【作者】 张玉琦；

【导师】 金震东； 李兆申； 邹多武； 高军；

【作者基本信息】 第二军医大学 ， 内科学， 2008， 博士

【摘要】 胰腺癌是恶性程度最高的肿瘤之一,发病率和死亡率位居我国恶性肿瘤的前10位,且有逐年增高趋势。预后极差,近50年来,尽管传统的治疗手段如手术、放疗、化疗或这些方法的联合应用取得许多重大进展,但对胰腺癌患者的预后几乎没有显著改善,胰腺癌死亡率依然近乎等于发病。表观遗传学(Epigenetics)改变导致的基因表达改变是肿瘤发生的重要机制,包括DNA甲基化(DNA methylation)以及组蛋白去乙酰化(Histone Deacetylation)等。DNA甲基化转移酶抑制剂(DNAmethyltransferases inhibitors,DNMTi)以及组蛋白去乙酰化酶抑制剂(histonedeacetylase,inhibitors,HDACi)被证明在多种肿瘤中具有治疗作用,可以使肿瘤中的抑癌基因重新表达,诱导细胞分化、抑制生长,诱导凋亡等,但其具体的分子作用机制仍不清楚。本课题旨在探讨DNA甲基化转移酶抑制剂5-AZA-dC(5-aza-deoxycytidine)和组蛋白去乙酰化酶抑制剂曲古菌素(trichostatin A,TSA)联合应用对胰腺癌的治疗作用。通过体外试验探讨对胰腺癌细胞增殖、凋亡和细胞周期及其相关基因的影响,同时检测增殖和凋亡相关基因表达的变化,体内试验观察对胰腺癌裸鼠移植瘤生长的抑制作用。初步探讨其在治疗胰腺癌的作用及其作用机制。一、HDAC1蛋白和DNMT1蛋白在人胰腺癌组织中的表达目的检测HDAC1蛋白和DNMT1蛋白在胰腺癌组织和癌旁组织中的表达情况,并对其在组织中表达的一致性进行相关性分析。材料和方法胰腺癌组织及相应癌旁组织标本23对。采用生物素-过氧化酶(streptavidin peroxidase,SP)免疫组织化学法检测胰腺癌和癌旁组织HDAC1蛋白和DNMT1蛋白表达,分析肿瘤组织与癌旁组织的阳性率差别;同一组织来源相邻切片进行配对,对HDAC1蛋白及DNMT1蛋白阳性率进行相关性分析。结果HDAC1蛋白及DNMT1蛋白均在胰腺导管细胞癌组织细胞核表达,染色为棕黄色,癌旁组织中多为阴性。胰腺癌与癌旁组织HDAC1蛋白表达阳性率分别为56.21%±10.26%和6.24%±5.28%,DNMT1蛋白表达阳性率分别为54.83%±23.33%和6.30%±10.78%,胰腺癌与癌旁组织阳性率均有极显著性差异(P=0.00)。相关性检验显示HDAC1蛋白与DNMT1蛋白表达相关系数0.713,有极显著统计学差异(P=0.00)。结论HDAC1蛋白及DNMT1蛋白在胰腺癌组织中的表达呈特异性,且其表达显著相关,提示HDAC1及DNMT1可能共同参与了胰腺癌的发生和发展。二、TSA、5-AZA-dC对人胰腺癌细胞株增殖、凋亡和细胞周期的影响目的研究TSA、5-AZA-dC对体外培养的人胰腺癌细胞株增殖、凋亡和细胞周期的影响,观察DNA甲基化转移酶抑制剂与组蛋白去乙酰化抑制剂是否具有协同抑制肿瘤的作用。方法分别以0.1、0.5、2.0μmol/L浓度TSA干预胰腺癌PaTu8988细胞株24小时,0.5、2.0、10.0μmol/L浓度5-AZA-dC干预胰腺癌PaTu8988细胞株72小时后,以及2种药物联合治疗,按照给药浓度不同分为联合低、中、高浓度组4组,分别给予5-AZA-dC 0.5、2.0及10μmol/L,干预48小时,最后24小时给予TSA 0.5μmol/L。采用WST-8法检测细胞增殖变化,流式细胞术检测细胞周期及凋亡的改变。结果1.细胞增殖试验:TSA0.1、0.5、2.0μmol/组细胞存活率分别为95.11%±4.36%、77.84%±13.58%、69.78%±7.04%,与对照组相比差异有显著统计学意义(P＜0.05)。5-AZA-dC 0.5、2.0和10.0μmol/L组细胞存活率分别为94.91%±1.51%、88.19%±3.28%、81.93%±4.89%,其中2.0和10.0μmol/L组与对照组相比差异有显著统计学意义(P＜0.01),三组间相比有显著统计学意义(P＜0.05)。联合治疗低、中和高浓度组细胞存活率分别为86.69%±9.07%、72.86%±4.19%及57.20%±9.67%,与对照组比较,低浓度组有显著统计学差异(P＜0.05)而另二组均有极显著统计学差异(P＜0.01),提示联合治疗较单独用药可以达到更好的治疗效果2.流式细胞术检测细胞周期:TSA组G2/M期细胞百分率、凋亡细胞百分率均显著高于对照组(均P＜0.01),表现为G2/M期阻滞及细胞凋亡率增加。5-AZA-dC组G2/M及S期细胞百分率均高于对照组(P＜0.05),亦表现为G2/M期阻滞。结论TSA及5-AZA-dC均能有效地抑制胰腺癌PaTu8988细胞株的增殖,TSA及5-AZA-dC均通过阻断细胞周期停滞于G2/M期,诱导细胞凋亡。三、TSA、5-AZA-dC对增殖、凋亡及细胞周期相关基因表达的影响目的TSA、5-AZA-dC干预胰腺癌细胞株出现细胞周期阻滞并诱导细胞凋亡,但其发生的分子机制尚不清楚,通过Real-time RT-PCR检测药物干预前后细胞周期及凋亡相关基因及蛋白表达的改变,并检测DNMT酶总体活性的变化,探讨抑制肿瘤细胞增殖、诱导凋亡的作用机制。材料和方法TSA及5-AZA-dC干预胰腺癌细胞株,抽提细胞株总RNA,Real-time RT-PCR检测细胞周期相关基因p21和Cyclin D1、凋亡相关基因Bax和Bcl-2的表达变化,Western Blot检测蛋白表达,抽提核蛋白检测DNMT酶总体活性。结果1.细胞周期相关基因改变:与对照组比较,TSA组p21表达明显升高,Cyclin D1表达则明显降低,有显著性统计学差异(P＜0.05);5-AZA-dC组p21表达升高,其中2.0μmaol/L及10.0μmol/L浓度有显著性统计学差异(P＜0.05),Cyclin D1表达则略降低,但无显著性统计学差异。Western Blot检测相应的蛋白表达与其基因改变呈一致性。2.凋亡相关基因改变:与对照组比较,TSA组Bax表达无显著变化,Bcl-2表达则呈下降趋势,2.0μmol/L浓度与对照组比较显示有显著性统计学差异(P＜0.05);5-AZA-dC组Bax表达呈升高趋势,但仅有10.0μmol/L有统计学差异(P＜0.05),而Bcl-2表达虽有降低,但无显著性统计学差异。Western Blot检测相应的蛋白表达与其基因改变呈一致性。结论TSA及5-AZA-dC通过上调p21和/或下调Cyclin D1基因表达,导致其相关蛋白发生表达量改变,调控胰腺癌PaTu8988细胞周期,抑制细胞增殖和分化,诱导细胞凋亡,从而抑制了胰腺癌PaTu8988细胞株的生长。四、TSA、5-AZA-dC对胰腺癌细胞裸鼠移植瘤的抑制作用目的评价TSA、5-AZA-dC对胰腺癌细胞裸鼠移植瘤生长的影响,探讨TSA、5-AZA-dC的抗肿瘤治疗价值。方法将人胰腺癌细胞PaTu8988接种至裸鼠背部皮下,随机分为对照组、TSA组、5-AZA-dC组和联合组4组,单次给药剂量分别为PBS 0.2ml、TSA1mg/kg、5-AZA-dC 3mg/kg和TSA 1mg/kg+5-AZA-dC 3mg/kg,以腹腔注射途径给药,隔日1次,共5次,观察裸鼠生命体征变化以及肿瘤大小的动态变化。实验结束后处死裸鼠切除肿瘤标本和脏器组织,进行免疫组化和HE染色,Real-time RT-PCR检测裸鼠移植瘤组织增殖及凋亡相关基因的表达。结果1.移植瘤生长曲线显示与对照组比较,TSA与5-AZA-dC均可使肿瘤生长速度减慢,肿瘤体积在各个时间点均小于对照组,联合治疗组对肿瘤的抑制作用最为明显,其次为TSA,各组与对照组相比差异具有显著性(P＜0.05),联合治疗组与对照组相比具有极显著统计学差异(P＜0.01)。2.细胞周期及凋亡相关基因mRNA改变:与对照组比较,TSA组p21明显升高(P＜0.01),Cyclin D1明显降低(P＜0.05),Bax无显著变化,Bcl-2呈下降趋势;5-AZA-dC组p21升高(P＜0.05),Cyclin D1略降低,Bax呈升高趋势,Bcl-2无显著变化。结论1.体内试验显示TSA、5-AZA-dC对人胰腺癌裸鼠移植瘤生长具有抑制作用,联合用药对肿瘤的抑制作用优于单独用药。2.TSA与5-AZA-dC通过增强p21基因和抑制Cyclin D1基因导致肿瘤生长抑制。更多还原

【Abstract】 Pancreatic cancer is one of the most malignant gastrointestinal tumors,characterized by a poor prognosis,absence of specific clinical symptoms and signs,prone to metastases and lack of response to conventional therapy.The morbidity and mortality of this disease are increasing in the whole worldwide.It is important to go on basic research and identify new therapeutic agents for fighting this devastating disease.There is growing evidence that gene expression govemed by epigenetic changes is crucial to the onset and progression of cancer.Epigenetics is the study of heritable changes in gene expression that are not coded in the DNA sequence itself.The best studied epigenetic mechanisms are DNA methylation and post-translational histone.DNA methylation and histone deacetylation are interconnected in gene silencing.Furthermore,a much more direct connection between DNA methylation and histone deacetylation exists by direct interactions between DNMTs and HDACs.HDAC and DNMT inhibitors have emerged as the accessory therapeutic agents for multiple human cancers,since they can block the activity of specific HDACs and DNMTs,restore the expression of some tumor suppressor genes and induce cell differentiation,growth arrest and apoptosis.The use of novel DNA methyltransferase inhibitors in clinical trials that allow monitoring of drug-induced DNA methylation changes should provide the foundation for improved epigenetic cancer therapies.This paper is to investigate the antitumor activity of HDACi(trichostatin A,TSA) and DNMTi (5-azadecitabine,5-AZA-dC) in pancreatic cancer.1.Expression of HDACI and DNMT1 proteins in Pancreatic Cancer TissuesObjective To detect the expression of HDAC1 and DNMT1 protein in pancreatic cancer and para-cancerous tissues,and investigate the correlation between HDAC1 and DNMT1 expression.Methods and materials Pancreatic carcinoma tumor tissue and paired para-cancerous tissues were obtained from 23 pancreatic carcinoma patients.Clinical data were collected. HDAC1 and DNMT1 protein expression was detected by streptavidin peroxidase immunohistochemistry.The correlation between HDAC1 and DNMT1 expression was analyzed.Results Positive cells were brown.Specific HDAC1 and DNMT1 staining were located in the nuclei of the cancer cells,while it was rarely seen in para-cancerous lesions. The positive rate of HDAC1 and DNMT1 protein expression in human pancreatic carcinoma tissues and para-cancerous tissues was 56.21%±10.26%vs 6.24%±5.28%and 54.83%±23.33%vs 6.30%±10.78%(P=0.000) respetively.Coefficient correlation of HDAC1 and DNMT1 expression between carcinoma tissues and para-cancerous tissues was significant at the 0.01 level(2 tailed).Conclusions In pancreatic carcinoma tissues HDAC 1 and DNMT1 protein was highly expressed,over-expression of HDAC1 protein and DNMT1protein might have an important role in the formation of tumor.2.Effects of TSA,5-AZA-dC on Proliferation and Apoptosis in Pancreatic Cancer Cell PaTu8988Objective To study the growth arrest and apoptotic effects as well as the inhibitory effects of TSA,5-AZA-dC in human pancreatic cancer cell line PaTu8988.Methods The growth suppression effect of TSA,5-AZA-dC on PaTu8988 were studied using Cell Count Kit(CCK-8);Apoptosis and cell life cycles were examined by Flow cytometry.Results 1.TSA and 5-AZA-dC inhibited the growth and proliferation of PaTu8988 cells in a concentration-dependent manner,which was showed by the growth curve.The proliferation ratios of cells treated with TSA(0.1,0.5,2.0μmol/L for 24h) were 95.12%±4.33%,77.84%±13.58%and 69.83%±7.04%respectively,5-AZA-dC(0.5,2.0, 10μmol/L for 72h) 94.91%±1.51%,88.19%±3.28%and 81.93%±4.89%respectively, therapeutic alliance groups(lower,mild and high dose) 86.69%±9.07%,72.86%±4.19%, 57.20%±9.67%respectively.2.The ratios of cell apoptosis was induced by TSA and 5-AZA-dC in a dose-dependent manner.The cells showed G2/M arrest after treated with TSA and 5-AZA-dC.Conclusions TSA and 5-AZA-dC inhibited PaTu8988 cell in the concentration -dependent manner.Increasing apoptosis of PaTu8988 cells may contribute to the anti-tumor properties of TSA and 5-AZA-dC.3.Expression of HDAC and DNMT genes in Pancreatic Cancer TissuesObjective To investigate the expression of p21,Cyclin D 1,Bax and Bcl-2 genes in as well as the inhibitory effects of TSA,5-AZA-dC in human pancreatic cancer cell line PaTu8988. Materials and Methods The expression of p21,Cyclin D1,Bax and Bcl-2 genes of cell lines PaTu8988 treated by TSA,5-AZA-dC was detected by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR).Protein was detected by western blot method,and the DNMT activity was detected by assay kit.Results 1.The relative quantitation(RQ) values of p21 and Bax genes of cells treated with TSA(0.1,0.5,2.0μmol/L for 24h) were obviously higher than control group,while Cyclin D1 and Bcl-2 were lower than control group.The expression of p21,Cyclin D1, Bax and Bcl-2 protein was at equal pace with mRNA.2.The DNMT activity of cell lines after treated with 5-AZA-dC was obviously decreased.Conclusions The inhibition and apoptosis of cell lines treated with TSA maybe caused by up-regulation of p21 and Bax genes expression or down-regulation of Cyclin D1 and Bcl-2 genes expression.4 Growth Suppression Effect of TSA and 5-AZA-dC on the Growth of Xenograft of Pancreatic Cancer in Nude MiceObjective To study the suppression effect of TSA and 5-AZA-dC on the pancreatic cancer xenograft models,in order to evaluate the potential role of HDACi and DNMTi as a novel anti-neoclassic agent for the treatment of pancreatic cancer.Methods 10 million PaTu8988 cells were injected into the back of BAL B/c nude mice.Once visible tumors were evidenced about 150mm~3,the animals were divided equally into 4 groups(6 animals/group) and treated with TSA,5-AZA-dC or control vehicle(PBS) by intraperitoneal injection once every 2 days for 5 times:Group A:control vehicle 0.2ml/animal;Group B:TSA 1mg/kg;Group C:5-AZA-dC 3mg/kg;Group D: TSA 1mg/kg and 5-AZA-dC 3mg/kg.The tumor volumes were measured every 5 days. The tumor xenografts and organs were removed,photographed and weighed.Using HE (hematoxylin-eosin) stain to show morphology,p21,Cyclin D1,Bax and Bcl-2 mRNA were measured by Real-time RT-PCR.Results 1.The growth of tumor xenograft was suppressed potently when administered with different drugs.Compared with control group,the mean tumor size in treated groups was reduced markedly at every point of test time.TSA caused profound tumor regression, some xenograft was even disappeared;5-AZA-dC can also decrease the tumor size,but the effects were not as remarkable as TSA.Antitumor result of combination TSA and 5-AZA-dC had much better effects than TSA or 5-AZA-dC.2.The relative quantitation (RQ) values of p21 and Bax genes of cells treated with TSA were obviously higher than control group,while Cyclin D1 and Bcl-2 were lower than control group.Conclusions TSA and 5-AZA-dC can attenuate the growth of pancreatic caner xenograft.The growth suppression effect of TSA in xenograft may partially due to its up-regulation the expression of p21 and Bax.TSA and 5-AZA-dC may exert a synergistic antitumor effect.更多还原