The Empirical Study On The Genomic Instability Of B16 Induced By Ionizing Radiation

Cancer has now become the first cause of death worldwide disease, both developed and developing countries, the incidence rate increased in varying degrees. Although a variety of treatment can prolong survival period, but still seriously affect the patients quality of life. Therefore, the ability to advance to monitor dangerous signal of human body and give intervention before tumor's formation has been a hot spot of cancer research.Cancer is a high heterogeneity, multiple genes involved syndrome. The main characteristics of cancer cells is genomic instability (genomic instability). Include:①micromutation;②chromosome microsatellite instability (MSI);③chromosomal instability;④regional chromosomal instability (regional chromosomal instability). So far found that 80% to 90% of the leukemia has clonal chromosome abnormalities, 70% of the leukemia has microsatellite instability.In 1994, Wada et al. analysised five microsatellite loci and confirmed the first time that when chronic myeloid leukemia (CML) acute transformation,the MSI significantly increased to 52.6%, that microsatellite instability and is closely related to CML acute transformation, analysis showed that the Alu sequence of BCR / ABL fusion gene of chronic myeloid leukemia is an absolute majority of the repeats, Alu sequences might be an important factor to the deletion mutation of chromosomal rearrangement. DNA mismatch repair defects are concerned with various parts of human tumorigenesis, including hereditary nonpolyposis colon cancer (HNPCC), gastric cancer, endometrial cancer, breast cancer, prostate cancer and so on. So, the incidence and the evolution of cancer are closely related with genomic instability.Ionizing radiation is a known cancer-causing factors can not only cause direct damage to DNA, but also lead to indirect effects to induce genomic instability. It may be used as early events of the multi-stage carcinogenic theory to increase the frequency of the change of genome, make the whole genome in a "critical" state, with the replication of cells accumulated enough mutations, leading to cancer. 1991, Kadhim et al. used theα-particle irradiated mice stem cells, for the first time proved that genetic instability of the radiation offspring cells. Little with mouse embryonic stem cell line 10T1/2 cells found: radiation-induced genomic instability may increase the likelihood of secondary injury to increase the risk of cancer formation. 2001, Brenna et al.used 60Coγrays to radiate the yeast strain to detect His3 + recombinant mutant frequency , showed that genomic instability is the early damage of cancer originating, which verifies the theory of cumulative damage. Further comfirmed radiation induced genomic instability is a multi-stage, multi-step theory of the key steps in cancer.The regional chromosomal instability has the largest frequency to happen and is the most important type of genomic instability. This recombine often occurred in the longer repeat regions of the DNA sequence, such as Alu repeat sequences, can lead to partial chromosome loss. The accommodation abnormal of chromosome homologous recombination can lead to regional chromosomal instability, According to this characteristics, Moynahan et al. designed the human glycophorin direct repeat sequences, and integration of the lacZ reporter gene sequence ,and successfully detected the genomic instability of the glycophorin fracture cluster clustering in vivo of mice. 1990s, Morgan, who established a GFP labeled genomic instability reporting system, using repeat GFP as a reporter factor, due to a gene inserted which makes the GFP inactivation. When homologous recombination occurred between the two repeat GFP, GFP-positive cells will be produced. Confirmed that the existence of cells delayed genomic instability. The research and testing of Genomic instability achieved greater breakthroughs.The GFP-based reporter plasmid of genomic instability reporting system which we used contains two un-functional repeats of GFP gene. When regional chromosome instability in the form of single strand annealing occurs, it causes recombination between the direct repeats to express functional GFP gene. The reporter makes the detectiong of regional chromosomal instability to come true. Used the liposome transfection, the genome instability report system marked by GFP will be established in B16 cells. After transfection, screening ,limiting dilution. The B16 cells with the reporter plasmid were irradiated with different doses of 60Co-γray (0Gy,2Gy,4Gy), and the appearance of green cells were counted by microscopy: Observed in the confocal microscope:①After irradiation 1st day, 2Gy, 4Gy appeared green fluorescent protein, the GFP quantity was increase in the 1st -3rd day, arrive at the peak in the 3rd day, and began to reduce in 5th day;②GFP expression and radiation dose were positively correlated: 4Gy > 2Gy> 0Gy group;③0Gy group didn't appear expression of GFP during the 1st -5th day.④Continue to train 0Gy group for 2 weeks, appeared spontaneous expression of GFP, the total cell number of about 0.00016% (2 / 1200000).The results suggest that: 1.The GFP marked regional chromosomal instability reporter had transfected B16 cell successfully. 2.The final concentration of G418 selection of B16 cell lines was 500μg/ml, and the maintain concentration was 250μg/ml. 3. Application of the limited dilution method can be successful make the B16 clonal growth which labeled by GFP genomic instability reporting system. The pick-up rate in 96 well plate was 13.3% (8/60 holes). 4. B16 cells can be successfully induced regional chromosomal instability by 60Co-γrays and can be detected by this GFP reporting system. 5. Different doses of 60Co-γray could induce different expression of GFP, GFP's expression was positively correlated with radiation dose, and irradiation time-dependent. 6. Ionizing radiation-induced regional chromosomal instability changes can be passed to offspring cells after irradiation.The study may have an impotant significance to explore the relationship between ionizing radiation and genomic instability and reveal the mechanism of cancer formation of ionizing radiation-induced genomic instability.