【作者】 赵谦；

【导师】 金锡御；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 研究背景与目的肿瘤分子遗传学研究普遍认为肿瘤的发生发展是多基因改变多步骤的过程。目前肿瘤的病因学主要包括:病毒、化学因素、物理因素、激素因素。膀胱癌在我国男性泌尿生殖系肿瘤中发生率第一,而且近年有发生率增加和年轻化趋势。较为明确的膀胱癌危险因素为:a、吸烟;b、化工染料(如苯胺);c、药物(如非那西丁、环磷酰胺)等,上述危险因素均为DNA损伤剂。一般认为,致癌物质及其代谢产物是通过尿液排泄的,这样它们可以直接作用于尿路上皮的粘膜层。那么DNA损伤剂是如何导致基因突变,最终导致肿瘤的发生,其分子机制一直不清楚。直到近年在生物细胞发现一系列的Y家族DNA聚合酶及相应的DNA损伤耐受机制,在这一领域才有了新的突破。Y家族DNA聚合酶一方面通过高灵活的碱基配对跨损伤复制DNA,以保证DNA复制的正常进行,另一方面缺乏错配碱基的校正阅读功能,以上均导致其对DNA复制有很高的碱基错配倾向。Polι属于Y家族的DNA聚合酶之一,是目前发现的保真性最低的聚合酶,其保真性较高保真聚合酶低1000-10000倍。因此,Polι在DNA跨损伤复制过程中,很有可能导致碱基突变而使基因发生点突变。如果这些突变基因为细胞维持基因稳定的基因(高保真聚合酶),就会导致基因产生级联式的突变,或导致抑癌基因、细胞生长因子基因等的突变,最终导致肿瘤的发生。本研究从Polι跨损伤复制DNA时可能导致基因突变角度出发,认为膀胱肿瘤的发生与发展是否有可能与Polι在组织的高表达有关。已有研究表明:人葡萄膜的黑素瘤的发展与高活性的表达Polι相关;肿瘤产生的缺氧诱导因子-1可能通过介导人肿瘤细胞Polι的表达而导致肿瘤的基因突变和遗传的不稳定性;在Polι高表达的乳腺癌细胞株中,对DNA的损伤更容易诱导基因突变。研究表明膀胱肿瘤高复发、高异质性的生物学行为与基因的高突变遗传背景有关,那么,膀胱肿瘤的这种生物学行为是否与膀胱肿瘤组织Polι高表达有关。因此,我们拟通过检测Polι在移行细胞癌组织的表达,探讨Polι在膀胱肿瘤发生的意义及其与膀胱肿瘤高复发、高异质性的关系。在研究Polι在乳腺癌细胞株的表达时,发现并克隆了Polι的剪接异构体,由于该异构体比Polι少第5个外显子,故命名为PolιS5,但对PolιS5的功能未做进一步的研究。人体蛋白编码基因是相对有限的,在mRNA转录水平,选择性剪接异构体的存在就为蛋白多样性的产生提供了一个重要机制。一个蛋白的不同剪接异构体,可以显示协调、增强等不同的功能,但也可能是拮抗性的功能。许多研究正在致力于建立剪接异构体的全面数据资料,其中包括与转录功能性相关的选择性剪接异构体、这些异构体在正常和病理生理状态下的特殊作用、以及它们怎样在更广泛的水平进行协调以获得细胞或组织特异性的功能。因此,对蛋白不同剪接异构体的功能进行研究就显得尤为重要。我们检测发现PolιS5在膀胱肿瘤细胞株同样有表达,那么PolιS5与Polι相比较,功能有何差异,对此我们进行了初步研究。本课题拟对PolιS5与Polι的功能进行对比研究,同时探讨Polι在膀胱肿瘤细胞株表达的意义,并进一步检测PolιS5与Polι在移行细胞癌组织是否高表达,与移行细胞癌组织的异质性生物学行为是否相关。本研究将对我们了解PolιS5的功能,进一步理解膀胱肿瘤的发生及其高异质性的分子机制,具有重要的理论意义。方法:1、PolιS5与Polι在膀胱肿瘤细胞株BIU87、T24的表达。培养膀胱肿瘤细胞株BIU87、T24,收集临床膀胱正常粘膜组织作为对照。临床标本膀胱正常粘膜组织15例来自第三军医大学西南医院泌尿外科研究所,正常膀胱粘膜组织取自距膀胱肿瘤2cm以上的正常粘膜,标本切取后均立即放入液氮冻存,防止RNA降解。采用Tripure提取组织和细胞总RNA,RT-PCR检测PolιS5与PolιmRNA在膀胱肿瘤细胞株BIU87、T24和膀胱正常粘膜组织的表达。2、pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5的转化和鉴定。pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5由第三军医大学生化与分子生物学教研室杨劲副教授赠送,利用氯化钙法将三个质粒分别转化入DH5α大肠杆菌,经过进行质粒小抽,初步鉴定后,进行测序,将测序结果利用生物信息技术BLAT(www.genome.ucsc.edu)进行鉴定。以证明pEGFP-C1-PolιS5的目的片段PolιS5比pEGFP-C1-Polι的目的片段Polι少Polι的第5外显子,为我们进一步对Polι与PolιS5的功能对比研究奠定基础。3、Polι与PolιS5对UVC导致的DNA损伤的功能对比研究。通过用激光共聚焦显微镜检测细胞在受到紫外线UVC照射后Polι及PolιS5在细胞核分布的改变分析其功能差异。培养HEK293细胞,利用阳离子脂质体瞬时转染的方法,将质粒pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5分别转染HEK293细胞,36小时后,用紫外线UVC(15J/m2)照射使细胞DNA损伤,12小时后用激光共聚焦显微镜观察在质粒pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5分别瞬时转染的HEK293细胞,细胞核绿色荧光蛋白在紫外线照射后分布的改变。4.PolιS5与Polι在临床移行细胞癌组织标本的表达。临床标本来自第三军医大学西南医院泌尿外科研究所,膀胱正常粘膜组织15例,膀胱肿瘤组织28例,肾盂癌组织11例;移行细胞癌组织I级13例,II级16例,III级10例。组织标本取自2005年5月2006年9月在西南医院行膀胱肿瘤切除术及肾盂癌根治手术的患者,经过病理检查证实,均为移行上皮细胞癌。在肿瘤原发灶上切取200mg左右组织作为癌组织标本;正常膀胱粘膜组织来自距膀胱肿瘤2cm以上的正常粘膜,作为对照。标本切取后均立即放入液氮冻存,防止RNA降解。采用Tripure提取组织和细胞总RNA,RT-PCR检测PolιS5与PolιmRNA在临床移行细胞癌组织标本的表达。结果:1、成功培养膀胱肿瘤细胞株BIU87、T24;通过RT-PCR检测发现,在BIU87和T24肿瘤细胞株有丰富的PolιmRNA表达,且较膀胱正常粘膜组织表达有非常显著差异(P<0.01);并发现PolιS5在膀胱肿瘤细胞株有表达,但在膀胱正常粘膜组织未检测到PolιS5的表达;在BIU87和T24肿瘤细胞株,Polι的表达较PolιS5表达丰富,并有非常显著差异(P<0.01)。膀胱肿瘤细胞株BIU87及T24中,Polι的表达显著增加,由于Polι高水平的表达可能导致DNA跨损伤复制过程中基因的高突变,因此Polι的高表达可能与膀胱肿瘤的发生相关;在膀胱肿瘤细胞株BIU87及T24中,发现PolιS5的表达,在文献中尚未见报道;PolιS5在膀胱肿瘤细胞株表达的意义尚待研究。2、成功将质粒pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5转化DH5α大肠杆菌,并进行质粒小抽,初步鉴定后将质粒进行测序,将测序结果利用生物信息技术BLAT(www.genome.ucsc.edu)证明: pEGFP-C1-PolιS5的目的片段PolιS5比pEGFP-C1-Polι的目的片段Polι少Polι的第5外显子,为我们进一步对Polι与PolιS5的功能对比研究奠定了基础。3、在质粒pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5瞬时转染的HEK293细胞的细胞核均有绿色荧光,且很均匀,质粒转染率几乎达100%。在紫外线UVC(15J/m2)照射三种细胞后,质粒pEGFP-C1转染的HEK293细胞核荧光仍然很均匀,pEGFP-C1-Polι、pEGFP-C1-PolιS5转染的HEK293细胞核均出现荧光聚集现象,同时发现质粒pEGFP-C1-Pol iotaS5(20%)转染的HEK293细胞核出现荧光聚集现象的细胞比例明显高于质粒pEGFP-C1-Polι(6%)转染的HEK293细胞核出现荧光聚集现象的细胞比例。说明:PolιS5与Polι在紫外线UVC导致的DNA损伤的跨损伤复制过程中可能起着相似的作用,而且PolιS5可能比Polι更敏感,结合DNA更紧密,但更详细的过程尚须进一步研究;PolιS5可能对UVC损伤较补骨脂素、顺铂导致的DNA损伤有一定的依赖性。4、通过RT-PCR检测,PolιmRNA在膀胱正常粘膜组织、膀胱肿瘤组织及肾盂癌组织均有表达, Polι在膀胱肿瘤及肾盂癌组织的表达显著高于膀胱正常粘膜组织的表达(P<0.01);Polι在I级移行细胞癌组织和II级移行细胞癌组织的表达显著高于膀胱正常粘膜组织的表达(P<0.05;P<0.01);在III级移行细胞癌组织的表达显著高于I级移行细胞癌组织(P<0.05)。但我们未检测到PolιS5在膀胱正常粘膜、膀胱肿瘤、肾盂癌组织的表达。结果表明:膀胱肿瘤组织和肾盂癌组织Polι的表达显著增加,可能与膀胱肿瘤的发生有关;移行细胞癌组织Polι的表达显著增加与膀胱肿瘤、肾盂癌的异质性相关;在移行细胞癌组织未发现PolιS5的表达,但不能排除PolιS5在其它组织的丰富表达。结论:1、在膀胱肿瘤细胞株BIU87及T24中,发现Polι的剪接体PolιS5;2、PolιS5与Polι在对紫外线UVC导致的DNA损伤后的复制过程中可能起着相似的作用,PolιS5可能比Polι更敏感,而且结合DNA更紧密,但更详细的机制尚须进一步研究;3、Polι在膀胱肿瘤细胞株、膀胱肿瘤组织和肾盂癌组织的高表达,可能与移行细胞癌的发生有关;4、Polι在移行细胞癌组织的高表达可能导致移行细胞癌的异质性生物学行为;5、在移行细胞癌临床组织标本未发现PolιS5的表达,但不能排除PolιS5在其它组织的丰富表达。更多还原

【Abstract】 Background and objective:The dangerous effects of transitional cell carcinoma such as smoking, chemical industry(aminobenzene), medicine(cyclophosphamide, phenacetin) are all DNA damage agents. Although DNA lesions can be removed by nucleotide excision repair and base excision repair processes, many lesions escape repair and present a block to continued transcriptional elongation by RNA polymerases and to replication by DNA polymerases. Human DNA polymeraseι(Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity. Due to the mismatch of the nucleotide, it suggests us that Polιprobably easily lead to genetic mutation. One of the hallmarks of cancer cells is genetic instability. In addition to large chromosomal changes involving thousands of bp, another form of genetic instability results in an increased mutation rate at the nucleotide level due to a perturbation in nucleotide synthesis or in cellular processes such as DNA repair and replication。So we think that the occurrence and development of transitional cell carcinoma maybe associate with the DNA translesion replication of Polιwhich is a mismatch repair polymerase different from other translesion synthesis DNA polymerase. Many studys have showed us Polιprobably easily lead to genetic mutation. Hypoxia-inducible factor-1-mediated Polιgene expression may be involved in the generation of translesion mutations during DNA replication. The Polιactivity was observed in seven out of eight malignant tumors, while it was absent in the normal uveal tract cells of the same patients. These findings serve as an additional confirmation of the Polιoncogenic potential.Yang J, et al found a Polι’s homologous isomer which is shorted of the fifth exon of Polιcompaired with Polι, and named it PolιS5, then, they cloned the Polιand PolιS5 gene and build their eukaryotic expression vector, but didn’t study the specific function of PolιS5. Recent analyses of sequence and microarray data have suggested that alternative splicing plays a major role in the generation of proteomic and functional diversity in metazoan organisms. Different splice variants of a given protein can display different and even antagonistic biological functions. Efforts are now being directed at establishing the full repertoire of functionally relevant transcript variants generated by alternative splicing, the specific roles of such variants in normal and disease physiology, and how alternative splicing is coordinated on a global level to achieve cell- and tissue-specific functions.We also detected the expression of PolιS5 in BIU87 and T24, so it is very important to study the function of PolιS5 futher and discuss the significance of the expression of Polιand PolιS5 in transitional cell carcinoma.Methods:1、Expression of Polιand PolιS5 in cell lines of transitional cell carcinomaCurrently, there are no reports describing the expression of Polιin cell lines of transitional cell carcinoma. To develop a more accurate picture of the abundance of Polιexpressed in these cells, we analyzed a series of transitional cell carcinoma cell lines. The cell lines of transitional cell carcinoma used in this study include T24 and BIU87. The clinical tissue samples, bladder normal membrana mucosa, as the control group, were obtained from the Institute of Urological Research of the Third Military Medical University. Expression of Polιand PolιS5 in cell lines of transitional cell carcinoma were detected by RT-PCR .2、The identify of pEGFP-C1-Polιand pEGFP-C1-PolιS5pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5 were presented by Dr. Yang Jin. Firstly, pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5 were transformed into Escherichia coli DH5αand were mini-extracted, then they were sequenced and identified by Blat (www.genome.ucsc.edu).3、The diference of function between Polιand PolιS5In order to improve our understanding of the biological function of Polιand PolιS5, we have analysed its cellular localization. The cDNA encoding enhanced green fluorescent protein(EGFP) was fused in-frame to the N-terminus of Polι(EGFP-C1-Polι) and PolιS5 (EGFP-C1-PolιS5), and then pEGFP-C1 vector, pEGFP-C1-Polιand pEGFP-C1-PolιS5 were transfected into HEK293 cell respectively. Due to the nuclear localization signal consensus motif in its amino acid sequence, EGFP were localized within the nucleus and appeared homogeneously distributed. The distribution of EGFP-Polιand EGFP-PolιS5 in the nuclear localization were detected by by Laser scanning confocal microscope. We irradiated the transfected cells with UVC(15 J/m2) and observed change of distribution of EGFP-C1-Polιand EGFP-C1-PolιS5 in the nuclear localization 12 h later.4、Expression of Polιand PolιS5 in tissue of transitional cell carcinoma.The clinical tissue samples were obtained from the Institute of Urological Research of the Third Military Medical University, including bladder normal membrana mucosa(15), bladder tumor(28), renal pelvic carcinoma (11),which include transitional cell carcinoma grade I (13), grade II (16) and grade III (10). Expression of Polιand PolιS5 were detected by RT-PCR .Results:1、Expression of Polιand PolιS5 in cell lines of transitional cell carcinomaIt was observed that the expression of Polιappears to be low in bladder normal membrana mucosa but significantly elevated in transitional cell carcinoma cell lines(P<0.01). The expression of PolιS5 was also detected in BIU87 and T24, but not in bladder normal membrana mucosa, and the expression of Polιwas much higher than that of PolιS5 in BIU87 and T24(P<0.01). The expression of Polιwas significantly elevated in transitional cell carcinoma cell lines and the over expression of Polιin cell probably lead to high genetic mutation when DNA was damaged. The function of PolιS5 will be still studied.2、The identify of pEGFP-C1-Polιand pEGFP-C1-PolιS5pEGFP-C1、pEGFP-C1-Polι、pEGFP-C1-PolιS5 were transformed into Escherichia coli DH5αand were mini-extracted, then they were sequenced and identified by Blat (www.genome.ucsc.edu).It was determined that the cDNA of PolιS5 in pEGFP-C1-PolιS5 is shorted of the fifth exon of Polιcompaired with the cDNA of Polιin pEGFP-C1-Polι. The next experience will be based on the result.3、The diference of function between Polιand PolιS5EGFP were localized within the nucleus and appeared homogeneously distributed. The EGFP-C1-Polιand EGFP-C1-PolιS5 were predominantly localized within the nucleus and appeared homogeneously distributed. In 2% of EGFP-C1-Polιtransfected cells, and in 4% of EGFP-C1-PolιS5 transfected cells, EGFP- C1-Polιand EGFP-C1-PolιS5 were localized in many intranuclear foci. This localization pattern was strikingly similar to observations with pol eta .Consequently, we asked whether the distribution of EGFP-C1-Polιand EGFP-C1-PolιS5 changes after DNA damage. We irradiated the transfected cells with UVC(15 J/m2) and observed the pattern of EGFP-C1-Polιand EGFP-C1-PolιS5 12 h later by Laser scanning confocal microscope. Percentage of EGFP-C1-Polιtransfected cells and EGFP-C1-PolιS5 transfected cells increased to 6% and 20% respectively. It were suggested that PolιS5 probably has the similar function with Polιin translesion sythesis and PolιS5 may be more sensitive to DNA damage that lead by UVC than Polι, but the more specific function will still be studied.4、Expression of Polιand PolιS5 in tissue of transitional cell carcinoma.It was observed that the expression of Polιappears to be low in bladder normal membrana mucosa but significantly elevated in tissue of transitional cell carcinoma(P<0.01). The level of expression of Polιwas associated with the grade of the transitional cell carcinoma. The expression of PolιS5 was not detected in tissue of transitional cell carcinoma. It was suggested that the over expression of Polιin tissue of bladder tumor and renal pelvic carcinoma may be associated with the occurrence of transitional cell carcinoma and the heterogenicity of transitional cell carcinoma.Conclusion:1. The expression of PolιS5, Polι’s splice isomer, was detected in BIU87 and T24;2. PolιS5 probably has the similar function with Polιin translesion sythesis and may be more sensitive to DNA damage that lead by UVC than Polιin function, but the more specific function will still be studied;3. The over expression of Polιin transitional cell carcinoma cell lines and tissue of bladder tumor and renal pelvic carcinoma may be associated with the occurrence of transitional cell carcinoma;4. The over expression of Polιin tissue of bladder tumor and renal pelvic carcinoma may be associated with the heterogenicity of transitional cell carcinoma;5. PolιS5 was not detected in clinical tissue of transitional cell carcinoma, but it could not exclude the expression of PolιS5 in other specific tissue.更多还原