【作者】 赵德平；

【导师】 徐志飞；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 研究背景肺癌已经成为世界范围内最主要的肿瘤死亡原因,大约31%与癌症相关的死亡是由肺癌造成的。在世界范围内,肺癌患者的5年生存率只有大约15%。目前肺癌存活率低主要在于基于症状的诊断方法对发现早期肺癌并不十分有效,3/4的患者在确诊时肺癌已发生转移。若能在早期检测到肺癌病变,患者5年生存率可提高到60-80%。目前肺癌的化疗仍主要是以铂类药为主的化疗,治疗失败的主要原因之一是肿瘤对化疗药的耐药,特别是对于复发性的肿瘤,耐药性较高(3)。NSCLC对铂类药的耐药是导致治疗失败的主要原因,核苷酸切除修复交叉互补组1(ERCC1)阳性表达与铂类药的耐药密切相关。Fanconi Anemia(范可尼贫血)是一种常染色体隐性遗传病,以先天性发育异常、进行性贫血和易发生肿瘤为特征。目前在范可尼贫血患者细胞中发现了15种变异基因,组成了FA-BRCA通路,简称FA通路。FA患者超高的肿瘤患病率提示了FA通路是一个肿瘤抑制通路。研究发现FA通路与细胞周期中分裂期的DNA同源重组和细胞周期检查点有关,FA通路功能障碍引起的DNA修复能力降低导致肿瘤发生的几率增高,同时又使肿瘤对于影响DNA交联的药物有高度敏感性。有研究发现ERCC1参与了FA通路的功能,可以激活FA通路。当通过基因敲除使ERCC1失活后,FA通路发生功能障碍,丝裂霉素C处理后没有出现FANCD2的泛素化。研究还发现当应用药物丁酸苯酯破坏FA通路的完整性之后,原先对顺铂耐药的头颈部肿瘤细胞又重新敏感起来。对于FA通路功能正常的肺癌细胞,应用丁酸苯酯破坏其FA通路后,若其对顺铂的敏感性提高,对于肺癌的化疗将提供一种新的思路。研究目的观察FA通路功能障碍在非小细胞肺癌(NSCLC)中的表达情况;FA通路功能障碍能否造成NSCLC细胞染色体不稳定。FA通路功能障碍能否增加NSCLC对顺铂的敏感性;苯丁酸钠能否增加FA通路功能正常的NSCLC对顺铂的敏感性。研究内容和方法选择50例经过铂类药物化疗2个疗程的NSCLC组织病理切片,免疫组化检测FANCD2泛素化的表达,另取5例正常肺组织切片作为对照;选择10株NSCLC细胞株,Western-blotting检测其经过顺铂处理后FANCD2泛素化程度;选择FA通路功能正常和受损的2株NSCLC细胞株(Ht182和A549),染色体核型实验观察其染色体断裂情况;以判断FA通路功能障碍在NSCLC中的表达情况。选择FA通路功能正常和受损的3株NSCLC细胞,FA通路功能障碍的Ht 182、UMC11以及FA通路功能正常的A549细胞,用不同药物处理后在不同时间点进行细胞计数观察其生存曲线;药物分组:生理盐水组;苯丁酸钠组;顺铂组;顺铂加苯丁酸钠组。上述三种细胞用顺铂处理7天后台盼蓝染色,显微镜下观察并计数细胞集落生长的情况。通过上述实验观察NSCLC细胞对顺铂的敏感性以及苯丁酸钠能否改变其敏感性。研究结果在50例NSCLC组织中,有14例(28%)未出现FANCD2泛素化表达,即其存在FA通路功能障碍。FA功能障碍的发生与癌细胞的分化程度有关(P=0.003),但与性别、年龄、病理类型和P-TNM分期无关。在10株NSCLC细胞株中,经顺铂处理后有4株细胞(UMC11、HT182、HOP62和Calu6)的FANCD2泛素化程度明显较低或未出现,即存在FA通路功能障碍;染色体核型分析表明FA通路功能受损的Ht182中,其染色体断裂出现的几率(44.74%)明显高于FA通路功能正常的A549细胞(2.27%), P<0.005。生存曲线实验表明,FA通路受损的Ht182和UMC11经顺铂处理168小时后,其生存率明显低于FA通路正常的A549细胞(P=0.027,0.016)。与生理盐水组相比,苯丁酸钠本身对于三种肺癌细胞的生存率未产生明显影响。经过苯丁酸钠处理后,FA通路功能正常的A549细胞对顺铂的敏感性明显提高(P=0.036),但未改变FA通路功能障碍的Ht182和UMC11对顺铂的敏感性(P=0.431,0.774)。实验结论本研究证实了在部分NSCLC中存在FA通路功能障碍。FA通路功能障碍发生几率与NSCLC的细胞分化程度有关,细胞分化程度越低,发生几率越高;但与年龄、性别、病理类型和临床病理分期无关。FA通路功能障碍的NSCLC细胞对顺铂的敏感性明显提高。应用苯丁酸钠可以提高FA通路功能正常的NSCLC细胞对顺铂的敏感性。更多还原

【Abstract】 BackgroundLung cancer has been the most important dead reason of all cancers in the world. About 31% of cancer related death is caused by lung cancer. The 5 year survival rate of lung cancer is about 15%. The low survival rate of lung cancer is mainly caused the late diagnosis based on symptom. 3/4 of the patients have metastasis tumor when diagnosed. If the lung cancer patients can be diagnosed in early stage, the 5 year survival rate can raised to 60-80%.Till today the chemotherapy of lung cancer is based on platins. The main reason of treatment failure is resistance of chemotherapy drugs, especially in recurrence tumor. Excision repair cross complementing 1(ERCC1) expression is found to have a close relationship to platinum resistance.Fanconi Anemia is a genetic disease characterized by malformation, anemia and easier to develop tumor. To date 15 variance genes were found in FA patients , the 15 genes composed the FA-BRCA pathway, which also is called FA pathway.. The super high incidence of cancer in FA patients revealed that the FA pathway is a tumor restriction pathway. FA pathway is found to related to S-phase homologous recombination and cell cycle checkpoint. Dysfunction of FA pathway will cause the lower ability of DNA repair, this rendered the high incidence of tumor and the high sensitivity to drugs which impact the DNA interaction. Experiments have found that ERCC1 is involved in FA pathway function and can activate FA pathway. When the ERCC1 is blocked by gene knock out, the FA pathway function will be discounted. Another experiment found that when destroy the FA pathway function by Sodium Phenylbutyrate, the head and neck cancer cells will be sensitive to cisplatin again. We expected to use Sodium Phenylbutyrate to destroy the FA pathway of NSCLC cells and this maybe will make NSCLC cells more sensitive to cisplatin. The idea may find a new way to settle the drug resistance problems of cisplatin. Object:To observe the FA pathway dysfunction expression rate in Non-small Cell Lung Cancer. Find out whether FA pathway dysfunction could make NSCLC cells more sensitive to Cisplatin, and whether Sodium Phenylbutyrate could change the sensitivity to Cisplatin in NSCLC cells.Materials and methods:50 NSCLC tissue samples after Cisplatin chemotherapy were collected, and the FANCD2 ubiquitination level was tested by immunohistochemistry. We also tested the FANCD2 ubiquitination level by Western-blotting in 10 NSCLC cell lines after Cisplatin treatment. Karyotype assay was used to observe the chromosome breakage in cell line A549 and Ht182.To observe the sensitivity to Cisplatin and Sodium Phenylbutyrate, cell survival assay were used in cell line A549, UMC11 and Ht182. And colongenic assay were selected to observe the ability of colony formation in the same three cell lines.Results:Among 50 NSCLC tissue samples, 14(28.0%) were found to have no FANCD2 ubiquitination expression. This means FA pathway dysfunction occurred in those 14 NSCLC tissues. The FA pathway dysfunction is related to the differentiation(P=0.003), but has no relationship to age, sex, P-TNM and histology. The FANCD2 ubiquitination level was obviously lower in 4 cell lines among 10. Karyotype assay shows that the chromosome breakage rate is much higher in the FA pathway dysfunction Ht182 cells(44.74%) than that in A549 cells(2.27%), which has a normal FA pathway(P<0.005).Cell survival assay showed that 168 hours after Cisplatin treatment, the survival rates of Ht182 and UMC11 were much lower than that of A549, the P level were 0.027 and 0.016 seperately. Controlled with NS, Sodium Phenylbutyrate did not change the survival rate in three NSCLC cell lines. Sodium Phenylbutyrate maked the A549 cells more sensitive to Cisplatin(P=0.036),but the same phenomenon was not seen in Ht182 and UMC11 cells.Conclusion:FA pathway dysfunction expresses in part of NSCLC cells, the expression rate is associated with poorly differentiated NSCLC cells. FA pathway dysfunction makes the NSCLC cells more sensitive to cisplatin. Sodium Phenylbutyrate can improve the sensitivity to cisplatin in NSCLC cells which have a normal FA pathway.更多还原