【作者】 吴飞翔；

【导师】 唐步坚；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2009， 博士

【摘要】 卵巢上皮癌是妇科恶性肿瘤中发病率占第三位,死亡率却居第一的恶性疾病。由于卵巢解剖上隐匿于盆腔深处,在发病早期缺乏特异性症状和体征,很难在早期发现和诊断,约60%-70%的卵巢癌患者确诊时已属于晚期。近30年来,虽然经过全世界妇科肿瘤专家的不懈努力,不断改进手术方式、现更新更好的化疗药物和增加新的化疗方案,但是5年生存率一直徘徊在20%-30%左右。铂类药物是治疗卵巢癌、肺癌等实体瘤的常用药物,而DNA损伤修复能力增强是导致卵巢癌铂类耐药的主要原因之一, DNA修复途径中的核苷酸切除修复、错配修复、碱基切除修复等均与铂类药物耐药有关,而核苷酸切除修复(NER)通路被认为是机体内修复DNA损伤的最主要途径。NER通路主要基因有:①DNA损伤的识别:涉及XPA、XPC等基因;②损伤部位DNA链的打开:由TFIIH的亚基ERCC3/XPB和ERCC2/XPD解旋酶打开DNA双链;③寡核苷酸链的切断:XPG和XPF-ERCC1复合体分别在损伤部位的3’端和5’端切断DNA单链;④PCNA、RPA等基因完成缺损部位单链片段的再合成和修补缺口。其中,损伤的识别/切除是通路中的限速或调节环节。在通路中,各个基因发挥着相应的独特作用,并按照严格的顺序加入和置换出NER过程,其中任何一个基因的功能异常即可导致整个通路对损伤修复的效率。目前国内尚未见有关DNA修复基因ERCC1、XPA、XPC、XPD、XPG与卵巢癌铂类药物敏感性的关系的报道,为了了解ERCC1、XPA、XPC、XPD、XPG等基因与卵巢癌铂类药物化疗敏感性的关系,我们在本实验中应用PCR及测序方法筛查ERCC1、XPA、XPC、XPD、XPG外显子单核苷酸多态性在卵巢癌耐铂类药物的细胞株和敏感细胞株中的分布差异,探讨了XPGHis46His及XPGHis1104Asp的多态基因型102例卵巢癌患者化疗敏感性的关系,并通过构建人XPG基因的shRNA表达载体,转染卵巢癌SKOV3耐DDP细胞,检测其对XPG基因的干扰效率。继而筛选出稳定干扰的细胞株,进行一系列功能实验,以了解XPG在卵巢癌耐药细胞株中的作用。为提高卵巢癌铂类药物化疗敏感性提供一些的实验依据。卵巢上皮癌铂类耐药与非耐药细胞间核苷酸切除修复酶类基因多态性的检测与比较目的:肿瘤细胞对铂类药物的化疗敏感性与个体的DNA损伤修复能力关系密切, ERCC1、XPA、XPC、XPD、XPG基因是核苷酸切除修复系统( nucleotide excision repair,NER)中与铂类药物抵抗有关的关键因子,本实验拟筛查ERCC1、XPA、XPC、XPD、XPG遗传多态与卵巢癌耐铂类药物的细胞株的关系,为下一步实验打下基础。方法:应用PCR及测序方法筛查ERCC1、XPA、XPC、XPD、XPG外显子单核苷酸多态性在卵巢癌耐铂类药物的细胞株和敏感细胞株中的分布差异,序列读解采用ChromaS软件,并结合NCBISNP数据库寻找和验证SNP位点,筛选出的SNP用于下一步实验。结果: DNA测序分析发现XPGHis46His及XPGHis1104Asp两个SNP位点在细胞株中存在分布差异。结论:核苷酸切除修复系统中XPGHis46His及XPG His1104Asp遗传多态可能与卵巢癌患者对铂类药物耐药性相关。XPG遗传多态与卵巢癌患者铂类药物敏感性关系目的:研究核苷酸切除修复基因XPG单核苷酸多态性与卵巢癌患者对铂类药物敏感性的关系。方法:对接受含铂类药物化疗的102例晚期卵巢癌患者进行临床疗效评价。以聚合酶链-限制性片段长度多态性( PCR- RFLP)的方法检测接受含铂类药物化疗的102例卵巢癌患者XPGHis46His及XPG His1104Asp的多态基因型,并比较不同基因型与化疗敏感性的关系。结果: His1104Asp多态在卵巢癌铂类药物化疗敏感组中分布与耐药组差异不显著。XPGHis46His在化疗敏感组T/T,T/C,C/C的基因型频率明显高于耐药组,二者差异有统计学意义(p=0.016),与携带XPG46T/T基因型比较,携带至少一个46C等位基因(即T/C和C/C基因型)的卵巢癌患者对铂类药物化疗敏感可能性增加3.096倍(95%CI:1.330-7.208)。XPG His46His及His1104Asp基因多态与临床病理特征之间的比较差异无显著意义(P>0.05)。T/T基因型组生存期短于T/C+C/C基因型组,差异有统计学意义(P<0.05);COX风险比例回归模型结果显示病理分级是卵巢癌患者的独立预后因素(P<0.05)。结论:核苷酸切除修复系统中XPGHis46His遗传多态可能与卵巢癌患者对铂类药物敏感性相关。RNAi阻断XPG基因表达对卵巢上皮性癌细胞顺铂耐药性影响的体外实验研究目的:通过构建人XPG基因的shRNA表达载体,转染卵巢癌SKOV3耐DDP细胞,检测其对XPG基因的干扰效率。继而筛选出稳定干扰的细胞株,进行一系列功能实验,以了解XPG在卵巢癌耐药细胞株中的作用。方法:针对XPG mRNA的不同区域构建了不同的siRNA载体,应用脂质体法转染SKOV3耐DDP细胞,实时定量PCR检验干扰效率,选择抑制率最高的siRNA构建shRNA表达载体,转染SKOV3耐DDP细胞,G418筛选后经western-blot鉴定确认获得稳定转染的SKOV3耐DDP细胞株,然后进行细胞生长特性、细胞周期变化、细胞药敏试验及细胞内药物浓度的实验。结果:用荧光定量PCR对不同siRNA干扰载体的干扰效果进行检测,实验结果显示:在XPG-733组XPG的相对拷贝数为1.050±0.0230,与对照组相比有统计学意义,抑制率为52.05%。构建shRNA表达载体转染SKOV3耐DDP细胞,获得的该组稳定干扰表达株,western-blot检验其XPG蛋白表达明显减少。流式细胞仪对细胞检测结果显示实验组处于增殖状态(S+G2+M)的细胞为34%,G1期为66%,阴性对照组则相应为41.3%和58.7 %,其处于增殖周期的细胞有减少的趋势。实验组与阴性对照组的IC50差别有均统计学意义(P<0.05),经化疗药处理后,用高效液相色谱仪检测其细胞内药物浓度,实验组细胞内药物浓度低于对照组细胞(P<0.05)。结论:采用构建shRNA表达载体对XPG基因进行干扰,SKOV3耐DDP细胞的生长特性、细胞周期无明显变化。干扰后SKOV3耐DDP细胞对顺铂的化疗耐药性明显下降,我们推测这种改变可能与促进细胞对DNA-DDP加合物切除修复能力下降相关。更多还原

【Abstract】 The morbility of epithelial ovarian cancer is the third in malignant gynecological tumors while the mortality is the first. Ovarian conceals cavum pelvis deeply. On the early pathogenetic stage, deficiency of characteristic symptom and physical symptom make the discovery and diagnosis difficult. About 60-70% of ovarian cancers are diagnosed at an advanced stage. Although in recent 30 years, chemotherapeutics modus were discovered, and modus operandi has been modified, the 5 years survival rate is still 20-30%.Platinum drug is frequently used in the therapy of solid tumors, such as ovarian and lung cancers, and DNA damage repair capability is one of the cause of platinum drug resistance in ovarian cancer. DNA damage repair, such as nucleotide excision repair (NER)、mismatch repair、mismatch repair excision repair, is involved in platinum drug resistance. DNA lesions are preferentially repaired by the NER pathway. The mechanism of NER is quite complexity, involving in driving the process from damage recognition and removal to DNA resynthesis. Mutations in NER genes can result in many kinds of disorders. In the NER pathway, XPC, RPA and XPA are involved in the recognition step, two subunits of TFIIH, XPB and XPD, exhibit helicase activity and unwind the DNA around the lesion. After binding of XPF-ERCC1, dual incision occurs by XPG and XPF-ERCC1, which cut 3’and 5’to the damage, respectively.After that,the damage of DNA is repaired by PCNA、RPA,they completed the DNA synthesis and ligation.The relationship between DNA damage repair genes ERCC1、XPA、XPC、XPD、XPG and platinum drug resistance in ovarian cancer has not been reported in domestically so far. To understand the roles of these genes in platinum drug therapy sensibility in ovarian, PCR and sequencing were performed to screen the single nucleotide Polymorphisms of exons of ERCC1、XPA、XPC、XPD、XPG in epithelial ovarian cancer cells which were resistant or sensitive to platinum-based treatment, and analysed the the relationship between polymorphisms of XPGHis46His、XPGHis1104Asp and clinical characteristics in 102 patients with ovarian cancer, and the expression vector of shRNA was constructed and transfected into ovarian cancer cell lines to establish gene knockdown cell lines, and then study on the effect of gene silencing in these cell lines. to provide insight into how to overcome Platinum drug resistance in overian cancer Analysis of genetic polymorphisms in nucleotide excision repair system inplatinum drug resistant and non-resistant ovarian cancer cellsObjective: The tumor cell sensitivity to platinum-based chemotherapy was highly associated with DNA repair captability. ERCC1、XPA、XPC、XPD、XPG are crucial genes in nucleotide excision repair system to the sensitivity of platinum-based chemotherapy. This study was to examine the correlations of polymorphisms of ERCC1、XPA、XPC、XPD、XPG in nucleotide excision repair system and the sensitivity of platinum-based chemotherapy in epithelial ovarian cancer.Method: PCR and sequencing were performed to screen the single nucleotide Polymorphisms of exons of ERCC1、XPA、XPC、XPD、XPG in epithelial ovarian cancer cells which were resistant or sensitive to platinum-based treatment. the results were analyzed by Chromas software and NCBI SNP data bank to search and authenticate the SNPs.At last, a few SNPs were chosen for further study.Results: XPGHis46His and XPG His1104Asp had polymorphisms in cells .Conclusion: The polymorphisms of XPGHis46His and XPG His1104Asp in nucleotide excision repair system may be associated with the resistance of epithelial ovarian cancer after platinum-based chemotherapy The relation between XPG genetic polymorphism and the sensitivity toplatinum-based chemotherapy in patients with ovarian cancerObjective:The goal of this study was to investigate the relation between nucleotide excision repair gene XPG mononucleotide polymorphism and the sensitivity to platinum-based chemotherapy in patients with ovarian cancer.Methods: XPGHis46His and XPG His1104Asp were genotyped by polymerase chain reaction restrictive fragment length polymorphism ( PCR-RFLP) method in the 102 patients with advanced ovarian cancer with platinum-based chemotherapy, and the correlation of genetic polymorphisms and clinical response were analyzed .Results: The polymorphism of His1104Asp was not significantly different between response group and un-response group. The frequency of T/T,T/C and C/C genotypes in drug sensitivity group was significantly increased as compared with that in the drug resistant group, and the differences had statistical significance(p=0.016). Individuals carrying at least one 46C variant allele (T/C and C/C genotypes) tended to have an increased sensitivity (3.096 times) to ovarian cancer patients with platinum-based chemotherapy as compared with those with the XPG46T/T genotype (95%CI:1.330-7.208). The polymorphism of XPG His46His and His1104Asp in various clinicopathologic characteristics of ovarian cancer was not significantly different (p>0.05).The survival time of T/T genotype group was shorter than T/C+C/C group(p<0.05); the COX risk risk regression model showed that the patho-grade was an independent factor for prognosis of ovarian cancer(P<0.05). Conclusion: Nucleotide excision repair gene XPGHis46His genetic polymorphism may be related to sensitivity to ovarian cancer patients platinum-based chemotherapy in ovarian cancer patients .Effect of XPG silencing on platinum drug sensitivity in epithelial ovarian cancer in vitroObjective: The expression vector of shRNA was constructed and transfected into ovarian cancer cell lines to establish gene knockdown cell lines, and then study on the effect of gene silencing in these cell lines.Method: Several siRNA vectors were constructed and wrer transfected into SKOV3/DDP cell line with highly expression of XPG. Real-time PCR was used to test the efficacy of interfering RNA.Western-blot was also employed to confirm the silencing efficacy. Then growth of cells, cell cycle, cell susceptibility tests and measurement of intracellular drug concentration were tested. Results: Fluorescence quantitative PCR results show that, XPG copy number in XPG-733 group was 1.050±0.0230, significantly higher than in other three groups. Western-blot demonstraded that XPG protein expression was significant decreased in XPG shRNA vector transfected SKOV3/DDP cell line. Inhibition rate was 52.05% in XPG-733 group. FCM test showed that 34% and 66% of cells in pGPU6/GFP/Neo-XPG-733cell lines were in phase of cell proliferation (S + G2 + M) and G1, respectively while oly 41.3% and 58.7%, respectively, in control group. The growth curve in pGPU6/GFP/Neo-XPG-733 group was almost coincidence with that in control group. The difference of the IC50 to DDP level between the tow groups was significantly different (p<0.05). The intracellular drug levels in DDP inexperiment cells were significantly lower than that of the control cells measured by high performance liguid chromatograph(p<0.05).Conclusion: There are no markedly changes of the growth characteristics and cell cycle in SKOV3/DDP cell line. However, knockdown of XPG increases the sensitivity to platinum drug in SKOV3/DDP cell line.We propose that this change may be relate to the degression of DNA-DDP- adducts excision repair capability.更多还原