【作者】 郑乃刚；

【导师】 董子明； 吴景兰；

【作者基本信息】 郑州大学 ， 病理与病理生理学， 2003， 博士

【摘要】 研究目的：肿瘤发生是一个有遗传和环境因素共同参与的多基因多步骤的发展过程，可分为启动、发展和演进三个阶段，涉及增变基因、癌基因及抑癌基因等的突变积累和恶性变细胞克隆的选择性扩增。实验表明一个增变基因的突变可导致细胞突变率的显著增高，早于癌基因及抑癌基因等的突变，其在癌变中的作用越来越受到重视。碱基切除修复是维护基因组稳定的重要机制之一，主要负责人体细胞经常发生的内源性的大量DNA小损伤，如氧化的碱基、烷化的碱基和单链DNA断裂等单个核苷酸的切除修复。而DNA聚合酶β(POLB)是碱基切除修复机制的关键分子。正常细胞DNA聚合酶β是低水平表达的。有报道多数肿瘤组织或细胞系，包括胶质瘤、淋巴瘤、乳腺癌、结肠癌、前列腺癌、卵巢癌和肝癌的DNA聚合酶β皆是高表达的。高表达的DNA聚合酶β修复保真性差，对错配碱基敏感性降低而耐受性增高，可导致基因组不稳定性、跨损伤修复和增变基因表型增加。在小鼠LN12细胞系内突变的DNA聚合酶β的表达可明显抑制野生型DNA聚合酶p的功能使细胞突变频率成倍增加。本课题组曾对人食管癌的DNA聚合酶p基因的突变情况进行的研究显示癌旁组织突变检出率(1/40)远低于癌组织(16/40)。但人食管癌组织、癌旁及正常组织的DNA聚合酶多基因表达情况如何?人食管癌Eca一109细胞系的DNA聚合酶p是否高表达?当应用分化诱导剂诱导Eca一109细胞分化时DNA聚合酶p表达水平是否可能向正常低水平调节?同时BER中必需参与的wP53和polp密切相关的耐药性如何?如将含野生型DNA聚合酶p基因 (wPolp)的表达质粒转染于Eca一109细胞而使细胞的帅olp表达进一步提高，是否会导致细胞进一步恶性化?还是通过功能调整是细胞呈现一、定恶性逆转化?这些问题对研究DNA聚合酶p与食管癌乃至与肿瘤的发生发展的关系具有重要意义，国内外尚未见报道。本文应用原位杂交、免疫组织化学、DNA转染等分子生物学技术对人食管癌组织、癌旁组织及正常粘膜的DNA聚合酶p进行了相对定量及定位研究，并对分化诱导剂及野生型DNA聚合酶p的表达质粒转染对人食管癌Eca一109细胞的生物学效应进行了探讨。 研究方法:1.组织标本制备及总RNA的提取:17例未经治疗食管癌患者的手术标本包括癌灶、癌旁及相邻正常的共51块组织，等分为2份，1份组织经4%多聚甲醛固定制备石蜡包埋切片;另1份组织按重量用Tri 201试剂提取总刚A，进行51个刚A斑点印迹。2.建立分化诱导剂处理的Eca一109细胞系和含wPolp的表达质粒(pcDNA3一wPolp)稳定转染Eca一109细胞系及相应各对照组细胞系.3.自p。DNA3一wPolp重组质粒经扩增、双酶切和电泳分离及提纯wPol p DNA.应用随机引物法制备生物素郑州大学博士学位论文摘要及地高辛标记的wPolp和Bi。一呻53及Bio--mP53的cDNA探针。应用体外转录法将wPol日DNA单酶切为线型模板，加SP6 RNA聚合酶、NTP及Bi。一1住，叮P进行体外转录制备反义RNA探针;并以DNA斑点印迹检测各标记探针的灵敏度。4.定位观察和表达信号相对定量:油镜下观察组织/细胞的各原位杂交信号或免疫反应性(POLB一1R、PCNA一IR及MRP一工R)的定位及其强弱级别。细胞信号/IR积分值结合RNA斑点印迹的TLC扫描数值相对定量。‘同时以不加标记探针和不加特异性抗体的标本分别作为原位杂交和免疫组化的阴性对照。5.应用DNA酸变性一甲绿一派郎宁GS染色计数增殖细胞比率。6.统计学处理应用t检验、XZ检验和相关分析等。 研究结果:1.人食管癌组织中polp基因表达信号定位于细胞质，相临正常组织内弱信号位于复鳞上皮的基层细胞，癌旁组织信号增强，癌灶组织内弱信号见于分化较高的癌巢，浸润的癌细胞信号较强，异型性巨癌细胞信号更强。DNApolp基因表达随着细胞恶性程度增加而增强。癌灶DNA po lp基因表达水平高于癌旁，p<0 .05;两者比正常组织表达显著增高，p<0 .001。结果表明人食管癌中DNA polp基因是高表达的。癌旁组织Polp基因较高水平的表达提示可能为癌变的早期事件。2.人食管癌组织中参与DNA早期损伤SSB修复的POLB和XRCCI的异二聚体表达定位于细胞核。在癌灶组织、癌旁组织和正常组织中POLB和XRCCI的表达水平及定位与polp基因表达水平及定位相似，其表达水平也随着细胞恶性程度增加而增强。两者在癌组织内的表达水平呈显著正相关r=0.9971，p<0.01。结果表明人食管癌中POLB和xRCCI的表达是高水平的。癌旁组织中参与DNA早期损伤SSB修复的POLB和XRCCI的较高水平的表达可郑州大学博士学位论文摘要进一步提示为癌变的早期事件。3.应用分化诱导剂诱导Eoa一109细胞趋向正常水平分化时，DNA polp基因表达向正常低水平下调，P<O.001;同时BER中必需的wp53基因表达上调，药物耐受性减低，P<O.05。这提示E。a一109细胞的DNA polp基因是高表达的。反之，顺铂处理的Eca一109细 {胞的polp基因表达上调，p<0 .001;wP53基因表达下调，药物耐受性增高，p<0 .001。这提示分化诱导剂比基因毒性药物可能具有较好的远期疗效。4.稳定更多还原

【Abstract】 Aim: Tumorigenesis occurs through multiple-steps of mutation accumulation of oncogenes and antioncogenes and through selection amplification of malignant cell clones. Recently the research works in the field of oncogenes and antioncogenes were expanded into the field about genes involved damaged DNA repair. The large amount of small DNA lesions, such as oxidized and alkylated bases and single strand break (SSB), weremainly repaired by DNA polymerase β in vivo through one single nucleotidebase excision repair (BER) pathway. The DNA polymerase β expressionlevel was very low in the normal tissues and cells; and possessed cell protection against apoptosis. Most of tumors, including glioma, lymphoma, breast cancer, colorectal cancer, prostate cancer, ovarian tumor and hepatic cancer and etc., exhibited overexpression of polbeta. The overexpressed polbeta with low fidelity repair could decrease sensitivity to the mismatched bases and increase tolerance to them, which could result in genomic instability, translesion repair and mutator phenotype. The polbeta with heterozygous of wild type (w) and mutant type (m) in which the mpolfl displayed dominantnegative effect over the wpolfi showed almost no BER activity. In our previous study there was a mutant frequency of 1/40 in the cancer-adjacent tissue, much lower than that of 16/40 in the cancer foci tissue in human esophageal cancer. Whether human esophageal cancer tissue could exhibit overexpression of polbeta and alteration of polbeta expression could occur earlier than that of sequence mutation? Especially the POLB and XRCC1, a heterodimer of POLB, involved in SSB repair of early damaged DNA could show altered expression in the cancer- adjacent tissue as an early event? Whether human esophageal cancer Eca-109 cell line could show overexpression of polbeta which could be down regulated during cell differentiation? How about the alteration of wp53 required in BER activity and anti-cancer drug resistance, when the cisplatin-treated cells were used as apositive control? If the pcDNA3-wpol β plasmid was transfected intoEca-109 cell line, the overexpressed polbeta could result in further mutator phenotype or normal tendency through functional regulation in the cell itself? As to the above-mentioned problems, especially associated with the localization of signals in human esophageal cancer, so far, no reports have been found.Methods: 1. 51 pieces of tissues, including cancer foci, cancer-adjacent and corresponding normal tissues were removed from 17 untreated patients with esophageal cancer. Each tissue was divided into 2 aliquots, one of them was fixed with 4% formaldehyde and paraffin-embedded specimens were prepared. The total RNA was isolated from another aliquot tissues by ’Trizol’ reagent, and 51 RNA dot blots were performed. 2. The Eca-109 cell line treated withdifferentiation drugs and stably transfected with pcDNA-wpol β Eca-109 cellline and their corresponding control cell lines were established respectively. 3. The wpol β DNA was isolated from pcDNA3-wpol6 recombinant plasmid after digesting with BamH1 and Hind III enzymes and electrophoresis. TheBiotin or Digoxgenin labeled wpolbeta cDNA probe, Bio-wp53 and Bio-mp53 cDNA probes were prepared by random primer method. The Bio-labeled wpolbeta antisense RNA probe was prepared by transcription in vitro. The recombinant pcDNA3-wpolβ plasmid was digested only by Hind III enzyme as the linerized DNA template. The transcription in vitro was carried out by addition of SP6 RNA polymerase, rNTPs and Bio-11-dUTP. The sensitivity of each labeled probe was detected by DNA dot blot. 4. Observation on the signal localization and relative quantitation of expression level: the signal/IR localization in the specimens prepared by in situ hybridization or by immunohistochemistry (POLB-IR, PCNA-IR and MRP-IR) were observed under oil-emersion microscope and the integrated value for demonstration of signal /IR intensity was combined with the TLC scanning value in the RNA dot blot or更多还原