【作者】 郭庆渠；

【导师】 吴育连；

【作者基本信息】 浙江大学 ， 外科学， 2009， 博士

【摘要】 1.研究背景与目的:胰腺癌(Pancreatic cancer)以早期发现困难,病情发展迅速为特征而成为预后极差的消化道恶性肿瘤之一。手术切除是唯一根治的手段。但是,多数患者就诊时已属晚期,只有10%～15%的患者有手术切除机会;手术切除的病人术后也多发生复发和转移,故此胰腺癌的预后极差,总体5年生存率低于5%,确诊后平均生存时间不超过6个月。化疗是胰腺癌重要的辅助治疗手段,对于改善病人生存质量,延长生存期有一定作用。目前,吉西他滨是胰腺癌化疗的一线药物,但总体有效率低于20%。主要问题在于多数胰腺癌病人对吉西他滨产生耐药。因此寻找胰腺癌吉西他滨耐药基因和增加吉西他滨的疗效是临床急需要解决的问题。Emodin是从自然植物中提取的一种活性物质,具有抗炎,抗肿瘤及抗菌作用,同时,Emodin是酪氨酸激酶Ⅱ的抑制剂。我们采用Affymetrix公司的HG U133A 2.0芯片检测未经处理的胰腺癌吉西他滨耐药细胞株(Panc-1)和非耐药细胞株(Bxpc-3)基因表达谱的变化,筛选鉴定出胰腺癌吉西他滨耐药基因,通过一系列实验探讨了Emodin增强吉西他滨对胰腺癌细胞株生物学行为的作用,并进一步分析Emodin增强吉西他滨治疗胰腺癌的作用机制。2.材料和方法:2.1.采用Affymetrix公司的HG U133A 2.0寡核苷酸基因芯片,检测胰腺癌吉西他滨耐药细胞株(Pant-1)和非耐药细胞株(Bxpc-3)基因表达谱,筛选出胰腺癌吉西他滨耐药基因。并利用荧光定量PCR对选择的芯片结果中差异表达的基因进一步验证。2.2.采用MTT试验调查不同浓度Emodin和吉西他滨在不同时间对胰腺癌细胞活力的影响,初步确定下一步实验浓度。MTT方法检测Emodin和吉西他滨联合作用48h对胰腺癌细胞活力的影响。2.3.采用流式细胞术,caspase3酶活性试验及检测PARP的剪切来观察Emodin和吉西他滨及联合用药对胰腺癌细胞凋亡的影响。2.4.对数期生长的Bxpc-3细胞每只0.1ml(7×10~6)接种于4-6周龄20g雄性BALB/C nu/nu裸小鼠背部皮下,制成胰腺癌种植瘤模型,待肿瘤全部长出后随机分为4组,分别给予生理盐水,Emodin(40 mg/kg),吉西他滨(125 mg/kg),及两药联合,每周二次腹腔注射,连续2周。每周测2次肿瘤大小及动物体重,研究Emodin在体内增强吉西他滨治疗胰腺癌作用。2.5.分别采用缺口末端标记法(TUNEL)和免疫组织化学检测Emodin在体内增强吉西他滨诱导胰腺癌细胞凋亡和抑制细胞增殖。2.6.采用RT-PCR、qRT-PCR和Western blot方法探讨Emodin对选择的胰腺癌吉西他滨耐药基因的影响。分析Emodin增强吉西他滨治疗胰腺癌的作用机制。2.7.统计学分析:数据表达为(?)±SE,进行双侧t检验或单因素方差分析(ANOVA,LSD多重比较),P＜0.05视为具有统计学意义。统计由SPSS 13.0统计包完成。3.结果:3.1鉴定胰腺癌吉西他滨耐药相关基因采用HG U133A 2.0寡核苷酸基因芯片检测两细胞株基因表达谱,鉴定相互2887各表达有差异的基因。与非耐药细胞株(Bxpc-3)比较,耐药细胞株(Panc-1)5倍及5倍以上差异基因有141个。高表达基因和低表达基因分别有103个和38个。3.2 qRT-PCR验证基因芯片结果为了验证基因芯片结果,应用qRT-PCR检测两种细胞中22个耐药细胞株(Panc-1)5倍以上过表达的基因。结果显示21个基因两种方法所得基因表达倍数大致相当。3.3 Emodin在体外增加吉西他滨对胰腺癌细胞生长的抑制为了探讨Emodin对胰腺癌细胞生长的影响,应用MTT方法检测不同浓度的Emodin(0-160μM)作用于胰腺癌细胞24h、48h和72h的效应。结果显示随着Emodin浓度和作用时间的增加,细胞活力呈下降趋势。随后的实验揭示,和单用药比较,给予48小时Emodin(Bxpc-3和Mia Paca-2,40μM;Panc-1,80μM)在体外可显著增强吉西他滨(Panc-1和Mia Paca-2,5μg/ml;Bxpc-3,0.51μg/ml)抑制胰腺癌细胞的活力(P＜0.05)。3.4 Emodin在体外增加吉西他滨诱导胰腺癌细胞凋亡胰腺癌细胞接受Emodin(Bxpc-3,40μM;Panc-1,80μM)、吉西他滨(Panc-1,5μg/ml;Bxpc-3,0.5μg/ml)和联合处理24h后,流式细胞术显示联合治疗引起凋亡细胞比例显著增多(P＜0.05),联合治疗后Caspase3酶活性及PARP剪切片断增加,提示Emodin能够增加吉西他滨诱导的细胞凋亡。3.5 Emodin在体内增加吉西他滨对胰腺癌种植瘤生长的抑制治疗结束后,联合治疗组的肿瘤体积显著小于对照组和吉西他滨组(P＜0.05),而所有动物无死亡,各组间的体重在治疗期间无显著差异,肝肾功能无差异,显示良好的耐受性。与对照组和单用药比较,联合治疗可以明显提高胰腺癌细胞的凋亡,并降低增殖基因Ki-67的表达(P＜0.05)。3.6 Emodin在体外增加吉西他滨疗效的机制采用RT-PCR、qRT-PCR和Western blot方法发现吉西他滨可以上调survivin的表达,而Emodin通过下调胰腺癌细胞吉西他滨耐药相关基因HS3ST1,AKRC13和survivin,达到增强吉西他滨疗效的作用。4.结论4.1利用基因芯片技术,筛选出了一些显著的胰腺癌吉西他滨耐药基因4.2 Emodin在体外对胰腺癌细胞有明显的诱导凋亡及抑制细胞生长作用,并能增强吉西他滨的效果。4.3 Emodin能够增强吉西他滨体内抑制胰腺癌生长作用。4.4 modin能调控胰腺癌细胞耐药基因的表达,通过调控胰腺癌细胞耐药基因达到增强吉西他滨作用。更多还原

【Abstract】 1.Background and objective:Pancreatic cancer is a highly aggressive malignant tumor,which has a characteristics of late clinical presentation,advanced stage and extremely poor prognosis.The curative treatment for pancreatic cancer is surgery,but only 10-15% of patients can be diagnosed in surgically resectable stages.Postoperative patients often accompany with recurrence and metastasis of tumor.Thus,the prognosis of patients with pancreatic cancer is extremely poor,with a 5-year survival rate of＜5% and a total median survival of 6 months.Chemotherapy is an importantly adjunctive therapy to pancreatic cancer patients,which is helpful to prolonging the median survival and improving quality of life.Currently,gemcitabine remains the first line chemotherapeutic agent available for treatment of advanced pancreatic cancer. However,single gemcitabine treatment has a response rate of less than 20%and is associated with drug resistance.Therefore,it is desirable to find predictor of gemcitabine chemoresistance and to develop strategies for overcoming gemcitabine chemoresistance.Emodin(1,3,8-trihydroxy-6-methylanthraquinone) is a natural anthraquinone derivative isolated from natural plant.Pharmacological studies have demonstrated that emodin possesses variously biological function,such as anti-bacterial,antiinflammatory, anti-cancer and a potent inhibitor of the Casein KinaseⅡ.Gene expression of nontreated pancreatic cancer gemcitabine-resistant cell lines (Panc-1) was compared with nontreated gemcitanbine-sensitive cell lines(Bxpc-3) by using GeneChip Human Genome U133A.A number of pancreatic cancer gemcitabine chemoresistant genes which were identified in the current study.Further studies showed that emodin enhanced the apoptotic effects of gemcitabine in pancreatic cancer cells. The mechanism by which emodin enhances the antitumor activity of gemcitabine in pancreatic cancer cells was investigated.2.Materials and methods:2.1 Oligonucleotide array hybridizationGene expression of nontreated pancreatic cancer gemcitabine-resistant cell lines (Panc-1) was compared with nontreated gemcitanbine-sensitive cell lines(Bxpc-3) by using GeneChip HG U133A 2.0 arrays.Pancreatic cancer gemcitabine chemoresistant genes were identified.Gemcitabine chemoresistant genes selected in microarray data were confirmed by Real-time PCR analyses.2.2 Cell growth assayEffects of emodin(0-160μM) and gemcitabine(0-50μg/ml) on growth of pancreatic cancer cells cultured for 24h,48 h and 72h were examined using MTT assay. The concentration of drug in next test was determined.Subsequent researches were done to examine the effect of a combination of emodin(Bxpc-3 and Mia Paca-2,40μM; Panc-1,80μM) and gemcitabine(Panc-1 and Mia Paca-2,5μg/ml;Bxpc-3,0.5μg/ml) for 48h on cell viability by MTT assay. 2.3 ApoptosisPotentiation of gemcitabine-induced apoptosis by emodin was evaluated by using Annexin V/propidium iodide flow cytometry,caspase 3 activities and PARP cleavage.2.4 Tumor growth and treatments7×10~6 Bxpc-3 cells collected in 100μl serum-free RPMI-1640 media in log phase growth were injected subcutaneously in the backs of 20 g athymic nu/nu male mice 4-6 weeks old.Once rumor masses became established and palpable,animals were randomized to receive intraperitoneal(IP) injections of vehicle(0.9%sodium chloride), emodin(40 mg/kg) alone,gemcitabine(125 mg/kg) alone,or emodin and gemcitabine in combination twice per week for 2 weeks.Tumor volumes and body weight were measured twice per week to evaluate the potentiation of antitumor activity of gemcitabine by emodin in vivo.2.5 TUNEL and immunohistochemical analysisPotentiation of apoptotic activity and cell growth inhibition of gemcitabine by emodin in vivo was investigated by TUNEL and immunohlstochemical analysis.2.6 Western blotting,RT-PCR and qRT-PCREffects of emodin in combination with gemcitabine on the expression levels of genes associated with gemcitabine chemoresistance in pancreatic cancer cells were analyzed by western blotting,RT-PCR and qRT-PCR.The mechanism by which emodin enhances the antitumor activity of gemcitabine was probed.2.7 Statistical analysisData were expressed as mean values±SE and analyzed by a two-tailed t-test or ANOVA followed by the LSD’s multiple comparison with P＜0.05 considered significant.Analyses were performed using SPSS 13.0 statistical software package. 3.Results:3.1 Identification of genes associated with gemcitabine chemoresistanceIn gene expression microarray analysis,the differential expression 2887 well-characterized human genes was identified between gemcitabine-resistant cell lines (Pane-1) and gemcitabine-sensitive cell lines(Bxpc-3).Compared with Bxpc-3 cells, 141 genes were identified that show significantly differential expression at least 5-fold in Pane-1 cells.Of these 141 genes,103 unique genes with high expression(≥5-fold) and 38 unique genes relatively lower expression(≥5-fold) were found.3.2 Validation of genes expression of gemcitabine ehemoresistance in pancreatic cancer cellTo validate the microarray results,expression patterns of 22 genes in Pane-1 and Bxpc-3 cell lines were subjected to quantitative real-time PCR(qRT-PCR).Among these results,expression patterns of 21 genes were consistent with those in the microarray data.3.3 Emodin potentiates growth inhibition induced by gemeitabine in pancreatic cancer cells in vitroTo investigate the effect of ernodinon cell viability,pancreatic cancer cell lines (Pane-1,Mia Paca-2 and Bxpc-3) were treated with increasing concentrations of emodin (0-160μM) for 24,48 and 72 hours.Cell viability was inhibited by emodin treatment in a dose-and time-dependent manner when compared with the control in tested cell lines. Subsequent researches were done to examine the effect of a combination of emodin and gemcitabine on cell viability by MTT assay.The results showed treatment with emodin (Bxpc-3 and Mia Paca-2 40μM,Pane-1 80μM) plus gemcitabine(Pane-1 and Mia Paca-2 5μg/ml,Bxpc-3 0.5μg/ml) for 48 hours resulted in significant cell growth inhibition compared with single agent alone in pancreatic cancer cells(P＜0.05).3.4 Emodin sensitizes pancreatic cancer cells to apoptosis induced by gemcitabine in vitroThe combination of gemcitabine(Panc-1 5μg/ml,Bxpc-3 0.5μg/ml) and emodin (Panc-1 80μM,Bxpc-3 40μM) resulted in a stronger apoptotic effect in comparison with either agent alone after 24 h treatment by using Annexin V/propidium iodide flow cytometry(P＜0.05).To further test the ability of the combination therapy to induce apoptosis,caspase-3 and PARP activation in both cell lines tested were evaluated. Co-treatment between emodin and gemcitabine caused an additional increase in caspase-3 activity(P＜0.05) and PARP cleavage in Panc-1 and Bxpc-3 cells by comparison with single agent alone after 24 h treatment.3.5 Potentiation of the effect of gemcitabine in vivo by emodinAt the end of four cycles treatment,the final tumor volumes showed significant decrease in the emodin+gemcitabine group compared with control(P＜0.05) or with gemcitabine alone(P＜0.05).More significantly,no apparent systemic toxicity occurred during the combination treatment,because neither mouse death nor damaged liver or kidney functions happened in these groups.TUNEL staining of tumor sections demonstrated a visibly higher level of apoptosis in tumors treated with combination therapy than controls or single treatment.Next study showed that emodin in combination with gemcitabine significantly downregulated the expression of Ki-67 in tumor tissues compared with the control group and single treatment group.3.6 The mechanism by which emodin potentiates the effect of gemcitabine in vitroOur results of western blotting,RT-PCR and qRT-PCR displayed the survivin expression was significantly up-regulated after gemcitabine treatment.Emodin enhances the effect of gemcitabine in pancreatic cancer by downregulating gemcitabine chemoresistant gene HS3ST1,AKRC13 and survivin.4.Conclusions:4.1 A differential gene expression pattern associated with gemcitabine chemoresistant phenotypes in pancreatic cancer cells was identified by using microarray.4.2 Emodin not only induces apoptosis and inhibits the growth of human pancreatic cancer cells but also increases the effect of gemcitabine in vitro.4.3 Emodin potentiates growth inhibition induced by gemcitabine in pancreatic cancer cells in vivo.4.4 Modulating several genes associated with gemcitabine chemoresistance may be the mechanisms by which emodin enhances the antitumor activity of gemcitabine in pancreatic cancer cells.更多还原