| Background Clear cell renal cell carcinoma(ccRCC) accounts for the most percentage( about 70-80%)in all renal cell carcinoma(RCC). Its morphous is usually irregular, and its cross-section is yellow commonly. It is mostly sporadic, unilateral, and unifocal. The ccRCC like other RCC responds very poorly or not at all to chemotherapy, hormonal therapy and radiation therapy. Nowadays, it is poor effective except the surgical operation which is better effective only in about 70% of early-stage and localized RCC. Its death ratio is still high because RCC is diagnosed difficultly in the early-stage. Now people understand poorly its pathogenesy, which is the key reason of poor effective management for RCC. In recent years, more people are studying the RCC in all worlds, the emergence of gene microarray provides an important tool. Microarray technology according to the principle of DNA double strands base complementation, denaturization and renaturation, many probes which are composed of known sequential oligonucleotide, cDNA or gene fragment are affixed in the surface of sustentaculum. The detectable target gene by fluorescent labeled and hybridization of the fragments to arrays are performed. Thearrays are scanned using a GeneArray scanner. The profiles of measured samples can be gained. Microarray technology can deal with lots of gene information high throughput and simultaneously. To explore the pathogenesy of ccRCC, we try to use the better advanced 60~70mer oligonucleotide microarray to identify the expression profile between ccRCC samples and normal tissue.Objectivesl.To detect some novel ccRCC specific gene through identifying the gene expression profile by using advanced long chain oligonucleotide gene microarrays.2. To explore the relationship between some known genes with ccRCC in order to determine the range of tumor specific gene in total, which is beneficial for studying ccRCC advancedly.3.To explore the new functions of some genes in order to provide the theory evidences for clinical diagnosis and therapy of ccRCC.MethodsCollecting four tissue samples of ccRCC, which were obtained from patients undergoing partial or radical nephrectomy at the Nan fang hospital, and three of them were ccRCC tissues , one was normal kidney tissues beyond 5cm away from the tumor edge to be the control group. All the samples were diagnosed by routine pathology and confirmed again before experiment, three neoplastic samples were graded I - II. Extracting total RNA from the tissues with one-step method, then underwent quality check, reverse transcription, two colour fluorescence labeling and purification to make the hybridization solution to hybridizate with Agilent Human 1A oligonuclotide array, then washing and scanned by Agilent2565BA scaner. The final parameters were analysed by Feature extraction software. Analysing the differential expression profile in the two kinds of tissues, kidney tumor specific gene were obtained.Results1. The total RNA was isolated from the three tumor tissues and adjacent normal tissue and was tested. The result was ideal and the A260/A280 values of total RNA were among 1.7 to 1.9 , the total RNA electrophoreses band 18s and 28s were clear, which showed the nucleic acid components were integrities, not degraded and not polluted by protein and organic solvent. The curves were standard and the samples were proved to be constituted of high quality of nucleic acid and not polluted and consistent with the request of experiments after analyzing again by Agilent Bioanalyzer2100.2. After the two microarrays have been hybridized and scanned, the shapes of every hybridization spot are regulation, and their spur are uniformity, no background color, no tailing and cavitas. It indicates that the effect of array hybridization is well.3. The gene expression profiles are analyzed and the ratio of Cy3/Cy5 is calculated in 3 cases carcinoma and adjacent normal tissue. If the ratio> 2, the gene is defined up-regulated; ratio<0.5, it is down-regulated. According to this standard, 204 genes of differential expression are identified, and 31 of them are up-regulated, 173 of them are down-regulated. The differentially expressing genes involve a variety of categories including ontogenesis and cancer suppressor genes, cytological signal transduction genes, DNA synthesis, repairing and recombination protein genes, DNA binding, transcription factors genes, cytological specific antigens and adhesive protein genes, ion channels and transportation protein genes, and metabolism related genes.Conclusion1. The microarray technology is an important means to detect the expression profile of clear cell renal cell carcinoma. There are 204 genes of differential expression, 31 of them are up-regulated and 173 are down-regulated. Thedifferentially expressing genes involve a variety of categories including ontogenesis and cancer suppressor genes, cytological signal transduction genes, cyclin genes, apoptosis protein genes, transcription factors genes et al.2. The clear cell carcinogenesis of kidney is related with the chromosomal abnormalities. It is the most common that a chromosome 3p and 14q loss and 5q gain frequently in cc-RCC.3. The differentially and significantly expressing genes: VHL, HIF-1A and EGFR, correlate with the pathogenesis of ccRCC clearly in the gene expression profile.4. Six down-regulated genes which are not enrolled in the genbank are discovered in the expression profile. Their construction and function will be further researched in the future.
|
PDF Full Text Request