| Gene chip is specially used for detecting nucleic acid, and it's also the most popular micro-array. No matter single or dual color fluorescence methods, gene chip all base on base-pair complementary theory. The most wildly used gene chip belongs to reverse hybridization technology. Lots of genes from cDNA library are fixed on the support and it can detect many genes'expression level simultaneously. The other kind of gene chip belongs to forward hybridization technology. PCR products from specimens are fixes on the support and it can detect SNP in many specimens on one chip simultaneously. Recently, gene chip is becoming more and more important in the cancer research.When researching nasopharyngeal carcinoma (NPC) based on the gene chip technology, we started from cDNA microarray. Gene expression profiles of 20 NPC specimens were analyzed, using inflammatory tissues as control. Utilizing bioinformatics and biostatistics, we screened a set of genes which may participate in the pathogenesis of NPC, for example, ribosomal protein S6 kinase. Some genes are reported for first time to be related with NPC. Furthermore, some bio-softwares are used to screen signal pathways which may play an important role in NPC pathogenesis. We found some crucial signal pathways, for example, regulation of Wnt receptor signal pathway, which have been reported to be related to pathogenesis in other cancers. These pathways may also affect NPC s development.Then, we classified specimens into two sets:radiation sensitivity set and radiation resistant set. Genes differently expressed in two sets were screened. These genes'expression levels may determine the subtype of NPC. We selected 12 differently expressed genes, including gene PPP1CC, etc. to construct discriminant of radiation sensitivity. This discriminant could help to detect the subtype of NPC. We also analyzed molecular mechanism of NPC radiation sensitivity by searching important pathways. Some signal pathways such like induction of apoptosis by intracellular signals, are thought to be related to NPC radiation sensitivity. Differently expressed genes in these pathways may the cause of different subtype of NPC. We also analyzed one case which was sampled before and after radiation therapy seperately. Through screening differently expressed genes in this case, we found many important genes, which may help us to understand the change of gene expression levels in radiation therapy.We researched the molecular mechanism of NPC's development and pathogenesis through system biology. Many important genes screened are involved in cell cycle, proliferation, apoptosis, and signal transduction. Some genes'functions remained unknown, further researches are needed.When studying NPC gene expression profiles, we found that NPC study was lack of suitable reference gene to normal expression data. While, some related work have been done in other cancers. We constructed model to calculate the most stably expression genes in chips as candidate genes. Real-time PCR and software geNorm were used to determine the most suitable reference gene in NPC. HPRT1 and YARS are considered as the most optimal genes for normalizing expression data in NPC.Development of cancer is always together with lost of chromosome. In order to test which parts of chromosome may lost in NPC, we used loss of heterozygosity method to determine 4 parts of chromosome which are including location D3S1300,D9S318,D11S4946 and D13S796. These 4 positions are not well known in early researches in NPC. After analysis, we found some positions lost in different level. Further researches show some important tumor suppressor genes such as multiple endocrine neoplasia type 1, are located in these places. This may give us some clues to research pathogenesis'of NPC.Besides, we studied the molecular mechanism of photodynamic therapy using hypericin as light sensitive agent in NPC therapy. Photodynamic therapy has been used in other cancer therapy in recent years. We try to use hypericin as light sensitive agent to testify its toxic effect on NPC cell line CNE-2. MTT, cell cycle and apoptosis methods are all used to measure the cell state. We found cell state obviously changed a lot. After culturing with hypericin and exposure to light, CNE-2 cells died a lot, and furthermore, cell cycle and apoptosis were both changed. We found that, cell number reduced in G1 period and increased in S and G2 period. Besides, apoptosis was observed in CNE-2 cells.In order to understand gene expression level of CNE-2 cells, we used illumine bead array to detect differently expressed genes in time course. We found some related genes and important pathways including response to wounding, response to external stimulus, response to hypoxia, etc. which reflects a big change in cell's function of damage and defense. These researches may help to understand detail molecular mechanism and signal transduction process when using photodynamic therapy to cure NPC in future.In this article, two kind of gene chips (cDNA chip and bead array) were used to research NPC gene expression levels. To understand more about hybridization dynamics principle of gene chip, we designed microarray-based dual-color fluorescence forward hybridization method. It can screen a large number of genomic DNA samples in parallel for a given SNP locus. The technique is especially suitable for genotyping some known candidate SNP loci in large-scale association study for complex disease. However, the hybridization dynamics about this technique hasn't been reported until now. In this study, by observing signal value (Cy5, Cy3) and ratio value (Cy5/Cy3), we discuss how spotting concentration of PCR products, PCR products length and fluorescence labeled probe length affect SNP genotyping. We have optimized the experiment conditions and parameters. This kind of chip can be used in SNP genotyping with high repeatability, accuracy and confidence. |
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