为CPE今后的临床应用打下基础。结果:1、前列腺组织石蜡切片免疫组化半定量分析显示Claudin-3在前列腺组织腺体周围细胞的胞膜中均有不同程度的表达;前列腺癌组中的表达显著高于良性前列腺增生组,但在前列腺癌的高、中、低分化组中表达无统计学差异。用Western blot方法对前列腺组织中Claudin-3蛋白的含量分析显示:前列腺癌组中的Claudin-3蛋白表达显著高于前列腺增生组。2、复苏、培养产肠毒素的产气荚膜梭菌标准菌株64615,并进行菌株的鉴定。提取细菌的基因组DNA,PCR扩增出全长cpe基因,并与质粒载体pET-28a连接,构建了重组的pET-28a-cpe质粒,酶切鉴定及测序结果表明,成功构建了原核表达质粒pET-28a-cpe。3、将质粒pET-28a-cpe转化入E.coli BL21感受态中诱导表达,并优化诱导表达的条件,最后通过Ni-NTA亲和层析进行分离纯化,纯化后的重组CPE蛋白浓度达到90%以上。4、进行人前列腺癌细胞株22RV1和PC-3的体外培养,并应用Claudin-3单抗进行免疫荧光染色,证实体外培养的人前列腺癌细胞株22RV1可以特异性高表达Claudin-3。将CPE作用于培养的22RV1,显示CPE对肿瘤细胞具有明显的细胞毒作用,细胞增殖明显降低。5、建立种植22RV1的前列腺癌荷瘤裸鼠模型。将CPE进行瘤内注射,发现肿瘤细胞坏死、肿瘤体积明显缩小,肿瘤生长明显延缓。结论:Claudin-3在前列腺癌组织中有特异性高表达,但与肿瘤的恶性程度相关性不明显;运用基因工程方法,成功构建了原核表达质粒pET-28a-cpe并转化E.coli BL21,诱导其表达CPE后,通过Ni-NTA亲和层析进行分离纯化,获得了较高纯度的CPE;CPE对特异性高表达Claudin-3的体外培养前列腺癌细胞株及前列腺癌荷瘤裸鼠模型均能产生明显生物学效应,发生细胞毒性作用,使肿瘤细胞坏死、肿瘤生长延缓。上述结果提示Claudin-3可能成为前列腺癌诊断和治疗的新癌标;本研究为CPE应用于肿瘤的临床研究及治疗提供了可能的前景。更多还原

【Abstract】 Prostate cancer is a most common malignant carcinoma and main reason for death in male in the world. Mortality attributable to prostate cancer is the second factor causing of cancer-related death in men of United States. The failure rate of local therapy for early stage disease combined with the limited duration of effective palliative management with hormonal manipulation for advanced metastatic disease beg the development of newer therapeutic agents and strategies. Recent advances in the use of combination chemotherapy for advanced hormone-refractory prostate cancer are encouraging but remain inadequate. Epithelial-derived tumors account for 90% of all malignant tumors, and their resistance to chemotherapy is a major clinical problem. Recent progress in combinatorial chemistry, proteomics, and genomics research has further advanced the development of effective drugs against carcinomas, but, for clinical application, it is also essential to develop selective and efficient drug delivery systems for these novel drugs.Tight junctions (TJs), which are points of intercellular contact and interaction, are characteristic and complex structures in the epithelia. TJs play a critical role in forming a barrier between apical and basal sides of the cell, and they are present on the lateral side of the cell where they mediate intercellular interactions. Loss of polarity is a typical feature of transformation in epithelial cells.Furthermore, abnormal localization of membrane proteins, including TJ components, adherence junction proteins, and apical and basal proteins, is observed during carcinogenesis. These findings indicate that abnormally localized membrane proteins may be useful for targeting drugs to carcinoma cells.Claudin is an approximately 23KDa transmembrane protein found in the TJs, and it plays a pivotal role in the barrier function of the TJ. There are more than 20 members of claudin family including Claudin-3 and Claudin-4, and they are expressed in a tissue-specific manner. Interestingly, the overexpression of claudins is frequently observed in the epithelium of ovarian cancer, hepatocellular carcinoma, malignant pancreatic cancer, and prostate cancer. Therefore, claudins are promising candidates for the targeting of anticancer drugs to carcinoma cells.Clostridium perfringens enterotoxin (CPE) is a single polypeptide with a molecular mass of 35 KDa that causes food poisoning associated with most human food-borne illnesses. CPE is made up of two functionally distinct domains: an approximately 22-KDa N-terminal domain that mediates cytotoxicity, and an approximately 13KDa C-terminal domain (C-CPE) that mediates binding allowing partial insertion of CPE into the target cell membrane with resultant initiation of massive small molecule permeability changes, osmotic cell ballooning, and lysis. However, only claudin-3 and 4 in the family of claudin proteins are the receptors for CPE to bind it and mediate toxin-dependent cytolysis. These findings suggest that CPE could be used for the targeting of claudins on epithelial carcinoma cells. Indeed, CPE has been successfully used to treat human ovarian and pancreatic cancers, both of which express high levels of claudin-3 or -4. The present study was designed to determine whether claudin-3 is expressed in malignant epithelial cells of prostate cancer to serve as a potential target for a CPE-mediated therapeutic strategy.Methods: The expressions of Claudin-3 protein in prostate cancer and benign hyperplasia of prostate were detected by immunohistochemistry and Western blot, and its contribution on pathological stage of tumor were analysed. The stand Clostridium perfringens 64615 which could produce Clostridium perfringens enterotoxin was incubated and identified. Then the full-length gene of cpe which was extracted and amplified from the whole-length DNA of the Cl. Perfringens was correctly inserted into the vector pET-28a by restriction endonuclease digestion with NdeI plus XhoI. The protein CPE was expressed after IPTG induction, then it was isolated and purified from the total proteins of E. coli BL21 transformed by the recombinant prokaryotic plasmid pET-28a-cpe depending on Ni-NTA affinity chromatography. The human prostate cancer cell line 22RV1 and PC-3 were cultured separately and detected by fluorescein stain. Changes of the cultured human prostate cancer cell line 22RV1 were observed after CPE was added into the 96 well plate. The Nude mice Balb/c were subcutaneously inoculated human prostate cancer cell line 22RV1. The mice of different groups were injected different dose of CPE in the local position of the inoculated tumor. The volume of the mice tumor were recorded and the pathological changes were observed by HE staining and immunohistochemistry.Results:1. Claudin-3 was expressed on the cell membrane of prostate inequablely. Compared with those sections of benign hyperplasia of prostate, the expression of Claudin-3 was significantly increased in the cases of prostate cancer by immunohistochemistry and Western blot.2. The stand Clostridium perfringens 64615 which could produce Clostridium perfringens enterotoxin was revived、incubated and identified. The full-length gene of cpe which was extracted and amplified from the whole-length DNA of the Cl. Perfringens was correctly inserted into the vector pET-28a by restriction endonuclease digestion with NdeI plus XhoI. The recombinant prokaryotic plasmid pET-28a-cpe was confirmed by partial nucleotide sequencing and restriction endonuclease digestion.3. The reconstructed protein was expressed after IPTG induction and isolated and purified from the total proteins of E. coli BL21. By induction of IPTG, the E. coli BL21 transformed by the recombinant prokaryotic plasmid pET-28a-cpe. Ni-NTA affinity chromatography was used for the Purification. The purity of the protein CPE was detected with SDS-PAGE, which was confirmed to have a purity of more than 90%.4. The human prostate cancer cell line 22RV1 could highly expressed claudin-3 detected by fluorescein stain compared with the human prostate cancer cell line PC-3. Cell line 22RV1 was inhibited by CPE mediating toxin-dependent cytolysis after CPE was added into the 96 well plate.5. The Nude mice Balb/c were subcutaneously inoculated human prostate cancer cell line 22RV1 successfully. After injecting CPE in the local position of the inoculated tumor, the volume of the mice tumor was reduced and the cellular necrosis of the inoculated tumor was observed by HE staining and immunohistochemistry.Conclusions: Claudin-3 protein is significantly highly expressed in prostate cancer. The protein CPE was successfully expressed in E. coli BL21 and purified by gene engineering. CPE could cause biological effect and mediate toxin-dependent cytolysis in cultured cells in vitro and in inoculated tumor of the Nude mice. Claudin-3 protein may become a new marker in the diagnose and treatment of prostate cancer. It is may be possible that CPE would be used as a new therapeutic strategy on the reasearch of clinic treatment for tumor in future.更多还原