| Ionization radiation is one of the most physical carcinogenesis factors and its potential hazard for human health is increasing with the wide use of nuclear energy. Because radiation carcinogenesis is the most important long-term post-irradiation effect, the study on its molecular mechanism is the key point of oncology and radiation biology which has great value and meaning. Many studies show that radiation can start and promote cell malignant transformation by inducing gene mutation or chromosome aberration and cell malignant transformation is a progressive process of gene mutation and gene expression change. So the study on the mechanism of radiation inducing cell malignant transformation should focus on the gene level and the target should not been limited in several known genes. The tumor form is a very complicated process involved in the change and function abnormity of many tumor related genes. Therefore, to clarify the mechanism of radiation carcinogenesis, we should study the differentially expressive genes between normal cell and malignant transformation cell and try to find related unknown genes from the whole viewpoint.Suppressive subtractive hybridization (SSH) is a powerful tool which bases on suppressive PCR and subtractive hybridization. By this way enriched differentially expressive genes can be attained and the difference of gene richness can be erased which is likely to isolate rare clone. The required mRNA is only 1～2ug and results can be attained within three or four days. This technique contributes to the research on the gene shift and expression program in tumor developing process.However, different gene analysis usually required a great deal of purified homogenous cells, which cause great research difficulties. By the common techniques to attain whole tissue, experiment results can only show the approximate gene expression in a cell population and those characters which cause cell classification even the sole difference are not clear because of the cell heterogeneity. Fortunately, single cell RT-PCR maybe resolves this conflict. Single cell Reverse transcription-PCR (SC-RT-PCR) can attain the whole cell cDNA by the amplification independent on special gene sequence. Those cDNA can be used to constitute difference cDNA library and screen differentially expressive genes. Based on this technique, a mini cell gene expression analysis system can be set up which will mitigate the difficulty of acquiring plenty of homogenous cells. And we can also study the whole genes expression in single cell by SC-RT-PCR combined with DNA chip. This technique also contributes to uncover the unique character of gene expression in homogenous cells.Aiming at above problems, we first set up a stable single cell RT-PCR experiment system including amplification the whole mRNA independent on special gene sequence and amplification special target gene. Second, we study the change of FHIT gene in BEAS-2B cell lines after different dosage γ-ray irradiation by SC-RT-PCR. Finally, we screen the differentially expressive genes related with radiation carcinogenesis in the γ-ray induced leukemia animal model by suppressive subtractive hybridization.In first part our experiment shows that the whole mRNAs in single cell were amplified effectively by SC-RT-PCR, the size of which was most between 0.2 kb to 2kb and the quality of which was up to ug level. According to the amplification results of human β-actin and DPH2L gene, we think SC-RT-PCR have fine differentiability sensitivity efficiency and fidelity to amplify special target gene. Second experiment results show that FHIT gene occur different mutation in different phase after different dosage irradiation and these mutations were all exon deletion mutations by DNA sequencing. According toχ2 analysis, in 24 hours after irradiation, the mutation rate was not different among all dosage groups(P>0.05). In 72 hours after irradiation, the mutation rate of 0.5Gy group and 1Gy group was different greatly with those of 16Gy group(P<0.05). In ten day... |
PDF Full Text Request