反复长波紫外线照射对人皮肤成纤维细胞DNA的损伤机制探讨

Mechanisms underlying repeated ultraviolet A radiation-induced DNA damage in human skin fibroblasts

目的探讨反复长波紫外线(UVA)照射对人皮肤成纤维细胞DNA损伤、修复及复制过程的影响及机制。方法自中山大学附属第三医院泌尿外科3例儿童包皮环切术后包皮组织中分离培养成纤维细胞,随机分为UVA组和对照组,UVA组接受连续UVA照射诱导慢性光损伤,对照组不接受UVA照射,通过MTT法、β半乳糖苷酶染色及流式细胞仪分别检测细胞活性、老化比率和凋亡率。逆转录法建立差异表达cDNA基因谱库,高通量测序,比较对照组与UVA组cDNA测序结果,分析差异表达基因。两独立样本均数比较采用独立样本t检验。结果 UVA组细胞活性(72.0%± 5.2%)明显低于对照组(96.0%± 3.7%),t = 6.51,P < 0.05;细胞老化率(79.7%± 5.2%)高于对照组(6.4%±0.8%),t = 24.12,P < 0.05;细胞凋亡率(29.0%± 3.3%)高于对照组(6.0%± 5.9%),t = 5.89,P < 0.05。在DNA错配修复过程、DNA复制和DNA碱基切除修复过程的关键酶中,细胞DNA连接酶(Lig)1表达量在UVA照射组下调为对照组的0.47 ± 0.13,核糖核酸酶(RNase)H2A下调为0.44 ± 0.07,解旋酶Dna2 下调为0.49 ± 0.11,两组间表达差异均有统计学意义(均P < 0.01)。UVA诱导人皮肤成纤维细胞慢性光损伤后,在DNA碱基修复过程,Lig表达下调导致DNA单碱基修复受阻,Lig及Lig1表达下调,导致DNA多碱基修复过程受阻;在DNA错配修复过程, Lig1基因表达下调,修复过程受阻;在DNA复制过程,RNaseH2A、Dna2、Lig1表达下调,复制过程受阻。结论反复UVA照射可改变皮肤成纤维细胞DNA碱基切除修复、DNA错配修复及DNA复制过程中关键酶的表达,从而影响皮肤成纤维细胞DNA修复及DNA复制过程。

Objective To evaluate the effects of repeated ultraviolet A (UVA) radiation on DNA damage, repair and replication processes in human skin fibroblasts, and to explore their mechanisms. Methods Fibroblasts were isolated from the circumcised foreskins of 3 children in the Department of Urological Surgery, Third Affiliated Hospital, Sun Yat-sen University, and subjected to a primary culture. Cultured human skin fibroblasts of 3rd - 10th passages were divided into 2 groups: UVA group treated with repeated UVA radiation to establish a chronic photodamaged cell model, and control group receiving no treatment. Cell counting kit 8 (CCK-8) assay,β-galactosidase staining and flow cytometry were performed to assess cellular proliferative activity, and determine the proportion of photoaged cells and apoptosis rate respectively. Reverse transcription reaction was performed to establish a differentially expressed cDNA library, which was then subjected to high-throughput sequencing. The cDNA sequencing results were compared between the control group and UVA group, and the differentially expressed genes were analyzed in Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The data were compared between the two groups by using two independent sample t test. Results Compared with the control group, the UVA group showed significantly decreased cellular proliferative activity (72.0%± 5.2% vs. 96.0%± 3.7%, t = 6.51, P < 0.05),but significantly increased proportion of photoaged cells (79.7%± 5.2% vs. 6.4%± 0.8%, t = 24.12, P < 0.05) and apoptosis rate (29.0%± 3.3% vs. 6.0%± 5.9%, t = 5.89, P < 0.05). Among the key enzymes involved in DNA mismatch repair, replication and base excision repair processes, the expression of DNA ligase 1 (Lig1), ribonuclease (RNase) H2A and helicase Dna2 in the UVA group was 0.47 ± 0.13, 0.44 ± 0.07 and 0.49 ± 0.11 times (all P < 0.01) that in the control group respectively. After the UVA-induced chronic photodamage in the human skin fibroblasts, After the UVA-induced chronic photodamage in the human skin fibroblasts, Lig expression decrease could block DNA single-base excision repair, Lig and Lig1 expression decrease could block DNA multiple-base excision repair, Lig1 expression decrease could block DNA mismatch repair, and RNaseH2A, Dna2 and Lig1 expression decrease could block DNA replication. Conclusion Repeated UVA radiation can change the expression of key enzymes involved in DNA base excision repair, DNA mismatch repair and DNA replication processes in skin fibroblasts, and then affect DNA repair and DNA replication processes in skin fibroblasts.