【作者】 宋波；

【导师】 唐建武；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 背景:转移是区别肿瘤良恶性的主要生物学标志,也是恶性肿瘤患者最常见的死因。揭示肿瘤转移的分子机制、提高恶性肿瘤患者的预后是近年来肿瘤学研究领域面临的迫切问题。由于恶性肿瘤绝大多数来源于上皮,而淋巴道是其主要转移方式,因此,淋巴道转移分子机制的揭示对于临床肿瘤患者的诊治具有更为重要的现实意义。肿瘤转移的发生受到多种因素的调控,是多种生物分子和信号通路在时间和空间上复杂作用的结果,但传统的研究方法一次只能检测一个或几个基因,不仅费时、效率低,而且可比性亦较差。上世纪 90 年代中期发展起来的高通量的基因芯片技术可同时对数以万计的基因进行快速而准确的检测,有效避免和弥补了传统技术的不足,结果客观可信。其中的表达谱芯片是应用最为广泛的基因芯片,它是对细胞内mRNA逆转录后形成的cDNA进行检测。Hca-F和Hca-P是一对高度同源但淋巴道转移能力显著不同的小鼠肝癌腹水型细胞株,其中 Hca-F为高转移细胞株,在有正常免疫功能的 615 小鼠体内,其淋巴结转移率在 70%以上;Hca-P 细胞为低转移株,其淋巴结转移率低于 30%。两株细胞源于同一亲本,具有基本相同的遗传背景,两者的差异主要集中于转移表型上,其差异表达基因可能参与肿瘤淋巴道转移机制的调控。 目的:本研究采用表达谱芯片比较高、低淋巴道转移力小鼠肝癌腹水型细胞株 Hca-F 和 Hca-P 的基因表达谱,以筛选出与肿瘤淋巴道转移相关的基因,为进一步阐明肿瘤淋巴道转移的分子机制奠定实验基础。 方法:TRIzol 试剂分别提取 Hca-F 和 Hca-P 细胞的总 RNA,用 T7-Oligo(dT)24引物(5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3’ )反转录合成双链 cDNA,Phase Lock Gel(PLG)—酚/氯仿纯化后以两种细胞的 cDNA为模板,使用 Affymetrix 公司的 Enzo RNATranscript Labeling Kit 进行体外反转录,同时进行生物素标记,合成生物素化的 cRNA 探针。合成好的 cRNA 探针经片段·2·化处理(大小约在50-200bp之间)后,分别在Affymetrix 公司的专用设备Affymetrix? Hybridization Oven 640 中与 Affymetrix GeneChip? MOE430A(包括 22,690 个转录本,对应于约 14,500 个小鼠已知基因和 4,371 个 EST)杂交 16 小时,杂交后的芯片经过洗脱、染色处理,使用 Affymetrix? G2500AgeneArray Scanner 对杂交信号进行扫描,芯片扫描后获得的数据利用 Affymetrix? Microarray SuitSoftware 5.0 计算和处理。在比较两张芯片的结果之前,先对每张芯片的数据进行归一化处理。以 Signal Log Ratio(在两张芯片相比较时代表了一个转录本变化的大小和方向)≥1.0 或≤?1.0(表明转录本的水平至少提高或降低 2 倍)和 Changep-value(代表了两张不同的芯片之间一个探针对表达水平相同或不同的可能性)≤0.05(表明实验组芯片表达水平高于对照组)作为筛选差异表达基因的标准,并应用生物信息学对检测结果进行分析。为确保芯片检测质量,在与小鼠 MOE430A芯片杂交前,cRNA 探针先与一张质量检测芯片“test chip”杂交,经过扫描和数据处理后,证实 cRNA 探针的质量没有问题后再用它与小鼠 MOE430A 芯片杂交。检测芯片内还设有β-actin 和 GAPDH 等看家基因对照,以及 bioB、bioC、bioD、cre 和过饱和的生物素化的寡核苷酸 B2 等外加的阳性对照,以检测基因芯片的质量。 采用分子信标探针,定量检测 Slc38a4、Cav 和 BC037006 等 3 个已知基因在两种细胞中的表达,以验证基因芯片的结果。每个基因重复三次,取三次实验所得 CT值的平均值,经过看家基因 GAPDH 校正后再进行比较。结果:动物实验证明,实验所用 Hca-F 和 Hca-P 细胞具有稳定的淋巴结转移能力,符合本实验对细胞模型的要求。cRNA 探针与“test chip”杂交后的扫描图可见,图上方的“Genechip TEST3” 字样和四角的点及中央的“+”字均清晰可见,整张扫描图内的点线分布较均匀,表明 cRNA 探针质量良好。基因芯片的检测报告表明,这两张芯片的背景值和噪音值都很均匀;其中的看家基因对照β-actin和 GAPDH 的 5′端和 3′端均被检测到且 5′端和 3′端的信号比值分别是 1.04、0.83 和 1.47、0.80,均明显低于 3.0 的标准值;外加的阳性对照 bioB、bioC、bioD和 cre 也被检测到且信号强度随着浓度由低到高呈现出由弱到强的趋势,表明基因芯片质量良好,杂交及检测体系正常,芯片检测结果可信。在总共 22,690 个转录本中,Hca-F 细胞检测到 12,048 个,占总转录本数的 53.1%;Hca-P 细胞检测到 11,881 个,占总转录本数的 52.4%。进一步利用 Affymetrix 公司提供的分析软件Affymetrix ? Microarray Suite Version 5.0对上述两个样品的芯片检测结果进行分析,并根据筛选标准进行筛选。结果发现 Hca-F 与 Hca-P 细胞相比,在 14,500个已知基因中,Hca-F 中表达上调的有 901 个(1,097 个转录本),占芯片中已知基因的 6.2%,在 4,371 个 EST 中表达上调的有 129 个(157 个转录本,数据在本文中未公布),占芯片中 EST 的 3%;Hca-P 中表达上调的已知基因和 EST 分别是更多还原

【Abstract】 Background: Metastasis is the major biological marker in differentiating themalignant tumor, and most deaths from cancer are due to metastasis. So understandingthe molecular mechanisms of metastasis and improving the prognosis of cancer patientsare the most important and urgent issues in the field of oncology research. Becausemajority of malignant tumors are carcinomas and lymph node metastases oftenrepresent the first step in the metastatic process, it is very important to make themolecular mechanisms of lymphatic metastasis clearly. At present, various genes andsignal pathways which are involved in this complicated process have been widelyaccepted. However, traditional gene analysis could only study one or a few genes at atime, and is costly and time-consuming, hardly explaining the whole process accuratelyand completely. The recent development of DNA microarray technology, a type ofhigh-density analysis for gene expression, has opened a new era in medical sciences. Itcan analyze the expression of many genes simultaneously. cDNA microarray is the mostpopular one, it detects the cDNA synthesized from cellular mRNA. Hca-F and Hca-Pare a pair of synogenetic mouse hepatocarcinoma ascites cell lines, presenting a specificpotential of lymphogenetic metastasis when inoculated subcutaneously in 615 mice,Hca-F showing a highly metastatic potential(>70%), while Hca-P a low one(<30%).Synogenetic and specific for lymphogenetic metastasis, with significant differentiationonly in metastatic potential, differentially expressed genes obtained maybe invovled inthe lymphatic process. Object: In order to obtain lymphatic metastasis-associated genes, thetranscriptional profiles of mouse hapatocarcinoma ascites cell lines Hca-F(highlymetastatic) and Hca-P(low metastatic) were compared using cDNA microarray. Method: Total RNA was isolated from F and P cells respectively using TRIZOL?reagent, cDNA was synthesized using the T7-Oligo(dT)24 primer(5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3’).Double-stranded cDNA was purified with Phase Lock Gel(PLG)-Phenol/chloroformextraction. Then in vitro transcription labeling was performed using the Enzo RNATranscript Labeling Kit (Affymetrix). The biotin-labeled cRNA was fragmentedrandomly to an average size of approximately 50-200 bases by mild alkaline treatment.Fragmented cRNA was hybridized to Affymetrix MOE430A array(containing 22,690transcripts, almost 14,500 known genes and 4,371 ESTs) for 16 hours in Affymetrix?Hybridization Oven 640. After washing and staining, the arrays were scanned using theAffymetrix?G2500AgeneArray Scanner. The data obtained through GeneChip?scanning was analyzed using Affymetrix? Microarray Suit Software 5.0. Before thetwo arrays were compared, the GeneChip? software conducted normalization andscaling of the data for each array. The mRNA expression level of a transcript is directlyrelated to the signal which is a quantitative metric calculated for each probe set andmeasures the mean difference of fluorescence intensity between perfect match andcentral mismatch oligonucleotides of a probe set. Signal Log Ratio, which estimates themagnitude and direction of change of a transcript when two arrays are compared, of atleast 1.0 or –1.0 (that indicates an increase or decrease of the transcript level by 2 foldchange) and Change p-value, which measures the probability that the expression levelsof a probe set in two different arrays are the same or not, ≤0.05( that means theexpression level in the experiment array is higher than that of the baseline array) werechoosen to select differentially expressed genes. The results were analyzed bybioinformatics. In order to confirm the quality, the cRNA probe was first hybridized toa “test chip” before to the MOE430A array. In addition, the hybridization solutioncontained a mixture of four control cRNAs for bacterial and phage genes(bioB, bioC,bioD and cre) to serve as comparison tools for hybridization efficiency between arrays.A biotinylated oligonucleotide B2, that specifically hybridized to features at the centerand co更多还原