UVB诱导皮肤细胞光损伤过程中线粒体DNA大片段缺失突变的研究

Large mitoehondrial DNA deletions in ultraviolet B-induced cutaneous photodamage

目的探讨UVB照射后，线粒体DNA（mtDNA）突变与皮肤细胞光损伤之间的关系。方法用UVB小剂量、多次照射人皮肤原代成纤维细胞（HSF）和成人原代角质形成细胞（HEKa），诱导其相对活性下降，分别提取基因组DNA，以普通PCR检测mtDNA中的4977bp缺失和3895bp缺失突变的发生频率，并以荧光实时定量PCR法定量检测4977bp缺失和3895bp缺失的突变水平。结果①两种细胞株中4977bp缺失和3895bp缺失突变的发生频率和表达水平均随UVB照射剂量的增多、细胞活性水平下降而逐渐升高（Pearson相关分析，P〈0．05）。②3895bp缺失突变的发生频率和突变水平最高分别达53．3％和（49．63±4．38）×10^-5相对拷贝，比4977bp缺失突变更能灵敏地反映UVB的累积辐射剂量。③UVB照射剂量达150mJ／cm^2时，HSF即出现3895bp缺失突变，4977bp缺失突变也在UVB剂量达200mJ／cm^2时被诱导出来，较HEKa更易被诱导出mtDNA突变。结论mtDNA突变的形成与累积与皮肤细胞接受的累积光照剂量密切相关，其中3895bp缺失较4977bp缺失更能反映细胞受到的光损伤程度，可作为一种皮肤细胞光损伤的生物学观察指标。

Objective To analyze the association between mtDNA mutations and photodamage after ultraviolet B （UVB） irradiation. Methods Primary human skin fibroblasts （HSF） and primary human epidermal keratinocytes of adult （HEKa） were irradiated by sub-lethal doses of UVB thrice a day for 4-5 days. Thereafter, genomic DNA was extracted from irradiated cells and conventional PCR was applied to detect the frequency rates of 4977 bp and 3895 bp mtDNA deletion. To quantitatively analyze the mutation levels, SYBR Green real-time PCR method was performed. Results In both cell lines, the frequency rates and relative copy number of deletions increased with the cumulative doses of UVB exposure （P 〈 0.05 ）. The prevalence rate of 3895 bp deletion peaked 53.3% and and relative copy number reached （49.63 ± 4.38） × 10^-5, showing a more intense response to the accumulation of UVB radiation than 4977 bp deletion. In HSF, the minimum cumulative dose of UVB radiation was 150 mJ/cm^2 for the induction of 3895 bp deletion, and 200 mJ/cm^2 for the induction of 4977 bp deletion. It seemed that mtDNA deletion was more readily to be induced by UVB radiation in HSF than in HEKa. Conclusions The development and accumulation ofmtDNA mutation are intimately related with cumulated UVB dose received by skin cells, and the 3895 bp deletion is more reliable in monitoring the photodamage caused by UV than 4977 bp deletion. Therefore, the 3895 bp deletion may serve as a biomarker for the detection of photodamage in skin cells. HSF appear to have an increased susceptibility to UVB radiation, which results in a higher frequency and level of mtDNA mutations compared with HEKa.