Exprimental Studies On Theraputic Stratagies Aiming At P53 Mutant By The Combination Of Radiation And Gene-therapy

The p53 gene,an important tumour suppressor,plays a key role in mediating cell response to various stresses,mainly by inducing or repressing a number of genes involved in cell cycle arrest,apoptosis,DNA repair and so on.p53 gene is located at 17p13,containing 11 exons and 10 introns,the total length is about 20 000 bp.mRNA length of p53 gene is 1182 bp,encoding a protein of 53 kDa.P53 protein composed of 393 of amino acids, existing as tetramer in vivo.More than 50%of the tumors are associated with p53 mutations, 74%of these are missense mutations.Single base pair mutations that alter the function of p53 gene and oncogenes occur frequently during oncogenesis.In cancer cells,p53 mutants might antagonizes wild type p53(WT p53 ) function and has intrinsic oncogenic properties,p53 mutants sometimes have a dominant negative effect on WT p53.Therapeutic strategies targeting p53 should be specific for the mutants without altering WT p53 function and ideally be broadly applicable to different mutations.The demonstration that small interferring RNAs(siRNAs) can be used to inhibit gene expression in mammals opens new avenues for gene-targeted therapies.According to the principle of RNAi,one p53 mutant,175H,which is closely related to tumorogenesis in chinese was selected,the apporiate retroviral RNAi vector Psuper-175HR containing specific hairpin RNA(shRNA) was constructed and the gene silencing effect of Psuper-175HR was detected.Simultenously,X-ray radiation was also applicated to compare the treatment effects between radiation alone and RNAi combined with radiation.1 Construction of the expression vector of p53 mutant mtp53 -175HThe wild-type p53 cDNA was used as the template,PCR site-directed mutagenesis was used to obtain p53 -175H cDNA sequence,gene recombinant technique was used to construct the PcDNA3.1-175H expression vector.Sequencing results show the successful construction of the expression vector.2 The establishment of cell model with p53 -175H genotype The PcDNA3.1-175H expression vector was transfected into H1299(p53 -/-) cells by liposome in vitro to establish the H1299-175H model.According to the instruction of invitrogen company,the vector and liposome were mixed and added onto H1299(p53 -/-)cells, then the cells were selected with G418(800 ug/ml) until the appearance of visible clones,the clones were augmented under the normal culture condition.Western blot was used to detect the protein expression of p53 -175H and the result shown the appearance of band at the position of 53 kD,suggesting the expression of p53 -175H protein.These proved that the expression vector mtp53 -175H is biologically active in the p53 -175H cell model.3 shRNA design and gene silencingThe shRNA sequence targeting p53 -175H was synthesized and inserted into retroviral vector Psuper to construct retro-Psuper-175HR.The retro-Psuper-175HR was then transfected into packaging cell 293T using calcium phosphate co-precipitation and the viral soup containing psy-viral particle was used to infect H1299-175H cells directly,western blot was used to detect the protein expression.As is shown,the expression of mtp53 protein was significantly reduced after the introuduction of retro-Psuper-175HR,illustrating that RNAi technology have a high efficiency of gene silencing though there has a high mtp53 expression level in this cell model.4 Changes of p53 expression and the effect of RNAi after ionizing radiation4.1 Time-course changes of p53 expression in the H1299-175H cells after ionizing radiationTime-course changes of mtp53 expression in the H1299-175H cells was detected by Western-Blot,and the protein was abstracted at 4h,8h,16h,32h after ionizing radiation with 4Gy X-rays.The expression of p53 protein was enhanced in H1299-175H cells in a time-dependent manner after irradiation.4.2 Dose-effect changes of p53 expression in the H1299-175H cells after ionizing radiationDose-effect changes of mtp53 expression in the H1299-175H cells was detected by Western-Blot,and the protein was abstracted at 16h after irradiation with 2,4,6,8Gy X-rays. The expression of p53 was enhanced in H1299-175H cells in a dose-dependent manner after radiation. 5 Effect of radiation together with gene silencing on mtp53 -175H after radiationThe Western-Blot was applied to detect gene silencing effect of psuper-175HR in H1299-175H cell model at 1h,5h,10h,16h after IR with 4Gy.Results shown no differences could be found between difference groups,there was no synergization between RNAi and radiation under this condition.In this study,PCR site-directed mutagenesis techniques,gene recombination technology and lipofection method was used to establish the H1299-175H cell model.The shRNA of p53 -175H was introuduced into H 1299-175H cells and mtp53 -175H expression was monitored by Western blot.From the experimental results,the RNAi interference effect can be seen resulting from the decrease of mtp53 expression.At the same time irradiation increased the expression of mtp53 in H1299-175H cells,while the increased expression was significantly reduced after the introuduction of RNAi vecotr.Thers has no significant differences between difference time interval groups.