| Summary: | 碩士 === 中山醫學院 === 醫學研究所 === 87 === 英文摘要
Fragile X syndrome is the most common cause of inherited mental retardation. Most of the patients have a mutation in the 5’-untranslated region of FMR1 gene, containing a (CGG)n repeat expansion, and a cytogenetically expressly folate-sensitive fragile site FRAXA in Xq27.3. Little is known about the molecular mechanism of the genetic control of the CGG trinucleotide expansion in the fragile X syndrome. It is known that mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats.
In the present study, cultivated human lymphocytes derived from the fragile X syndrome patients and normal individuals were characterized with cytogenetics, southern blotting and western blotting. The lymphocytes were further damaged by hydrogen peroxide, bleomycin, ethyl methanesulfonate, 4-nitroquinoline N-oxide, etoposide, mitomycin C, and methotrexate; and the DNA damage were assayed by comet assay（single-cell gel electrophoresis）. The effects of folate depletion on DNA stability in human lymphocytes were also reported. DNA strand breaks, uracil misincorporation, and DNA repair capability were determined by variants of the comet assay.
The comet assay is a sensitive method for detecting DNA strand breaks at the level of individual cells. Cells embedded in agarose are lysed, electrophoresed, and fluorescently stained. Breaks in the DNA release its supercoiling and allow DNA to extent toward the anode, resembling a comet.
Our results showed that spontaneous DNA damage in FRAXA lymphocytes were slightly higher than normal lymphocytes. H2O2 induced slightly higher DNA strand breaks in FRAXA lymphocytes, which showed decreased repair ability to H2O2-induced DNA strand breaks. Folate depletion in human lymphocytes significantly increased DNA strand breaks and decreased repair capacity for H2O2, especially in FRAXA lymphocytes. BLM, 4-NQO, EMS, MMC, and etoposide induced similar level of DNA damage both in normal and FRAXA lymphocytes.
We also found that uracil misincorporation were not detectable using comet assay when the cells cultured in low folate mdium for up to 10 days There was no significant DNA repair observed both in mitomycin C treated normal and fragile X syndrome lymphocyte cell lines after 48 hours incubation. These results suggest that CGG repeat instability in the fragile X syndrome may be not due to endogenous or inducible DNA repair pathway deficiency.
It is well characterized that the comet assay is sensitive as a technique to evaluate the DNA damage among a variety of cell types, induced by a variety of physical and chemical agents. From the study of 18 cancer patients and 16 normal control individuals, our results showed that spontaneous DNA damage in cancer lymphocytes were higher than normal lymphocytes. In comparison with other methods, the comet assay is relatively robust and economical in its use of material.
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