【作者】 温泉；

【导师】 赵玉沛；

【作者基本信息】 中国协和医科大学 ， 外科， 2006， 博士

【摘要】 研究背景：胰腺癌属高度恶性肿瘤，虽然手术切除能够提高胰腺癌患者的生存率，但多数患者就诊时已属晚期，只有20％的病人可以行手术治疗，术后5年生存率不超过5％。除手术外，放射治疗是目前治疗胰腺癌、预防术后复发、延长病人生存时间和提高生存质量的重要辅助治疗手段之一。但放射治疗对不同胰腺癌患者的疗效差别较大；而在同一个患者的治疗过程中，随着放射治疗的进行，疗效也逐渐下降，即出现了放射治疗后期肿瘤的辐射抗性与加速再增殖现象。因此如何提高放射治疗的疗效是目前胰腺肿瘤治疗领域研究的热点之一。随着肿瘤放射生物学的研究深入，目前已发现p53、p21、Bc1-2家族和survivin等基因及其表达产物与胰腺癌放射生物学特性之间有密切的关系。如，携带survivin、突变型p53基因的胰腺癌细胞对放射耐受；通过降低survivin的表达可降低了胰腺癌细胞的耐放射性。多数研究结果提示，部分促进肿瘤生长基因的高表达和抑癌基因的低表达或缺失可能与肿瘤的耐放射性有关。为了预测胰腺癌的放射治疗效果和对耐放射进行干预，我们前期工作对已建立的胰腺癌细胞株和耐放射亚株作基因芯片高通量筛查，发现在耐放射亚株增高和低表达的基因中，S100A2在耐放射细胞株与亲本株中表达差异较大，于是选其作进一步验证和深入研究。钙结合蛋白S100A2是S100A2蛋白家族中唯一的与肿瘤生长呈负相关的蛋白，由于它在正常组织中表达，而在相应的肿瘤组织和细胞中表达水平低或缺失，且表达S100A2的肿瘤侵袭及转移率低、预后好，故目前普遍认为S100A2基因为抑癌基因。既往研究表明S100A2的表达水平与细胞周期有密切关系；另外还发现S100A2参与细胞内的氧化还原反应、与氧化损伤有关；其增强的靶基因可能为抑癌基因RUNX3；其抑制的靶基因可能为促进肿瘤生长的肿瘤相关基因，其中EGFR和NFKB2与肿瘤的耐放射性有关。S100A2以上诸多特性均与肿瘤的方射生物学特性有关(如，细胞周期各时相的比例对细胞对放射线的敏感性影响较大)，因此推测其表达情况可能影响胰腺癌的放射生物学特性。但S100A2的表达情况是否与胰腺癌的放射生物学特性相关，目前尚未见报道。有必要进一步研究。研究目的：研究S100A2与胰腺癌放射生物学特性的关系。研究方法：1．检测胰腺癌细胞株及其继发耐放射亚株中S100A2 mRNA及蛋白质表达水平的差异2．检测通过去甲基化药物增强S100A2表达后相应胰腺癌细胞株放射生物学特性的改变。3．特异增强S100A2表达后胰腺癌细胞株耐放射亚株放射生物学特性的改变。4．特异使S100A2沉默后胰腺癌细胞亲本株放射生物学特性的变化。研究结果：1．胰腺癌细胞株SWl990耐放射亚株较亲本株基因S100A2的表达水平明显降低(2～3倍)。2．耐放射细胞株经5-氮杂-2-脱氧胞苷处理后S100A2的mRNA、蛋白质水平均较用药前明显升高(2～5倍)，G2期比例增高，且处理后的细胞株耐放射性明显降低。3．转基因S100A2后，SWl990耐放射亚株的G2期细胞比例明显升高，其放射耐受性明显降低。4．RNA干扰降低SW1990亲本株S100A2的表达后，其G2期细胞明显降低，G1期细胞比例升高，其耐放射性增强。研究结论1．胰腺癌细胞株SW1990继发耐放射亚株基因S100A2的表达较其亲本株明显降低。2．5-氮杂-2-脱氧胞苷使基因S100A2在胰腺癌细胞株SWl990继发耐放射亚株中重表达。3．S100A2的表达影响胰腺癌细胞株SW1990的细胞周期，主要是提高G2期细胞的比例、降低G1期细胞的比例。4．S100A2在胰腺癌继发耐放射细胞亚株中的低表达是引起其继发耐放射特性的机制之一。5．S100A2通过影响细胞周期而影响胰腺癌细胞的放射生物学特性，不排除其它机制参与。更多还原

【Abstract】 Background : Pancreatic cancer remains a common and lethal cancer with a median survival ofapproximately 6 months. Surgical resection of the primary tumour is only possible in about10% of cases as many patients have locally advanced or metastatic disease at the time ofpresentation.Only 20% of the patients are candidates for curative resection at diagnosis. For themajority of patients,treatment outcomes continue to be poor, joint treatment is important,including surgical treatments or chemotherapy, radiotherapy or interventional radiologicaltechniques. Radiation therapy is one of the important adjuvant treatments. However, clinicaltrials showed that radiotherapy response varies individually, and there were clinical reports ofradiation resistance. Our study planned to investigate different expression and significance ofgenes between parental pancreatic cancer cell lines and radioresistant sublines, to predict theclinical effectiveness of radiotherapy and to use it in intervention of radioresistant pancreaticcancer. We have successfully established radioresistant human pancreatic cancer celllines( radioresistant subline , SW1990—R). Using oligonucleotide microarrays , we haveidentified up-regulated genes and down-regulated genes to radioresistant sublines,in whichgene S100A2 were down-regulated..In this study, we determine to relate changes in the geneS100A2 expression to radio biological responses of pancreatic cancer cell lines.OBJECTIVE: The purpose of this study was to discover whether S100A2 expression changesis associated with second radioresistanc of pancreatic cell line.METHODS:①Test S100A2 mRNA and S100A2 protein in human pancreatic cell line(SW1990 and radioresistant subline, SW1990-R).②Radioresistant subline SW1990-Rcells were treated with 0.1, 1.0, 6.25, 12.5,25 and 50μmol/L 5-Aza-2’-deoxycytidinerespectively. The expression of the gene S100A2 mRNA and S100A2 protein was detected byreverse transcription polymerase chain reaction (RT-PCR) and fluorescence quantitativeanalysis . The cell cycle were analyzed by flow cytometry.The growth of the cells wasobserved by a cloning efficiency assay (Radioresistant subline SW1990-R were treated with 0.1, 1.0, 6.25, 12.5,25 and 50μmol/L 5-Aza-2’-deoxycytidine respectively ,then were exposed to0,1,2,3,5,7,and 10 Gy x-ray radiation , then cultured for 2 weeks.) .③Transfect gene S100A2to radioresistant subline SW1990-R cells, and then test gene S100A2 expression , cell cycleand radio biological responses of gene S100A2 trasfected cells.④Knock out gene S100A2 inparental SW1990 cells , and then test gene S100A2 expression , cell cycle and radio biologicalresponses of gene S100A2 kocked out cells.RESULTS:①Expression of S100A2 in radioresistant subline SW1990-R is lower than inparental cell line.②5-Aza-2’-deoxycytidine increased expression of S100A2 in radioresistantpancreatic cell line .Expression of S100A2 is associated with G2 phase of cells, S100A2increased in cells at G2 phase. Cloning efficiency assays showed that survival rate ofradioresistant cell line was lower by Rexpression S100A2.③S100A2 expression wasupregulated in transfected gene S100A2 radioresistant subline SW1990. Expression of S100A2is associated with G2 phase of cells, S100A2 upregulation in cells at G2 phase (P＜0.05).Cloning efficiency assays showed that survival rate of radioresistant subline SW1990-R waslower by Rexpression S100A2 (P＜0.05).④S100A2 expression was downregulated in geneS100A2 kocked out parental SW1990 cell line, which reduced the proportion of knocked-outgene cells at G2 phase (P＜0.05). Cloning efficiency assays showed that survival rate ofknocked-out gene cells was higher by downregulating the expression of gene S100A2 (P＜0.05).CONCLUSION:①Expression of S100A2 in radioresistant subline SW1990-is lower than in parentalline SW1990.②5-Aza-2’-deoxycytidine may reexpress gene S100A2 in radioresistant sublineSW1990-R, which diminished the radioresistance of radioresistant subline SW1990-R.③Expression of S100A2 is associated with G2 phase of cells, which affects the radio biologicalresponses of pancreatic cancer cell line.④Don’t exclude that S100A2 affects the radiobiological responses of pancreatic cancer cell line through other mechanisms.更多还原