| Among all the environmental carcinogenic factors, physical, chemical and biological ones are predominant. And ionization radiation is one of the most commonly-seen physical factors. With the more and more widely adoption of nuclear energy in economics and military, the underlying damage of ionization radiation to human being increases. The effect of ionization radiation is a kind of far after-effect in oncogenesis. The study on the molecular mechanics of radiation oncogenesis is a key to oncology, radiobiology, prophylactic medicine and radiation protection. Radiation oncogenesis is similar to other oncogenesis in that it is a very complicated process related to the change and dysfunction of many tumor related genes, such as p53, p16, AMT, Rb, hRAD50. The products of tumor related genes, including proteins and enzymes, participate in the transcriptional control, message conduct, damage repair, cell life control, multiplicative division, energy metabolism, and apoptosis, etc. With the furthering in the study, the experts are accompanied by some puzzles. The spectrum of tumor related genes varies from race, individual, tissue type, and inducing factor. The genes differ in the different stages of cellular malignancy transformation. Among all the accompanying changes in genes during oncogenesis, it is difficult to distinguish which gene dominates, which is secondary to abnormity in DNA repair and genome instability. These all make the study on oncogenesis very complex. And there are some questions asking for prompt answers. First, the spectrum of changes in the many tumor-related genes during the process of oncogenesis of normal cells after radiation should be studied. Secondly, from the many genes, which one plays the key role? And furthermore, it must be elucidated which new genes are important in oncogensis. So to screen the specific gene in the oncogenic model after radiation is crucial.The newly developed gene array technique, aiming for the screen of tumor related genes, has the characteristics of high efficacy. And it can screen thousands of genes during one experiment. The technique was developed by Doctor Stephen Fodor in 1996. Afterwards, it developed rapidly and was used widely in the neoplasm study.The shortcoming is that there is false positive and that the specificity is not very high. For the problem of false positive, it can be avoided by the increase in the times of experiments and the used of fluorescent real-time quantitative PCR. And for the problem of specificity, it is related to the choice of EST and can be avoided by preparation of specific cDNA or by replenish with other screen result (such as suppression subtractive hybridization, SSH). SSH is an effective tool in the study of gene differential expression, basing on suppressive PCR and subtractive hybridization. The cDNA from SSH is rich in differential expression gene. And the deference in abundance between target genes is balanced. So the method is especially useful for the separation of clone with low abundance. And it has the characteristics of high sensitivity and good reproducibility. SSH has become a powerful tool in the study on the differential expression between two cell colonies in molecular biology and has facilitated the study on the gene switching and expression process in oncogensis. SSH has prompted the studies on cellular inheritance, growth, differentiation, and cancerization. In the clone of fully length mRNA of new gene, the cDNA library screen basing on nucleic acid hybridization technique has been a kind of reliable method and widely used. But it has the shortcoming of time and energy consuming. Especially in some instance, its usage is limited when the full length of mRNA is not available. The RACE technique basing on PCR has in some degree solved the problem of library screen. And the development of SMART RACE technique makes it possible even for a small laboratory to get the full length of new mRNA sequence.Our study attempted to adopt gene clip and SSH in the screen for the differential expression genes in the radiation induced carcinogensis model. And the full length mRNA was got of part of the new EST. From the genes, some were chosen to study on the effect on the radiation induced carcinogensis. And the basis for the further study was provided thus. Methods:1. Firstly the 60Co was adopted as radiation source to establish the y-rayinduced cell model as human bronchus epithelium malignant transformation toprovide the specimen for further study.2. The technique of gene microarray was used to screen the genes related to radiation induced hymus type leukemia malignant transformation model of rat. And the effect of some of the differential gene was analyzed.3. Te method of SSH was used to screen the differential expression genes of the radiation induced malignant transformations cellular model. The sequence of the differential fragment was got and its bio-informatics was analyzed. Then fluorescent real time quantitative PCR was used to detect some of the screened differential expression genes and to determine their changes. The new EST was registered in the GenBank to preliminarily analyze the bio-informatics and to choose the needed full length sequence.4. The pi6 gene was chosen from the differential expression genes screened by gene clip. And study was made on pi6 protein expression and gene mutation in the y-ray induced leukemia model of rat.5. The technique of SMART RACE was adopted to clone the full length mRNA of the new EST sequence T54 (GenBank ID: EB643248). And the function was preliminarily analyzed.Results:1. The establishment of radiation induced cellular malignant transformation model:(1) Flat plate clone experiment suggested that the reproductive activity was significantly increased of the 35-time-passage cells after 22Gy radiation.(2)Serum antibody experiment suggested that in the radiation group the clone formation increased of the 45-passage cells. In the soft agar colony formation experiment, The colony formation ability of the 50-passage cells increased significantly (13.10%o). The p-value was less than 0.01 as compared with the control group (2.86%o).(3) In the 8 athymic mice implanted with 22Gy 50-passage cells, there was one rat which developed neoplasm at the nape after 50 days. In the 8 athymic mice implanted with 22Gy 60-passage cells, there was four which developed neoplasm. It was believed to be the reliable indicator to determine the cellular malignanttransformation.2. The gene clip experiment indicated:The results of the two clips from the animal model were similar. There were 319 genes with more 3 folds of differential expression between the neoplasm group and the control group. Among them, the gene expression increased of 111 genes and decreased of 208 genes. And 147 genes had been reported. The other 172 genes had not been reported of their function.3. The cDNA library was successfully established of the malignant transformation cellular model by suppression subtractive hybridization:The clones were amplified with the common primer. Then 40 clones were chosen to be sequenced from the library of increased expression and decreased expression respectively according to the length of insertion element. Among the sequenced 80 clones, the definite sequences were got of 73 clones. There were 41 sequences with decreased expression in the transformation cells. Blast analysis indicated that there were 6 clones with known sequence and 20 EST sequences which were unknown genes and 7 void sequences and 8repeated sequenced ones. And 32 sequences had increased expression in the transformation cells. Blast analysis indicated that there were 14 clones with known sequence and 9 EST sequences which were unknown genes and 9 repeated sequenced ones. All of the 29 unknown sequences were registered into the GenBank with ID: EB643220, EB643221,EB643222......EB643248. Some of the genes were accessed by fluorescentquantitative PCR of their changes. The results indicated that in the radiation transforming group the expression of MY06, HACE1, ZNF143, HNRPH1 increased significantly (there were 3.49, 29.38. 12.99.5.00 folds increase compared with that control group respectively) and the expression of PCBP2, RPL15, TCERG1 decreased significantly (there were 1.89, 48.77. 11.95 folds decrease compared with that control group respectively).4. The results of gene clip suggested that the expression of pi 6 decreased in the radiation induced neoplasm group. The causes were study further which indicated: (1) in the leukemia model, there were 13 cases of depletion of the exon 2 of pl6 genewith an incidence of 33.3%;(2) there were 9 cases of methylation in the priming domain of the exon 1 with an incidence of 23.1%;(3) there was no basic radical mutation;(4) the expression of pl6 protein decreased about 52.4%.5. The new gene cloned (Lcrp369) had a full length of 1038bp. Preliminary bio-informatics analysis found that Lcrp369 located in the 14p22. The function of Lcrp369 was unknown yet. Its coding region included 7 exons and 6 introns. The full length of mRNA was 1038bp with a PolyA "tail" of 21bp. The encoded protein included 244 amino acids. The protein located in the cell nucleus. It had DNA binding site, N- glycosylation site, cAMP/cGMP dependent protien kinase phosphorylation site, casein kinase phosphorylation site and N-myristoylation site. The secondary structure was presumed to a-helix. Its molecular weight was 28Kd and the isoelectric point was 6.19. Digital tissue expression spectrum analysis indicated that Lcrp369 was widely expressed in many tissues and tumors. The full length of Lcrp369 had been registered into the GenBank with ID of DQ525179.Conclusion:1. The radiation induced bronchial epithelium malignant transformation model was successfully established, providing basis for study on the molecular mechanism of radiation oncogenesis.2. The results of gene clip and SSH indicated that radiation oncogenesis was a very complicated process involving many genes with a lot new genes of unknown function. The reported genes with significant change in expression was classified into: proto-oncogene and anti-oncogene;signal conduction related gene;cell cycle related gene;DNA binding gene, transcription initiation factor;cell surface antigen and adhesion molecule gene;metabolism related enzyme and kinase gene;immunoglobulin gene;DNA synthesis, repair and reconstruction protein gene;cytoskeletal protein gene;and others. Study on these new genes will be helpful for the understanding of tumorigenesis.3. The amount of pi6 protein decreased in the radiation induced tumors. The decrease was caused by exon 2 depletion and promoter region methylation. The decrease in pi6 expression participated in the radiation induced tumorigenesis andmay be the meta- or late- event in radiation induced tumorigenesis.4. Preliminary results from bio-informatics analysis suggested that the new gene of Lcrp369 can be expressed in several normal tissues and several tumors. The potential acting sites, including N- glycosylation site, cAMP/cGMP dependent protein kinase phosphorylation site, casein kinase phosphorylation site and N-myristoylation site, were all related to tumor's unbalanced growth, adhesiveness and cellular signal transduction. Some of the genes had been listed as tumor metastasis promoting genes. All these suggested that Lcrp369 played some role in radiation induced carcinogenesis. And its function needs further study.
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