| Nuclear energy had been widely used in the national economy and military affairs territory, the potential risk of the ionization radiation was increasing uninterruptedly. Of all the risks , radiation carcinogen is the most important aftereffect .Thus the research of the molecular mechanism of the radiation carcinogen became the focal point to the radiobiology, atom prevention medicine , prophylactic medicine , radiation protection medicine, the research results of which involved the utilization of the energy source , environment and warfare. All of these results make its research posses important social, military , economic and political significance.Amount of study had shown that radiation could make gene mutation or chromosomal aberration, both of which could push or switch on the cell malignancy conversion .The cell malignancy conversion was mainly the results of the accumulation of gene mutation and chromosomal aberration . Therefore in order to clarify the mechanism of the cell malignancy conversion induced by radiation , we should study it from the gene level. The present research was limited to the expression and mutation stat of few known important gene, anti-oncogen, such as p53, c-myc, H, K, H-ras, Rb telomerae and so on in various models. As these research had one-sidedness, we might not know the whole process of the cell malignancy conversion through several gene. Meanwhile radiation carcinogen and the cell malignancy conversion induced by radiation themselves had tolerable complexity and heterogenous. Thus clarifying the mechanism of radiation carcinogen need identify the differential expressed gene between the normal cells and the corresponding malignancy cells from the whole point of view, and search the unknown gene that took part in the process of the cell malignancy conversion. This was an imagine before several years, but with the quick development of the molecular genetics technology, especially the emerge of mRNA differential display technology , the identification of the differential expression between the different functional status cells became impossible.mRNA differential display technology was a new analysis method of the cell gene expression that developed in the ninety, it was also called differential display reverse transcription PCR(DDRT-PCR). The rudiment of which was performing te-vefse tcarocripkm ?CR. -with the 3'tip anchor primer and the 5'tip random primer, amplifying the 3' distal end of mRNA . In this experiment , as the 5' tip random primer was shorter, which could match pair with various cDNA , thus we might obtain a lot of cDNA parts. Using the PCR product labeled by isotope, we can obtain a strip spectrum by sequencing the gel electrophoresis and isotope self-developing. If we change the fabrication of the primers, the about 15000 kinds of mRNA of the mammal can be displayed. Compared the mRNA dactylogram of the different functional status cells , the differential expression gene can be identified. The virtue of this method was as following: sensitive 5u.g RNA was sufficient to 80 kinds of primer fabrication, which could overjet the majority of cells mRNA ; excellent reproducibility: we can compare more than two kinds of RNA samples simultaneously , which might be widely use in identifying the specified gene differential expression in certain process and finding the new tumor-correlated gene. The foundation of this technology became the effective model for studying the gene breaker and the expression routine in the process of the tumor development , thereby accelerate the research of the molecular mechanism of the tumor occurrence.But the classical DDRT-PCR method used the PCR product labeled by the radioactive isotope , even though the sensitivity of which was higher, the radioactive isotope was harmful to anthropometric and might make the DNA strips mistaken cutting, these could affect a series of subsequent experiments. Ideal labeled molecular should give consideration to sensitivity , safety and convenient simultaneously. Some researchers labeled the PCR products with fluorescein instead of isotope, but the sensitivity of the fluorescein was not high and could present artificial positive strips. An et al, reported that they detected the PCR products with chemiluminescence, Chen et al, reported they used cardiox instead of isotope . But all these methods need special detecting instruments or their sensitivity were not high, both of which could prevent this technolgy development and application.The three mainly technology involveded in the mRNA differential display technology was as follows: PCR, Electrophoresis and Image developing detect technology. Certainly it also included other process such as: reclaiming, reamplifyingand so on. From all the data about the DDRT-PCR technology, we can find that the results of this technology is not favorable, which mainly consists in that the results of the image developing is not perfect. The process of the conventional DDRT-PCR technology was as follows: applying the radioactivity isotopeisotopes in labeling the PCR products, sequencing the gel electrophoresis (metamorphic polyacrylamide gradient gel electrophoresis) and isotope self-developing the gel. The sensitivity of the isotope self-developing is so high that we can not control the developing time, which may result in higher background, broader straps. These straps can adhere each other and deliminate unclearly, which make we unable read the gel results exactly. Moreover cutting the different DNA strips is contrast to the the developing film, which might result in mistaken cutting and even affect the subsequent experiments.As the isotope is harm to the human body, the isotope experiments should be made in special laborary. It is trouble to treat the disposal too. All of these unconvenient factors make people search other technology. Resent years someone apply the argetodye technology in developing. The history of DNA argetodye was long in time , which was a kind of more sensitive technology. The minimum detectable content of nucleic acid is 2ig^g.the operation of this technology is simple, and the favorable virtue of which is that we can read all strips directly from the gel. Thus we can cut the objective straps conveniently.But the results was not perfect , which mainly consists in the unclean banding pattern, the severitier pulling trails, the unclear delimitation of the straps and the unsatisfating readability. The perfect labeled molecular should give consideration to sensitivity, safety and conveniece. Some researchers labeled the PCR products with fluoresceins instead of isotopes, but the sensitivity of this method is not sufficient and can result in artificial positive straps. An reported that they detected the PCR products with chemiluminescence, and Chen reported that they replace isotopes of cardiox. All these methods need special detecting instruments and the sensitivity is not enough , which interference this technology develop and apply. Our trials try to improve the DDRT-PCR technology, mainly in regulating the electrophoresis selection and strain technology in order to find a more stable and perfect experiment method.Time resolved fluoroisnmunoassay (TRF) is a new nonradioactivity nucleic acid assay technology , the marker of which was lanthanum element. The fluorescence decay time this element was long, the migration of the Stock's was great, you couldeliminate the interaction of the the unspecial background fluorescence as possible as you can by postponing the measuring time. Thus the measurement sensitivity can rival to the method of the radionucide labeled .The virtue of it also included quick, convenient , high sensibitity ,stability , these made this method used widely in the nucleic acid quantu assay . At present there had been nucleic acid assay reagent kit that labeled by Eu3+ provided overseas.this kit can be used in nucleic acid hybridization assay and PCR assay. Our lab apply it in the nucleic acid and PCR product quantu detect with the National Nature Fund financial assistance , and obtain satisfying results.In this study we want to create the radiation carcinogen nude mouse tumor metastasis models and improve the present DDRT-PCR technology, find a more stable and perfect differential expression method. The radiation carcinogen nude mouse tumor metastasis models act as the investigation objects,We analyze the difference of the gene expression in different time phase during the process of the radiation carcinogen, identify the differential expressed gene between the normal cells and the corresponding canceration cells across the board, search the new unknown gene that involve in the process of the radiation carcinogen. The purpose of this research is to clarify the function of this method , and provide some theory basis for investigating the molecular mechanism of the radiation carcinogen. In addition , we are trying to set up the method of detecting the biomacromolecule strips of the gel electrophoresis with the time resolved fluoroisnmunoassay, and provide the theory basis for sequencing the DNA difference strips of the gel caraphoresis with this method.The research results were as follows:l)we established radiation carcinogen nude mouse tumor metastasis models.2) we set up the DDRT-PCR test method, and the propene polymer sequencing gel electrophoresis method successfully, we also use the argento-dye technology to replace the frequently used autoradiography. The resolution and the reliability , the operation of this new method are all better than the autoradiography .3) we compared the thymus and the bone marrow cells mRNA of the radiation carcinogen animal groups , the corresponding un-carcinogen animal groups and the normal unexposured animal groups, we also screened successfully 8 differential expressed gene using 24 pairs primers and amplified them.4) we detected the thyroxine sodium strip labeled by Eu in the SDS-PAGE with the Time resolved fluoroisnmunoassay, and compared the results dyed by Coomassie light... |
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