Exploration On The Pathogenic Genes Of Chordoma With Gene Microarray

Exploration on the pathogenic genes of Chordoma with gene microarrayChordoma stemmed from embryonic notochordal remnants of the primary malignant bone tumors, Mainly occurred in the axial skeleton . In recent years, through the locus, MI analytical techniques and oncogenes, The initial location of tumor suppressor gene on chromosome found mainly in the change of chordoma chromosome 1p36 and 7q33. With Development and application of gene chip microarray analysis, library technology, information technology, biotechnology,DDPCR,RFLP,In oncogene amplification, tumor suppressor gene mutation and microsatellite instability analysis. Of chordoma, the initial mechanism reveals progress。However, the use of experimental study of tumor and normal tissue samples, and the organization does not have homology chordoma, Screening of flux and smaller, it's hard to reflect the full chordoma genes related to the situation.This study used the experimental group as fresh chordoma three cases, one case of fresh fetal nucleus pulposus as the control group, Total RNA was extracted, purified by reverse transcription double-strand cDNA synthesis. After further purification of the synthesis of biotin-labeled cDNA and fragmentation. With the test group and control group with biotin-labeled cDNA microarray hybridization. ScanArray3000 scanning and DNA sequencing analysis software and terns, Chordoma is to be differentially expressed genes in the nucleus pulposus and the fetus. Design of differentially expressed genes quantitative PCR primers and immunofluorescence test The differentially expressed genes of bioinformatics analysis and the incidence of related mechanisms revealed chordoma . Objective :1,the use of fetal nucleus (in the development of biological homology) as a control mechanism for the pathogenesis of chordoma.2,the level of genome research in the pathogenesis-related genes chordoma screen.3,the completion of the initial exploration of chordoma mechanism for the genetic diagnosis of chordoma, gene therapy target genes.4,for further chordoma disease-related genes and gene knockout studies lay the initial foundations transfectionMethods :1, chordoma fetal nucleus with the collection : Laboratory specimens were 10 cases of chordoma from December 2003 to August 2006during the Second Military Medical University, Chang Hospital orthopedic surgical resection specimens. Implantation surgery removed immediately frozen in liquid nitrogen tank standby. All samples were organized by the Changhai Hospital were diagnosed as chordoma immune pathology. Fetal nucleus pulposus for the period March 2005 to August Changhai Hospital, Second Military Medical University, obstetrics and gynecology, labor fetal nucleus pulposus , the samples were collected immediately after the induction cans frozen in liquid nitrogen and placed reserve.2, chordoma,fetal nucleus pulposus with biotin-labeled RNA extraction and cDNA synthesis : the total RNA isolation is conducted by QIAGEN?'s total RNA isolation kit.The synthesis of the double-chain cDNA,the biotin-labled cRNA and the fragmentation of the cRNA were strickly followed by Affymetrix? GeneChip? U133A expression analysis experiment method.(for detailed experiment procedures,please see the experiment guildlines of Affymetrix? GeneChip? U133A).3. Hybridization of the gene microarray: the prepared biotin-labled cRNA(15μg*4) were hybridized overnight with 4 U133A chips. The following procedures such as the wash,staining were restrictly followed by the standard experimental guild line of Affymetrix? GeneChip? U133A and were accomplished by the automatic gene-chip washing sation. The 4 processed chips were scaned by Affymetrix scanner 3000. The scanning result were analysis by Microarray Suite5.0 software.4. Data analysis: the differently expressed genes between chordoma and fetal nucleus pulposus were screened by Microarray Suite5.0 software. Using an entrance limit of≥2.0 or≦-2, there are 10 groups of differently expressed genes which are composed of 5 groups of up-regulated and 5 of down-regulated genes. Among the 6 groups of genes, the common differently expressed genes were found(up-and down-regulated included,for the details of the genes ,please see the Results as follows.). The analysis of the differently expressed genes were conducted by comprehensive use of Affymetrix analysis center ( www.affymetrix.com/analysis ) ,GenBank database(www.ncbi.nlm.nih.gov/Genbank)and swissprot database.5. real-time PCR confirmation:the total RNA isolation of the 9 chordoma samples were accomplished by one-step method using TRIzol reagent (GIBCO/BRL .CO); the design of the primers: logging in GENEBANK wetsite,design the pairs of the primers using the selected 10 differently expressed genes as stencil-plates. The synthesis of the primers were accmpolished by ShangHai BioChip co.ltd; SYBR? Green I real-tim RT-PCR were conducted by ABI Prism 7000. ( for details,please see the the SYBR? Green I real-tim RT-PCR experimental instructions) Results1. the differently expressed genes between chordoma samples and fetal nucleus pulposus samples: Using an entrance limit of≥2.0,there were 47 up-regulated and 153 dowm-regulated genes identified by microarray which were commonly differentially expressed among all the three chordoma samples in comparison with the fetal nucleus pulposus samples, the detailed results are as follows: 968 genes were up-regulated while 888 were down-regulated insample 1;878 genes were up-regulated while 896 were down-regulated in sample 2;1150 genes were up-regulated while 803 were down-regulated in sample 2;The cluster analysis chart of the 200 commonly differently expressed genes were made (fig.8). Among the 47 commonly up-regulated genes, we randomly chose TPD52,XAGE1,MAGE-1,MMP-7,MMP-9 for confirmative experiment in resected samples. Meanwhile, in the 153 commonly down-regulated genes, 5 genes:P16INK4a,FHIT,RNF-11,CASP9,c-myc were randomly chosen for real-time PCR confirmative experiment.2. the result of the real-time PCR confirmative experiment of the 10 genes: The array results of the 10 genes were further confirmed in the 9 clinically resected chordoma samples by the real-time RT-PCR. The results indicate that all the samples have the same change of the gene expression as the microarray shows at the level of mRNA(fig.13),which in turn further confirm the reliability of the method of gene microarray.Conclusion1. Many genes such as the oncogenes, tumor suprressor genes, tumor-related genes were involved in the pathogenesis of chordoma. Many tumor-related genes such as TPD52,XAGE1,MAGE-1,MMP-7,MMP-9,P16INK4a,FHIT,RNF-11,CASP9,c-myc which were reported to have stong relationship with other tumors of different histogenesis were find in our study to be significantly differently expressed in the malignancy. Further investigation of the commonly differently expressed genes in our study will help to find the truth of the origin of this malinancy.2. The genome array is a powerful tool in the study of the pathogenesis of themalignancies which will help us to screen the tumor-related genes at an unprecedented level of the the human genome. It is helpful to apply gene array to find the laws of the gene expression and to study the gene-gene interactional relationships in this tumor..