| Summary: | 碩士 === 中山醫學院 === 毒理學研究所 === 88 === The present study developed a "multiplex long quantitative polymerase chain reaction" (multiplex long QPCR) to investigate the difference of sensitivity of ultraviolet (UV) irradiation and repair capacity in the p53 gene of various cell lines as well as blood lymphocytes of lung cancer patients and control subjects. Under the optimized conditions of intial template 100-200ng and PCR cycle number 25-30, multiplex long QPCR can quantify the damage and repair efficiency in the p53 gene. Furthermore, a "reverse transcription (RT)-PCR assay" was used to measure the expression level of the repair genes and the p53 gene in various cell lines and blood lymphocytes of patients with different chemo-responses. Under the optimized conditions of PCR cycle number 30-33, RT-PCR can simultanely quantify the mRNA expression of several genes.
The application results of multiplex long QPCR in cell lines with known repair capacity indicated that the initial damage of the p53 gene is similar between normal lung cell MRC5, the partial repair-deficient XPC cell; and repair-deficient XPA cell in the dose range 10-20 J/m2 of UV irradiations. However, the differences in repair were pronounced at 8 hr post-UV at 15 J/m2. The repair efficiencies of UV damage from the p53 gene were 100%, 50%, and 0% in the MRC5, XPC, and XPA cells, respectively. As to measurement of the blood lymphocytes from 12 lung cancer patients and 14 normal subjects, lymphocytes were irradiated with 12 seconds by 3 J/m2/sec dose rate of UV irradiation. The averaged initial damage of UV irradiation were 0.25 and 0.32 damage per p53 fragment in lung cancer patients and normal subjects, respectively. Their repair efficiencies in the p53 gene at 16 hr post-UV were 51.6% and 35.6%.
The application results of the RT-PCR in cell lines with known drug resistance indicated that the induction levels of the mRNA expression in the ERCC1 and hMSH2 repair genes in HeLa-CPR drug resistant cell were 2-17 fold and 9 fold, respectively, during 3-9hr post-UV treatment at 15 J/m2. The induction levels were significantly higher than that in the HeLa parental cell, which has no mRNA induction post-UV irradiation (P < 0.05). In addition, the mRNA levels of the ERCC1 and hMSH2 repair genes as well as the p53 gene in 44 chemotherapeutic lung cancer patients were analyzed. The 23 chemoresistant cases had a 40% higher mean expression level of both hMSH2 repair gene and p53 gene than did the 21 effective cases (P < 0.05).
In conclusion, damage formation and repair efficiency of p53 gene may differ in various cell lines and blood lympfocytes of individuals. In addition, the increased mRNA expression levels of repair genes and p53 gene may correlate with chemoresistance. The present study also demonstrated the multiplex long QPCR and the RT-PCR assays were simple, rapid, reproducible, and easy to apply to the various cell lines and readily accessible tissue specimens such as blood. The assays provide tools for the advanced study of the correlation of DNA repair with tumorgenesis and chemoresistance in the future. The results can be also used to identify individuals at high risk of developing cancer that may associate with genetic alteration in the repair process.
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