cDNA芯片在癌症研究中的应用

【作者】 魏庆；

【导师】 谢毅；

【作者基本信息】 复旦大学 ， 遗传学， 2006， 博士

【摘要】 cDNA微阵列是将cDNA文库中大量的基因序列固定于载体上，可用于同时检测比较生物样品中多个基因的表达状况。近年来基因芯片被越来越多生物学家用来分析比较癌症组织与相应正常组织之间基因表达的差异，以期研究癌症机制和帮助临床治疗。 在方法学上，为了提高cDNA芯片的性能，我们采用不对称PCR的方法来制备DNA靶分子，并和传统的方法在杂交动力学方面进行了比较。由于基因芯片上的cDNA序列包括编码链和非编码链，这使得它们会和样本中的cDNA发生竞争性的杂交，增加了实验的复杂性，降低了杂交效率和灵敏度。为了解决这个问题，我们使用不对称PCR来制备大量的单链cDNA靶分子，用于制作芯片。在实验中，我们系统的比较了ssDNA和dsDNA的杂交行为，而且对不对称PCR的参数进行了优化，使它能够有效的制备高重复性，精确性和可靠性的ds-ssDNA芯片。 在癌症研究的课题中，本人对3种癌症，即急性白血病、脑膜瘤和前列腺癌的表达谱进行了分析。结合生物信息学、统计学和分析软件，筛选出了一批十分具有研究价值的基因，许多还是在癌症中首次报道。从系统和整体的角度，对急性白血病的发生发展机制进行了探讨，构建了一条脑膜瘤发生发展的通路，对在前列腺癌中差异表达，且和激素相关的基因的网络相互作用进行了阐述。进一步的，结合样本的临床资料情况，对急性白血病中难治易治的机制进行了分析，对脑膜瘤中各级别间的差异进行了探讨，对前列腺癌中雄激素依赖和非依赖的机制进行了研究。更深一步的，依据这几种癌症各自的特点，对前列腺癌中激素和血管生成相关等方面，对脑膜瘤中男女病人比例差异、辐射影响和HIV病毒因素等方面进行了分析和探讨。最后，对这3种癌症的表达谱数据还进行了Meta组合分析，找到一组在3种癌症中都显著差异表达的基因，这些基因的异常表达反映了癌症细胞中细胞周期、增殖、生长、凋亡和信号传导等方面异常的现象。其中一些基因的功能还尚不清楚，因此更值得深入研究。更多还原

【Abstract】 cDNA microarray (gene chip) is a set of short Expressed Sequence Tags (ESTs) made from a cDNA library of a set of known (or partially known) gene loci, which could be used to detect the expression status of thousands of genes in samples. Recent years, more and more biologists use cDNA microarray to analysis the difference of expression profiles between cancers and homologous normal tissues, in order to find the mechanism of cancers and help clinic.In order to improve the performance of cDNA microarray, we try to use asymmetric PCR instead of conventional PCR to prepare cDNA targets, and Compared hybridization behavior between these 2 approaches. Double stranded targets on the cDNA microarray contain representatives of both the coding and noncoding strands, which will introduce hybridization competition with probes. Here we compared the effect of double and single strands of targets on the signal intensity and the ratios of Cy5/Cy3 within the same slide. The results show that the single stranded targets can increase the hybridization efficiency without changing the ratio of Cy5/Cy3. Based on these results, we established a new strategy by generating cDNA targets with asymmetric PCR instead of conventional PCR to increase the sensitivity of cDNA microarray. Furthermore, we validated the feasibility of this approach. The results indicate that cDNA microarray system based on asymmetric PCR is more sensitive without decreasing the reliability and reproducibility as compared with that based on conventional symmetric PCR.In the study of cancers, we analyzed the expression profiles of acute leukemia, meningioma and prostate cancer. Combined with bioinformatics, statistics and analysis softwares, we picked out a lot of worthy genes, and many of them were first reported in cancers. In a holistic and systematic view, we discussed the progress mechanism of acute leukemia, found a hypothetic pathway of meningioma progression, and described the network interaction of a set of genes which differentially expressed between androgen independent prostate cancer and androgen dependent prostate cancer significantly and related to hormones. In further research, according to the clinic information, we discussed the difference of refractory and non-refractory acute leukemia, subtypes in mengingioma, and androgen independent and androgendependent prostate cancer. Last but not the least, according the specialties of these cancers, we discussed the differentially expressed genes of prostate cancer in hormone and angiogenesis and of meningioma in sexual difference, radiation and HIV effect. Finally, we did a Meta analysis of these expression profiles and found a set of genes significantly differentially expressed in acute leukemia, meningioma and prostate cancer. These genes reflect the disorder status of cancer in cell cycle, proliferation, growth, apoptosis and signal transduction etc. Some genes’ functions are still unknown, which are worth to be further studied.更多还原