【作者】 张惠丹；

【导师】 方瑾；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2009， 博士

【摘要】 前言肿瘤的发生是分子上逐步累积的多基因改变的过程,通过基因的筛查可以对肿瘤的发生和发展进行有效的诊断和预测,从而实现肿瘤的早期诊断和早期治疗。单核苷酸构象多态性（Single-nucleotide polymorphisms,SNPs）/基因突变和DNA甲基化是最常见的遗传学和表观遗传学改变形式,经常用作研究疾病的重要分子标志物。迄今DNA测序仍是分析检测基因的金标准,然而其耗时长、价格昂贵等特点不适宜大样本检测。尽管目前已经出现多种检测基因突变和甲基化的方法,但仍需进一步提高检测速度和准确性,并降低成本。温度梯度凝胶电泳（temperature gradient gel electrophoresis,TGGE）技术是一种稳定、高效的测序前基因突变筛查技术,利用不同构象的分子具有不同的50%解链温度（Tm）来进行分离和检测,其最大优势就是无需事先了解突变类型,只要其Tm值处于施加的温度梯度范围内,就能有效检测已知和未知突变。但现阶段传统的TGGE技术中温度梯度形成系统复杂,重现性差,而且检测灵敏度较低。微全分析系统（Miniaturized total analysis systemsm,μTAS）是基于物理学、分析化学、生物学等多学科研究领域而发展起来的微生化分析工具,其目的是通过分析仪器的微型化和集成化,极大限度地把分析实验室的功能转移到便携的分析设备中,因此又被称为微流控芯片（microfluidic chip）或芯片实验室（Lab on a chip,LOC）。微流控芯片具有分析速度快,灵敏度高等优势,样品消耗可降低至纳升级,易于集成化自动化的特点更适用于对珍贵临床试样的大样本分析,增加速度,减少污染。微流控芯片电泳分析系统是在毛细管电泳基础上发展起来的一种更为优化的分析工具。其原理是以电场为驱动力,借助于离子或分子在电泳迁移或分配上的差异,从而实现对复杂试样中多种组分的分离。由于芯片的微通道面积-体积比显著增大,焦耳热能很快向四周溢散,所以可施加平板凝胶电泳难以达到的高场强,从而实现对样品的快速、高效的分离检测。近年来大肠癌发病率迅速上升,而基因突变和DNA甲基化常发生在肿瘤早期,对大肠癌的早期诊断具有重要意义。本研究通过微流控温度梯度毛细管电泳系统的建立可对大肠癌细胞系和各种临床样本中K-ras基因突变和p16基因启动子甲基化进行快速高效检测,为肿瘤相关基因的大规模筛查提供了更为切实有效的分析工具。材料与方法一、微流控芯片TGCE基因突变检测系统的建立1、用标准光刻和湿法刻蚀技术在玻璃基片上刻蚀出十字形微通道网络,采用热键合方法将其与盖片进行封接,在微通道末端粘接储液池,制成玻璃微流控芯片。搭建共聚焦型激光诱导荧光检测装置,设定电泳进样及分离电压。2、基于倾斜式热辐射温度梯度形成系统的建立将一程序控温铝块放置于玻璃微流控芯片上,通过在芯片与铝块接触面的上游边缘放置一条绝缘丝线,则分离通道自上游向下游会因获得的辐射热逐渐增加而温度逐渐升高,从而形成一个连续的温度梯度。3、四种具有不同片段长度和较大Tm值范围的点突变模式样品分别在有效温度梯度范围为3.0cm的5℃温度梯度(1.7℃cm^-1)和10℃温度梯度(3.3℃cm^-1)下进行微流控芯片TGCE检测。电泳有效分离距离为4.5cm,每次电泳分离时间少于7min。二、微流控芯片TGCE检测K-ras基因突变1、微流控芯片TGCE检测6种大肠癌细胞系K-ras基因突变。2、根据密码子12突变与否,分别按不同比例将野生型HT29和突变型SW480细胞进行混合培养:1:1,4:1,16:1,64:1,128:1,256:1,512:1,考察系统对突变型K-ras的检出限。3、对83例大肠癌患者石蜡切片进行检测,考察微流控芯片TGCE的筛分系统与直接PCR产物测序相比对大肠癌的筛查是否有意义。4、微流控芯片TGCE检测20例大肠癌患者的肿瘤组织和粪便样品中K-ras基因突变情况,与PCR产物测序法比较检出率和符合率。5、定量分析突变型比例采用AutoCAD软件获得同源双链和异源双链下的峰面积后,计算异源双链峰面积占总峰面积的百分比就可以得到突变型的比例,用克隆测序获得的突变型比例对芯片检测结果进行校正,获得回归方程。三、基于微流控芯片TGCE基因甲基化检测方法的建立1、亚硫酸氢盐-微流控芯片TGCE甲基化检测系统的建立利用亚硫酸氢盐可特异性地将未甲基化的胞嘧啶修饰转化为尿嘧啶（C→U）,而对甲基化的胞嘧啶无修饰作用的特点,将靶序列原来甲基化位点的甲基化状态差异转化成了核苷酸点突变（C→T）,将能够进行基因突变检测的温度梯度毛细管电泳（TGCE）应用到了甲基化检测中。2、检测甲基化模式样品分别从具有不同p16基因启动子甲基化模式的大肠癌细胞系获得完全甲基化和非甲基化模式片段。通过甲基转移酶修饰法和去甲基化法获得多种具有不同甲基化位点的p16基因启动子片段,分别与非甲基化片段以等比混合变性复性后,在5℃和10℃温度梯度下进行微流控芯片TGCE检测。复杂甲基化样品PCR产物直接变性复性在5℃和10℃温度梯度下检测。3、微流控芯片TGCE检测p16基因启动子甲基化情况已知的4种大肠癌细胞系和未知的3种大肠癌细胞系的该基因甲基化状态。4、系统检出限的考察根据p16基因启动子甲基化与否,将非甲基化HT29细胞系和甲基化SW480细胞系的PCR产物等比混合、变性复性后稀释50倍、100倍、200倍、400倍和800倍进样电泳检测,确定系统对DNA分子的检出限。分别按不同比例将非甲基化LS174T细胞系和甲基化SW480细胞系的PCR扩增片段按下列比例进行混合:1:1,4:1,16:1,64:1,128:1,256:1和512:1,微流控芯片TGCE检测确定其对甲基化片段的检出限。5、亚硫酸氢盐-微流控芯片TGCE检测20例大肠癌患者的肿瘤组织和配对粪便样品的p16基因甲基化状态。结果一、微流控芯片TGCE基因突变检测系统的建立本研究以从4种单点突变质粒中扩增出的具有较大变性温度范围的PCR产物作为实验材料,首次将TGCE过程移植到微流控芯片上,建立了一种简单、高效、低成本的快速检测基因突变的分析技术平台。该装置在3cm的加热区域内通过热辐射差异形成了芯片分离通道内稳定而重现性好的温度梯度,并且随着丝线直径的变化,温度梯度能够在3-10℃的范围内任意调节。应用该体系,实现了在最大10℃梯度(3.3℃cm^-1)内对DNA点突变进行检测,由于一般DNA片段的变性温度都位于10℃梯度内,使得这一方法应用于检测未知突变成为可能,而不必知道具体的突变位置和突变类型。二、微流控芯片TGCE检测K-ras基因突变基于倾斜式热辐射的微流控芯片TGCE系统对基因突变检测的有效性进一步通过检测6种大肠癌细胞系K-ras基因突变得到了验证。该系统对比例混合细胞中突变型K-ras的检出限可达1/513。用克隆测序检测的突变型K-ras比例对芯片电泳检测的突变型比例进行校正,以从电泳结果计算精确的突变型比例,回归方程为y=0.993x-1.387。对84例石蜡切片K-ras基因检测结果显示,微流控芯片TGCE的检出率明显高于PCR产物直接测序。结合临床资料分析,当肿瘤位于直肠时,以及浸润深度达全层时K-ras基因突变率显著增加。对20例大肠癌肿瘤组织和配对粪便样品进行微流控芯片检测的结果显示,K-ras基因突变的检出率,以及粪便样品与肿瘤组织中K-ras基因突变的符合率,均明显高于PCR产物直接测序。三、基于微流控芯片TGCE基因甲基化检测方法的建立用亚硫酸氢盐修饰法使基因甲基化的差异转化为基因多点突变,建立了基因甲基化检测的亚硫酸氢盐-微流控温度梯度毛细管电泳法（bisulfite-μTGCE）。分别在5℃和10℃温度梯度下完成了具有1个、2个、5个、12个、30个和35个甲基化位点的甲基化模式样品和复杂甲基化样品的检测。应用此方法,完成了7种大肠癌细胞系p16基因启动子甲基化的检测。该系统检测灵敏度可达0.5ng/μl,对比例混合细胞中甲基化片段的检出限可达1/257。对20例大肠癌肿瘤组织和配对粪便样品进行电泳检测的结果显示,该方法能对临床样品进行有效的甲基化检测,粪便样品与肿瘤组织中甲基化基本符合,但尚需进一步扩大样本量。结论提出了一种基于倾斜式热辐射的芯片温度梯度形成新模式,建立了高效微流控温度梯度毛细管电泳基因突变和基因甲基化检测系统,实现了对基因突变和基因甲基化的高效检测,为肿瘤相关基因的筛查提供了有效的分析工具。更多还原

【Abstract】 IntroductionCurrently,great attention has been focused on the molecular diagnosis,therapy and prophylaxis of gene-based diseases such as cancers.Single-nucleotide polymorphisms（SNPs）/mutations and methylation,being the most common form of genetic and epigenetic variation,often offer markers of the highest resolution for tracking disease genes.DNA sequencing is considered the gold standard for DNA analysis,while the high-cost and laborious operations prevent it from being employed universally.Nowadays,although many techniques have been developed for detecting mutations and methylation,increasing efforts are being aimed at developing techniques that offer greater speed,accuracy,and cost-effectiveness than what is currently available.Temperature-gradient gel electrophoresis（TGGE）offered an effective approach for detecting SNPs and mutations without resorting to more sophisticated DNA sequencing procedures.In TGCE,where electrophoresis is processed under a continuous temperature gradient,the DNA fragments consisting of homoduplexes and heteroduplexes are separated according to the difference of their melting temperatures. Compared to other presequencing screening methods,the most outstanding advantage of TGCE is that it can detect mutations without knowing their exact nature,as long as the Tm’s of the samples studied are sufficiently close to the applied temperature gradient range.However the traditional gel electrophoresis and the complexity of temperature gradient system causes poor reproducibility and low detection sensitivity. Microfluidic analysis system is a micro-biological and chemical analysis tools based on physics,analytical chemistry and biology.An integrated microfluidic device incorporates many of the necessary components and functionality of the typical room-sized laboratory on a small chip,also called“lab-on-chip”.Microfluidic chip offers several advantages over traditional gel electrophoresis,the most important of which are high-throughput capabilities and high sensitivity,reduced sample and solvent consumption,increased portability,and reduction in analysis time.More recently,TGGE has been implemented in capillary electrophoresis（TGCE） and microchip formats（chip-based TGCE）as an alternative to slab-gel electrophoresis. The advantages of higher separation efficiency with chip-based systems were achieved mainly owing to smaller injected sample lengths and higher dissipation of Joule heating, allowing application of higher field strengths compared with CE.In this work,we developed a simple,cheap and robust system for establishing stable and reproducible spatial temperature gradients on a simple glass microfluidic chip for TGCE separations,without resorting to complicated equipment and fabrication techniques,and which is easily adaptable to standard microfluidic CE chips.The chip-based TGCE system can successfully detect the K-ras mutation and p16 methylation,providing a more effective analytical tool for tumor-associated genes screening.Materials and Methods1、Establishment of chip-based TGCE system for DNA mutation analysis（l）Microchips were fabricated by using standard photolithography and wet chemical etching techniques.The etched plate and the cover plate were bonded together via fusion bonding.A home-build LIF detection device and a PMT detector were employed for detection.（2）A home-made thermostated heater plate was used to provide gradient heating along the separation channel of the glass chip.The heater plate was mounted on the chip,and a continuous thermal gradient could be formed by slanting the heater relative to the plane of the glass chip by inserting a thin spacer at one end of the plate.During the electrophoresis analysis,the DNA sample was electrokinetically introduced into the sample channel and separation channel.（3）Four known point mutation samples were detected under the temperature gradient of 5℃(1.7℃cm^-1)and 10℃(3.3℃cm^-1)in less than 7min.In this study,a much shorter heating length of only 3.0 cm（4.5 cm separation length）was employed, and found to be sufficient for resolving all samples studied.2、K-ras mutation detection with chip-based TGCE（l）The point mutation of K-ras gene from 6 colon cancer cell lines was tested by chip-based TGCE.（2）According to coden 12 of K-ras gene,a mixed model of wild and mutation with proportions of 1:1,4:1,16:1,64:1,128:1,256:1,512:1 was established and was detected by chip-based TGCE,observing the detection limit for mutant K-ras.（3）K-ras gene of 84 Paraffin-embedded tissues from patients with colorectal cancer was detected by chip-based TGCE system.Compared with sequencing,the relationship between K-ras mutation rate and colorectal cancer screening was analysized.（4）K-ras gene of 20 tumor tissues and paired fecal samples from patients with colorectal cancer were detected by chip-based TGCE system.Compared with sequencing,detection rate and accordance rate for K-ras mutation were analysized.（5）AutoCAD software was applied to get the peak area of homoduplexes and heteroduplex.To determine the ratio of mutant K-ras,the peak height for mutant K-ras was divided by the sum of the peak heights for wild and mutant.The non-adjusted proportion of mutant K-ras from chip-based TGCE and the ratio of the mutant clones to the total number of sequenced clones were used to calculate an exact proportion of mutant K-ras. 3、p16 methylation detection with chip-based TGCE（l）Genomic DNA is treated with bisulfite prior to PCR amplification using primers encompassing differentially methylated sequence,whereby multi-mutations are introduced by conversion of cytosines to thymines in the unmethylated CpG sites and no conversion in the methylated ones,finally all methylation patterns can be identified by electrophoretic peak profile.（2）Fragment of p16 with various numbers of methylated sited were mixed with unmethylated fragment,and then were detected by chip-based TGCE under the temperature gradient of 5℃and 10℃in a shorter heating length of only 3.0 cm。Complex methylated samples were detected under these temperature gradients.（3）The p16 promoter methylation from 5 known colon cancer cell lines and 4 unknown colon cancer cell lines were tested by chip-based TGCE.（4）According to promoter methylation of p16,PCR products from unmethylated and methylated colon cell lines were mixed at ratio of 1:1 and were diluted 50 times, 100 times,200 times,400 times,800 times to detect the detection limit for DNA molecules.A mixed model of unmethylated and methylated PCR products with proportions of 1:1,4:1,16:1,64:1,128:1,256:1,512:1 was established and was detected by chip-based TGCE,observing the detection limit for methylated fragment.（5）To further study the applicability of this chip-based TGCE system to clinical samples,p16 promoter methylation of 20 tumor tissues and paired fecal samples from patients with colorectal cancer were detected by chip-based TGCE system.Results1、The slantwise radiative heating system could provide stable and uniform temperature gradients required for performing mutation detection on chip-based TGCE with simple instrumentation.Four known point mutation samples were successively detected under the temperature gradient of 5℃and 10℃in a shorter heating length of only 3.0 cm.The recordings showed good reproducibility in peak migration time（1.9% RSD,n=5）and peak pattern. 2、The point mutation of K-ras gene from 6 colon cancer cell lines and all kinds of clinical samples were successively tested by the present system.The detection limit for mutant K-ras is 1/513.3、Various mathylation model samples were successively detected with this chip-based system.The recordings showed good reproducibility in peak migration time （RSD 1.7%under 5℃temperature gradient,RSD 0.9%under 10℃temperature gradient,n=5）and peak pattern.The detection limit for DNA molecules is 0.5ng/μl, and the detection limit for methylated pl6 is 1/257.The methylation state of pl6 gene from 7 colon cancer cell lines and all kinds of clinical samples were successively tested by chip-based TGCE.ConclusionsIn this study,we showed the effectiveness of a robust and simply constructed slantwise radiative heating system for chip-based TGCE.Efficient and reproducible detection of genetic point mutations and methylation in DNA samples was achieved on the basis of stable and uniform spatial temperature gradients established along the separation channel.The system allows effective mutation and methylation detection within a wide temperature range of 10℃within a short separation length of only 3 cm. This is important for broadening the range of applicability for detection unknown mutations and methylations.Therefore,the approach shows promise in developing dedicated microfluidic equipment for early diagnosis of cancer diseases involving known gene mutations and methylation.更多还原