| Objective: PIF1gene is widely considered to be radiation-sensitive genes, through thestudy of PIF1gene is involved in the biological function of DNA damage and repair inducedby ionizing radiation,to explore the molecular mechanisms of the PIF1gene involved in DNAdamage repair. Using a technique called RNA interference "knock the full-length PIF1geneof low", further study PIF1helicase physiological function. The construction of threerecombinant plasmids, which were then transfected into HeLa cells in cell lines to sievepressure stability around the expression by liposome transfection method, for more in-depthstudy of PIF1gene.Methods: Carries on the γ radiation exposure of different dosages to the training HeLa cell,examines the change in of PIF1gene with Western blot and Real-Time PCR expressionquantity in the expression level of PIF1gene, to understand and study the possible function ofPIF1gene in DNA damage repair of the function. Using RNA interference technology"silent" PIF1full-length genes, by Western blot and Real-Time PCR to detect knocking at lowcell PIF1gene transcription and translation of knocking on low level effects. To knocking lowthe cell line carries on the γ radiation exposure of different dosages,Value added of live cellcount by understanding cell capacity, measurement of cell cycle by flow cytometry, cloneformation rate and so on experiment to observe the knockdown cell lines with wild-typestrains before and after γ ray irradiation DNA damage repair and cell cycle changes. From theoriginal pCR2.1-TOPO-PIF1plasmid, amplification of the gene PIF1, PIF1N and PIF1C byusing the PCR method, the target gene and vector pcDNA3.1(-) were digested connection,constructed three recombinant plasmids [PcDNA3.1(-)-PIF1, PcDNA3.1(-)-PIF1N,PcDNA3.1(-)-PIF1C]. By liposome transfection method to these three recombinant plasmidstransfection into HeLa respectively, sieve pressure turned the expression of cell lines.Results:①HeLa cells were treated with different doses of γ rays through the Western blotdetected the expression of PIF1at different time points have decreased firstly and thenincreased, By the method of Real-Time PCR detection of PIF1gene in mRNA level alsoappeared at different time points have decreased firstly and then increased.②ThroughReal-Time PCR and Western blot methods detect knocking at low cell lines when comparedwith the control cells, transcription and translation has a different degree of knocking on lowlevel. After knocking low the cell line carries on γ radiation exposure processing of differentdosages, Viable cell counts were found in Sh-PIF1-Hela knockdown cells than Sh-NC-Helacontrol cell growth rate will slow a lot and increased apoptosis. The colony formationefficiency experiment discovered that with increase of cell exposure dose, the appreciation ofcell reduces, forms the clone number to reduce, knocks compared with the low cell andcomparison cell, the survival capability of cell reduces. Measurement of cell cycle found knockdown cells arrest at G2/M phase was prolonged by flow cytometry.③Successfullyconstructed three recombinant plasmids, and the transfected three overexpression cells,through the Western blot method did not detect the expression of PIF1protein was increased,but the relative expression method of using Real-Time PCR to measure the full-lengthPcDNA3.1-PIF1and PcDNA3.1-PIF1C end of the mRNA is increased to a certain extent, andthe PcDNA3.1-PIF1N end the relative expression of mRNA did not increase. Whether toconstruct cell line stably transfected depends on the further experimental verification.Conclusions:①PIF1gene with radiation sensitivity, and DNA damage repair.②Construction of the success of the stably transfected cell lines at low (Sh-PIF1-Hela). Foundto be closely related to PIF1gene and apoptosis.③The three recombinant plasmids has beensuccessfully constructed. |
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