沈阳药科大学本科毕业论文论文题目:化合物A联合化合物B对人肝癌HepG2细胞作用机制研究起止时间:2014年10月26日—2015年6月20日姓名学号:学院专业:年级班级:指导教师:XXX副教授实习单位:XXX大学生命科学与生物制药学院药理系2目录中文摘要······································································1英文摘要······································································2第1章绪论··································································31.1细胞凋亡·····························································31.2立题依据·····························································5第2章实验部分······························································62.1实验材料·····························································62.2实验方法·····························································72.3实验结果····························································12第3章结论与讨论···························································173.1结论································································173.2讨论································································18参考文献·····································································21致谢·········································································23沈阳药科大学本科毕业论文目录3摘要原发性肝癌是世界常见的恶性肿瘤之一,也是我国的高发恶性肿瘤,由于不易被早期诊断,进展迅速,术后容易复发、转移,因此预后较差。目前肝癌仍以外科手术为公认首选和主要的治疗手段,但由于很多肝癌患者确诊时已是中晚期,失去了手术治疗的机会并于诊断后1年内死亡,手术切除率30%,术后总体5年生存率仅30%~40%。对于可手术的肝癌患者,术后复发、转移是影响治疗效果及预后的主要因素,对于不能手术的中晚期肝癌患者尚缺乏有效的治疗方法。传统的化疗、放疗对肝癌疗效欠佳,目前仍缺乏行之有效的化学预防和治疗靶位[1]。因而本课题是对化合物A联合化合物B对人肝癌HepG2细胞作用机制研究。本实验首先用MTT法分别测得不同浓度的A和B单独处理HepG2细胞及不同浓度的A和B联合用药处理HepG2细胞死亡的影响。结果表明不同浓度的A和B单独处理HepG2细胞,增值抑制程度表现为时间剂量依赖效应,并且在A6μmol/L联合B100μmol/L协同作用最为显著。流式细胞仪检测细胞凋亡率显示,联合用药组凋亡率升高更为明显。WesternBlot法检测蛋白显示,促凋亡相关蛋白Bax、FADD增加,抗凋亡蛋白Bcl-2表达减少。关键词联合用药,细胞凋亡,作用机制沈阳药科大学本科毕业论文英文摘要2AbstractHepatocellularcarcinoma(HCC)isthemostcommoncancerintheworldandanincreasedmalignantillnessinourcountry.Becauseofitsaggressivebiologicalbehavior,rapidlyprogressesanddelayeddiagnosis,thismalignancyhasagrimprognosisevenfollowingsurgicalresectionandhashighdisease-recurrence.SurgeryisthefirstandmostcommontherapyofHCC.Unfortunately,thediseaseisusuallydetectedatanadvancedstage,andthemajorpatientslosethechanceofsurgery,diewithin1yearafterthediagnosisoftheirdisease.Relativesurvivorare30%and30%~40%respectively.Thehighdiseaserecurrenceandmetastasisaretheimportantreasonsforeffectandprognosis.Curativetherapiesareleasteffectiveforsurgicallycases.Forthesepatients,medicaltreatments,includingchemotherapy,chemo-embolization,ablation,radiotherapy,remaindisappointing.Clearly,thereisanurgentneedfornewtherapiesforthisaggressivediseasebecauseeffectivepreventandtargetsoftherapyareunavailable.ThereforethisissueisthemechanismoftheJointDrugB,DrugA,onHepG2cellsissignificant.ThisarticleassessedtheeffectsofdrugAalone,drugBaloneandcombinationofdrugAanddrugBonHepG2cellswithMTTassay,suggestingthatAandBhaveamaximalcombinationaleffectatthedoseofA6μmol/LandB100μmol/L,respectively.WesternBlottinganalysisindicatesthatpro-apoptosisproteinsincludeBAXandFADDareincreasingandanti-apoptosisproteinBCL-2isreducing.KeywordCombination,apoptosis,themechanismofaction沈阳药科大学本科毕业论文第一章绪论3第一章绪论1.1细胞凋亡细胞凋亡定义、意义、形态学特征、作用通路、机理1.2立题依据先介绍下肿瘤的危害,再介绍下肝癌HCC(hepaticcellcarcinoma)的危害。虽然有些临床治疗药物具有一定的疗效,但同时也会加重肝脏的损害。因此治疗肝癌药物与保肝药物合用起到减毒增效的作用对肝癌患者具有极其重要的临床意义。药物A是一种独特的多靶点抗肿瘤药物,既抑制肿瘤细胞增殖又抑制了肿瘤血管的形成,从而抑制肿瘤的转移。DrugA属于多酶抑制剂,能抑制RAF-1、BRAF丝氨酸/苏氨酸激酶的活性,以及血管内皮生长因子(VEGF-2、VEGF-3)、血小板衍化生长因子(PDGF)、受体酪氨酸激酶KIT、FMS样酪氨酸激酶-3(FLT-3)等多种受体的酪氨酸激酶活性。DrugA具有抑制肿瘤细胞增殖和血管形成的双重作用,即它一方面可以抑制受体酪氨酸激酶KIT和FLT-3以及RAF/MEK/ERK途径中丝氨酸/苏氨酸激酶,抑制肿瘤细胞增生;另一方面它可以通过上游抑制受体酪氨酸激酶VEGFR和PDGFR,及下游抑RAF/MEK/ERK途径中丝氨酸/苏氨酸激酶,抑制肿瘤新生血管的形成和切断肿瘤细胞的营养供应而达到遏制肿瘤生长的目。因此它可以起到抗肿瘤血管生成和抑制肿瘤细胞增值的双重作用。但在治疗的同时,药物A也会使患者的转氨酶短暂性升高(22%)、酯酶增加等,因此能加重对肝肾功能的损沈阳药科大学本科毕业论文第一章绪论4害。药物B能够抑制肿瘤受体型酪氨酸激酶(receptortyrosinekinase,RTK)的活性,包括表皮生长因子受体1(epidermalgrowthfactorreceptor1,EGFR)和胰岛素样生长因子1受体(insulin-likegrowthfactor1receptor,IGF-1R)及其下游信号分子的活化。此外,药物B可以选择性清除羟自由基(•OH),且对NF-κB有明显的抑制作用。研究表明,药物B有明显的保护和稳定肝细胞的作用,用于治疗急慢性肝炎、肝硬化、肝中毒等病。对肝炎患者症状、肝功能均有明显的改善。在临床上的应用为对肝脏的保护作用。临床试用表明该药作用强,疗效显著。第二章实验部分2.1实验材料一、细胞人肝癌HepG2细胞购于AmericanTypeCultureCollection(ATCC,Rockville,MD,USA)。接种在含10%胎牛血清、2%谷氨酰胺的1640培养液中,在37℃,5%CO2培养箱中培养。二、药品及试剂DrugA、DrugB于无菌条件下,DMSO溶解后,用1640培养液稀释至所需浓度。1640培养基,美国Gibco公司(GrandIsland,NY,USA);胎牛血清,购于北京元亨圣马生物技术研究所;胰蛋白酶、谷氨酰胺、青霉素、链霉素、Hepes、二甲基亚砜(DMSO)、甲基噻唑蓝(MTT)、碘化丙啶(PI)、丹磺酰戊二胺(monodansylcadaverine,MDC)、RNase、ProteinK、琼脂糖、溴酚蓝、二氨基联苯胺和ECL试剂盒购于美国Sigma公司。抗p-ERK、ERK和β-actin的抗体,及辣根过氧化酶标记的二抗,购于美国SantaCruzBiotechnology公司(SantaCruz,CA,USA)。三、仪器二氧化碳培养箱(NuAir,Plymouth,MA,USA),酶联免疫分析仪(Tecan,Salzburg,沈阳药科大学本科毕业论文第二章实验部分5Austria),6孔、24孔与96孔培养板(Nunc,Roskilde,Denmark),倒置相差显微镜(Leica,Bensheim,Germany),荧光显微镜(Olympus,Tokyo,Japan),流式细胞分析仪(BectonDickinsonFACScan,Franklinlakes,NJ,USA),DYY-2型稳压稳流电泳仪,DYY-III28A型蛋白质电泳仪,DYY-III40B电泳槽(北京六一仪器厂)。2.2实验方法一、不同浓度的DrugA和DrugB单独处理HepG2细胞及DrugA和DrugB联合用药处理HepG2细胞死亡的影响四甲基偶氮唑[3-(4,5-dimethylibiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide,MTT]是一种能接受氢原子的染料。可作用于活细胞线粒体中的呼吸链,在琥珀酸脱氢酶和细胞色素c的作用下tetrazoliu环开裂,生成蓝紫色的结晶甲臜(formazan),甲臜结晶的生成量仅与活细胞数目成正比(死细胞中琥珀酸脱氢酶消失,不能将MTT还原)。用DMSO溶解甲臜后,在一定波长下用酶标仪测定光密度值,即可定量测出细胞的存活率