生物芯片和纳米中药对肝胆肿瘤早期诊治的研究 毕业论文.doc
华 中 科 技 大 学 博 士 学 位 论 文 博士学位论文生物芯片和纳米中药对肝胆肿瘤早期诊治的研究学位申请人: 学科专业:外科学指导教师: 答辩日期:2013年5月101A Dissertation Submitted to Huazhong University of Sicience and Technology for the Degree of Doctor of MedicineResearch on early diagnosis and treatment of hepatobiliary tumors by biochip and Nano-traditional Chinese medicine Ph. D. Candidate : Wang BingSupervisor : Prof. Zou ShengquanMajor : General SurgeryTongji Hospital Tongji Medical CollogeHuazhong University of Science and Technology Wuhan, 430030 P. R. ChinaMay,2013独创性声明本人郑重声明,本学位论文是本人在导师指导下进行的研究工作及取得的研究成果的总结。尽我所知,除文中已经标明引用的内容外,本论文不包含任何其他个人或集体已经发表或撰写过的研究成果。对本文的研究做出贡献的个人和集体,均已在文中以明确方式标明。本人完全意识到本人将承担本声明引起的一切法律后果。学位论文作者签名:日期: 年 月 日学位论文版权使用授权书本学位论文作者完全了解学校有关保留、使用学位论文的规定,即:学校有权保留并向国家有关部门或机构送交论文的复印件和电子版,允许论文被查阅和借阅。本人授权华中科技大学可以将本学位论文的全部或部分内容编入有关数据库进行检索,可以采用影印、缩印或扫描等复制手段保存和汇编本学位论文。保密 ,在_年解密后适用本授权书。本论文属于不保密。(请在以上方框内打“” )学位论文作者签名: 指导教师签名: 日期: 年 月 日 日期: 年 月 日中国科技部国际合作司资助项目纳米中药高靶向治疗消化道恶性肿瘤的相关研究(编号:2010DFA31870)This study was supported by a grant from The Chinese Ministry of Science and Technology(NO. 2010DFA31870)目 录主要略缩词一览表1前 言2中文摘要4Abstract9本研究创新点16第一部分 生物芯片早期诊断肝胆肿瘤的探讨17Part MeDIP芯片分析和建立胆管癌差异甲基化谱17引言18材料和方法19结果22讨 论28参考文献29Part 无透镜显微镜联合细胞诊断芯片集成系统的构建31前言31材料与方法32结果35讨论37结论38参考文献38第二部分 雷公藤内酯醇新型固体纳米粒治疗肝胆肿瘤的实验研究40Part 雷公藤内酯醇新型固体纳米粒对肝癌生长抑制的研究40材料与方法41方法42观察指标43结果44讨论50参考文献52Part 甲基化酶抑制剂作用于胆管癌的实验研究55材料与方法56结 果58讨 论61参考文献62Part 组蛋白脱乙酰化酶抑制剂作用于胆管癌的研究65材料与方法66结 果68讨 论70参考文献72Part 地西他滨联合丙戊酸钠增强TP-SLN对人胆管癌细胞杀伤作用的初步研究74前言74材料与方法75结 果75讨论76第三部分 应用无透镜显微镜联合细胞诊断芯片集成系统初探纳米中药对胆管癌细胞作用机制77前言77材料与方法77结果78讨论78综述 表观遗传学治疗的现状与展望79附录 发表文章96致谢97主要略缩词一览表缩写英 文 全 称中 文 译 注CCAMeDIPCholangiocarcinomamethyl - DNA immunoprecipitation胆管癌甲基化DNA免疫共沉淀MSPmethylation specific PCR甲基化特异性多聚酶链反应BSPbisulfite sequence PCR亚硫酸氢盐修饰后测序法DACDecitabine地西他滨VPAValproate acid丙戊酸DNMTDNA methylation transferaseDNA甲基转移酶HDAChistone deacetylases组蛋白脱乙酰化酶GFP-LC3Green fluorescence protein -tagged MAP-LC3绿色荧光蛋白标记的MAP-LC3PIpropidium iodide碘化丙碇CCK8cell counting kit-8细胞计数试剂盒PBSphosphate buffered saline磷酸盐缓冲液生物芯片和纳米中药对肝胆肿瘤早期诊治的研究前 言胆管癌(Cholangiocarcinoma, CCA)是一种起源于肝内或肝外胆管上皮细胞的消化道恶性肿瘤,其侵袭性强。最近来自美国和英国的一项调查研究显示:过去十年间全球的胆管癌发病率和致死率均呈上升趋势。胆管癌约占所有消化道恶性肿瘤的3%。其预后极差,诊断时往往已是进展期。尽管有许多改进了的治疗方法已经出现,还有一些新的靶向疗法也正在兴起,但外科手术仍是迄今为止最有效的治疗方法。然而,却只有少于1/3的胆管癌患者适合手术切除。另一项调查显示:切除瘤体后的肝内胆管癌、肝门部胆管癌和肝外胆管癌的5年生存率分别为22-44%、11-41%和27-37%。其余治疗方法,诸如传统的放化疗、肝移植等对已手术切除和未能切除的胆管癌患者的5年生存率没有实质性地提高。胆管癌的早期诊断和有效治疗均极为棘手的原因主要还是对其独有的生物学特性的研究不够深入透彻,且基础研究的成果不能很好地应用于临床。因此,寻找特异性强、灵敏度高的早期诊断方法,加强胆道肿瘤治疗理论基础研究及研发对胆管癌治疗行之有效的方法显得尤为必要和迫切。本课题组一直以来从事胆道肿瘤的基础和临床研究。在前期研究中,我们从表观遗传学的角度,采用基因甲基化芯片,筛选出胆管癌细胞和正常胆管上皮细胞间基因启动子区CpG岛差异性甲基化谱,并对其中最具特异性的一些基因(家族)进行生物学验证。结果证实,芯片筛选出的这些位点无论在组织还是在细胞层面,其甲基化差异均较显著。我们已经并正在对这些基因进行深入的功能研究,试图探明这些基因与胆管癌发生发展过程中的哪些生物学行为有关,为后期的早期诊断标志物的筛选和有效治疗奠定基础。近年来,一些芯片集成无透镜成像系统应用于细胞计数及检测新杆状线虫(体长达1mm)的活动等研究中。一般的光学显微镜是通过光学物镜实现成像,而这种无透镜系统是直接将生物样品的“影子”投射在CCD或CMOS光传感器中。这套系统的分辨率取决于传感器的像素及衍射效应。本课题组利用德国IBMT研究所先进的芯片实验室平台,与IBMT合作构建了一种方便携带使用、成本低廉、快速分析、样本和试剂消耗少的细胞诊断芯片。该组件可用于动态定点监测细胞形态变化和细胞的荧光图像,为后期诊断芯片的进一步开发提供重要的参考数据。此外,在胆道肿瘤的治疗方面,本课题组一直扎根于探索纳米中药对胆管癌的靶向治疗,在“863”课题和国家科技部课题的支持下,我们取得了丰硕的成果。本文中,为了达到更好的疗效,我们采用了一种新型的纳米材料-新型固体脂质纳米粒。数据证实其对胆管癌细胞和肝癌鼠移植瘤均有明显的减毒增效作用。中文摘要第一部分 生物芯片早期诊断肝胆肿瘤的探讨Part MeDIP芯片分析和建立胆管癌差异甲基化谱目的:利用全基因组启动子区CpG岛甲基化位点检测芯片技术,分析人胆管癌细胞株与人正常胆管上皮细胞株间的基因甲基化差异,建立胆管癌基因差异甲基化谱,为寻找特异性胆管癌早期诊断标志物奠定。方法:采用NimbleGen HG18 CpG Promoter 芯片(Roche,Germany)分别检测人胆管癌细胞株TFK-1和人正常胆管上皮细胞株BEC全基因组启动子区CpG岛甲基化位点,并分析比较两细胞株间的差异甲基化位点。并应用Molecular Annotation System (MAS)软件分析差异甲基化位点对应的基因的功能。采用亚硫酸氢盐测序法(BSP)检测HOX基因甲基化水平。采用荧光定量PCR和western-blot法检测目的基因在细胞水平的表达情况。免疫组化检测目的基因在胆管癌和癌旁组织中的表达差异。甲基化PCR(MSP)检测甲基转移酶抑制剂干预前后,目的基因甲基化的变化情况。结果: 1MeDIP芯片结果显示:相比于BEC细胞株TFK-1细胞株中有2103个CpG岛表现为差异性高甲基化; 2在所有差异性高甲基化CpG岛中有97个基因属于HOX家族基因,这些基因涉及细胞分化、周期改变、粘附、侵袭与转移以及血管生成等多个肿瘤发生机制; 3SignalMap软件分析这97个HOX家族基因,发现甲基化率最高的前15位基因是HOXA5、HOXA2、HOXA11、HOXB4和HOXD13。BSP证实其各自的甲基化率为:HOXA5(95.38%)、HOXA2(94.29%)、HOXA11(91.67%)、HOXB4(90.56%)和 HOXD13(94.38%); 4PCR和Western-blotting结果显示:在肝癌、胆管癌、胰腺癌和大肠癌等消化道肿瘤细胞株中,胆管癌细胞株TFK-1中HOXA5表达量最低。免疫组化显示:相比于癌旁和正常胆管组织而言,胆管癌中HOXA5表达量明显降低。MSP证实:在甲基转移酶抑制剂干预后,HOXA5在TFK-1中甲基化程度明显降低。结论:1MeDIP芯片是筛选胆管癌早期诊断标志物的有效方法之一; 2DNA甲基化可能是HOXA5在胆管癌中表达降低的重要原因,且HOXA5高甲基化可作为胆道肿瘤的早期诊断标志物之一。关键词:MeDIP芯片、甲基化谱、胆管癌、HOXA5。Part 无透镜显微镜联合细胞诊断芯片集成系统的构建目的:微流控芯片实验室(Lab-on-a-chip)技术越来越多地用于细胞毒性检测和药物测试。该技术主要利用蚀刻技术到以各种材料制作的芯片上的通道和孔洞构成的“网络”构建微型实验室。实际应用时,压力以精细控制的方式推动钠升的体积流过通道,从而实现在单个集成的系统上进行样品处理、混合、稀释、电泳、定点追踪以及色谱分析、染色和检测。本实验的目的是利用德国IBMT研究所先进的芯片实验室,构建一种方便携带使用、成本低廉、快速分析、样本和试剂消耗少的细胞诊断芯片。方法: 本实验利用接触式光学平板印刷技术的原理构建了一种无透镜荧光显微镜/光学显微镜联合细胞芯片集成系统。生物样本被放置在一个包含有一个1.5 l漏斗状的腔及底部为1 m厚的硅氮化合物的MEMS芯片内。再将芯片装载在一个5兆彩色CMOS成像元件阵列上,最后在两者间覆盖一个光滤片。利用此系统培养鼠源性纤维细胞L929和人胆管癌细胞TFK-1。同时,分别用Erythrosine B和FITC进行细胞染色处理后,再比较此系统与传统荧光显微镜的成像效果。结果:1该系统监测L929细胞及TFK-1细胞影像的对比度和清晰度与4x物镜下的影像基本一致;2定点追踪观察单个或少量细胞形态变化,效果良好;3L929经红染或绿色荧光染色后,该系统显示细胞形态清晰。结论:该组件可用于动态定点监测细胞形态变化和细胞的荧光图像,为后期诊断芯片的进一步开发提供重要的参考数据。关键词:细胞诊断芯片、无透镜成像系统、胆管癌。第二部分 雷公藤内酯醇新型固体纳米粒治疗肝胆肿瘤的实验研究Part 地西他滨和丙戊酸钠单用对人胆管癌生长抑制作用研究目的:探讨地西他滨(DAC)和丙戊酸钠(VPA)单用对人胆管癌细胞株TFK-1、QBC939、HUCCT、CCLP1细胞增殖的影响,并探讨其可能的机制。方法:1采用CCK8法检测DAC、VPA单独使用时对细胞生长的抑制率; 2应用流式细胞仪检测DAC、VPA单独使用后细胞周期的变化; 3经Hochst33342/PI染色,光镜下观察经DAC、VPA单独使用后细胞形态的变化,同时应用流式细胞术检测处理后细胞凋亡情况;4光镜下观察经DAC、VPA单独使用细胞120h后,细胞分化的情况;5构建GFP-LC3质粒并转染细胞,再加入DAC、VPA单独使用处理72小时,荧光显微镜观察细胞自噬情况; 6体内实验采用裸鼠TFK-1细胞皮下种植瘤模型,观察DAC、VPA单独使用对瘤体生长及生存率的影响。 结果:1DAC、VPA单独使用对人胆管癌细胞的增殖抑制作用呈时间和剂量依赖性; 2流式细胞术检测显示,随着药物浓度的增加和作用时间的延长,胆管癌细胞呈现明显的G2/M或G0/G1期阻滞; 3经DAC、VPA单独使用后,光镜下可见凋亡的细胞呈明显的胞体固缩、核固缩、核碎裂及凋亡小体形成。流式细胞术结果表明:细胞凋亡呈剂量依赖性; 4光镜下观察到DAC、VPA单独使用120h后,细胞形态呈树突状突起; 5荧光显微镜下观察到DAC、VPA单独使用后,细胞有明显的自噬现象; 6体内试验证明:当应用DAC或VPA后,总给药时间为2周时,其对裸鼠TFK-1细胞皮下移植瘤的生长有明显的抑制作用。而且,治疗组的裸鼠生存期较对照组明显延长。结论:体外实验和体内实验均证实, DAC或VPA对胆管癌细胞均具有明显的生长抑制作用。关键词:地西他滨、丙戊酸钠、胆管癌、细胞增殖、周期和凋亡、细胞分化、印迹基因。Part 雷公藤内酯醇新型固体纳米粒对肝癌生长抑制的研究目的:1观察雷公藤内酯醇新型固体纳米粒(TP-SLN)经尾静脉注射给药对SPF级BALB/C小鼠的急性毒性研究;2观察TP-SLN对肝癌细胞株H22体外和荷瘤体内的抗肿瘤作用研究方法:1雷公藤内酯醇(TP)和TP-SLN分别作用于H22细胞24h、48h和72h后,CCK-8法检测细胞的存活率;2SPF级BALB/c小鼠50只(雌雄各半), 按照0.65 mg/kg,0.773 mg/kg,0.919 mg/kg,1.189 mg/kg,1.300 mg/kg单次尾静脉注射给予TP-SLN,共5个剂量组。观察小鼠单次给药的症状体征,统计死亡率、体重等相应指标的变化; 3建立H22细胞BALB/c小鼠皮下移植瘤模型,分为生理盐水空白对照组,空白固体脂质纳米粒组,雷公藤内酯醇(TP)组,TP-SLN组,顺铂阳性对照组,隔日尾静脉给药后观察记录肿瘤的体积、体重、一般生长活动状况。12天后处死所有小鼠,剥离肿瘤组织进行称重。结果:1体外实验中,TP-SLN对H22细胞生长抑制呈时间和剂量依赖性,作用明显且强于TP;2TP-SLN对于SPF级BALB/c小鼠尾静脉注射途径而言,其LD50值为0.891 mg/kg,95%的可置信区间是0.814 mg/kg0.971 mg/kg。并且在该剂距范围内,TP-PM对于BALB/c小鼠而言,死亡率呈现良好剂量效应关系;观察发现:TP-PM对于SPF级BALB/c小鼠而言,在注射后的4hrs内小鼠均未出现死亡,随着时间的延长,部分小鼠症状逐渐加重抖动,共济失调,进而活动减少,活动抑制直至死亡; BALB/c小鼠死亡时间集中在24-48 hrs, 96 hrs 后绝大部分存活动物恢复正常的运动、呼吸等,体重上升。3与生理盐水空白对照组比较,TP、TP-SLN及顺铂均引起肿瘤体积下降和体重减轻。抑瘤率分别为 22.4%、49.2%、51.5%。TP、TP-SLN与生理盐水空白对照组相比,小鼠的生活状态(体重、反应能力等)均有一定的改善。顺铂组生活状态无明显改善。 结论:与TP相比,TP-SLN体内、外对肝癌细胞的抑制增强且稳定、持久,副作用减弱。关键词:TP、TP-SLN、H22细胞、抑瘤作用。Part 地西他滨联合丙戊酸钠增强TP-SLN对人胆管癌细胞生长抑制作用的初步研究 目的:探讨DAC联合VPA能否增强TP-SLN对人胆管癌细胞杀伤作用。方法:DAC联合VPA预处理TFK-1细胞3天后,再加入TP-SLN处理24h、48h和72h,采用CCK-8法检测细胞存活率。结果:与未预处理组相比,地西他滨联合丙戊酸钠预处理后,TP-SLN对人胆管癌细胞增值抑制作用明显增强结论:地西他滨联合丙戊酸钠预处理增强TP-SLN对人胆管癌细胞增值抑制效应。关键词:TP-SLN,DAC、VPA第三部分 应用无透镜显微镜联合细胞诊断芯片集成系统初探纳米中药对胆管癌细胞作用机制目的:应用无透镜显微镜联合细胞诊断芯片集成系统观察纳米中药与胆管癌细胞间相互作用方法:应用无透镜显微镜联合细胞诊断芯片集成系统培养TFK-1细胞72小时后,培养基中加入TP-SLN,采集处理24h、48h和72h后的细胞图像结果:无透镜显微镜联合细胞诊断芯片集成系统动态观察到纳米药物与细胞的相互作用后,细胞形态发生变化,但纳米材料荧光信号很弱。结论:无透镜显微镜联合细胞诊断芯片集成系统可应用于观察纳米药物与细胞相互作用过程。关键词:无透镜显微镜联合细胞诊断芯片集成系统,TP-SLN,TFK-1细胞。Research on early diagnosis and treatment of hepatobiliary tumors by biochip and Nano-traditional Chinese medicineAbstract1. Exploration to early diagnosis of cholangiocarcinoma by biochip Part Analysis and establishment of methylation profiles of cholangiocarcinoma by MeDIP chipAims: To analyze gene methylation differences between human cholangiocarcinoma cell line and normal bile duct epithelial cell line by genome-wide promoter region CpG island methylation sites microarray technology and establish differentially methylated spectrum of cholangiocarcinoma so as to lay basis of early diagnose to cholangiocarcinoma.Methods: NimbleGen HG18 CpG Promoter chip (Roche, Germany) were used to detect human cholangiocarcinoma cell lines TFK-1 and normal human bile duct epithelial cells BEC genome-wide promoter CpG island methylation sites,. Annotation System (MAS) software was applied to analysis the function of the gene,which has differentially methylated loci. bisulfite sequencing (BSP) was used to detect HOX gene methylation level. PCR and western-blot method were applied to detect target gene expression at the cellular level. Immunohistochemical detection were used to examine target gene expression differences in cholangiocarcinoma and adjacent tissues. Methylation PCR (MSP) was used to detect the target gene methylation changes before and after methyltransferase inhibitor intervention Results:1.There were 2103 CpG islands difference hypermethylation performance between BEC cells and TFK-1 cells. 2.There were 97 genes belonging to the HOX family genes among all the difference methylation of CpG islands,which involved in multiple tumor mechanism,such as cell differentiation, the cycle of change, adhesion, invasion and metastasis and angiogenesis3. SignalMap software was used to Analyze of the 97 HOX family genes, the top 15highest methylation rates of genes are HOXA5, HOXA2, HOXA11, HOXB4 and HOXD13 et al. BSP confirmed their respective methylation: HOXA5 (95.38%), HOXA2 (94.29%), of HOXA11 (91.67%), upregulation of HOXB4 (90.56%) and HOXD13 (94.38%); 4. PCR and Western-blotting results showed that: among liver cancer, bile duct cancer, colorectal cancer cell lines, the expression of HOXA5 of cholangiocarcinoma cells was the lowest. Immunohistochemistry showed that: compared to adjacent normal bile duct, bile duct cancer HOXA5 expression was significantly reduced. The MSP confirmed: After methyltransferase inhibitors intervention, HOXA5 in TFK-1 significantly reduced the degree of methylation.Conclusion: 1.MeDIP chip is one of the effective method of screening early diagnostic marker of cholangiocarcinoma; 2.DNA methylation may be important reason for HOXA5 expression decreased in cholangiocarcinoma, and HOXA5 hypermethylation can be used as one of early diagnostic marker for biliary tract tumors Keywords: MeDIP chip, methylation profiles, bile duct cancer, HOXA5.Part Construction of on-chip integrated lensless microscopy module for cell-based sensors Aims: Lab-on-a-chip systems are increasingly applied in cell-based assays for toxicology and drug testing. In this paper, by use of the advanced trchnology of IBMT institute (Germany), we worked together to construct an on-chip integrated lensless microscopy module using a direct projection method for optical monitoring of the shadow images of adherent growing mammalian cells.Methods:We present an on-chip integrated lensless fluorescence imaging module applying the principle of contact/proximate optical lithography. The biological samples or solutions are sustained in disposable sterilized microfluidic chips with 1 m thick silicon nitride (Si3N4) membranes. These chips are assembled on the surface of a 5 megapixel colored CMOS image sensor array with 1.75 µm pixel size, which is coated with an additional interference filter. The function is demonstrated by the growth monitoring of L929 and TFK-1 cells cultured in cavity chips with Si3N4 substrate for 2 days and by checking the colorimetric staining of cells with a compromised membrane.Results: 1. The pixel resolution is comparable with a 4 × objective microscope. 2. It is possible for point-of-care applications to observe the morphology of single cell. 3. The image quality of the module with a Si3N4-chip for L929 cells is good. Conclusion: the module can be used for point-of-care observation.It can be offered some important informations for further development of dignosis chip. Key words: dignosis based on cell-chips, lensless microscopy module, cholangiocarcinoma.2 Research on treatment to hepatobiliary tumors by TP-SLNPart Effect of valproic acid or Decitabine on inhibition growth of human cholangiocarcinoma cells and its mechanismAims: To investigate the effect of valproic acid or Decitabine on inhibition growth of human cholangiocarcinoma cells in vitro and in vivo and its possible mechanism. Methods: cell growth inhibition rates were determined by CCK-8 assay; Cell cycle and cell apoptosis were analyzed by flow cytometry after treated with various concentrations. Cell autophagy was observed under fluorescence microscope. The effect of valproic acid (VAP) or DAC on growth of cholangiocarcinoma in vivo was determined in cholangiocarcinoma cells mice xenograft model. Results:The growth inhibition rate of cholangiocarcinoma cells in VPA or or DAC-treated group decreased in a time- and dose- dependent manner. After treated with VPA or DAC, cell cycle of cholangiocarcinoma cells was arrested at G2/M or G0/G1 phase. The apoptosis rate was significantly higher in treated group than control group. Cell autophagy was observed after treated with VPA or DAC in cholangiocarcinoma cells. The growth of implanted tumor of cholangiocarcinoma cells was significantly slowed down after mice were treated with 300mg/kg/6 times a week VPA or 0.8 mg/kg/6 times a week DAC for 2 weeks. Conclusion VPA or DAC can inhibit growth of cholangiocarcinoma in vitro and in vivo, which is probably related with cell cycle arrest, cell apoptosis, and partially with autophagy.【key words】 VPA; DAC; cholangiocarcinoma; cell cycle and apoptosis; autophagy; xenograftPart Inhibition of the New Triptolide-loaded Solid Lipid Nanoparticles to H22 Hepatoma CellsAims:1. To investigate the acute toxicity and degree of the animal's death of triptolide loaded polymeric micelles via the tail vein injection in SPF grade BALB / c mice, then calculate its LD50, which will provide dose design basis for the long-term toxicology experiment.2.To investigate the anti-tumor effects of triptolide-loaded solid lipid nanoparticles(TP-SLN) on the H22 hepatoma cell line, and provide the basis for further clinical application as anticancer drugs. Method:1. H22 cells were incubated with triptolide (TP) and TP-SLN for 24 h, 48 h and 72 h. Cell viability was then measured by the CCK-8 assay2. 50 SPF grade BALB / c mice (half male half female) are given TP-PM in accordance with the 0.65 mg/kg, 0.773 mg/kg, 0.919 mg/kg, 1.189mg/kg, 1.300