无机砷及其代谢产物对皮肤成纤维细胞DNA甲基化酶影响的研究
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摘要
目的：观察亚酸钠、砷酸钠、DMA(5价)、MMA(3价)对人皮肤成纤维细胞的增殖抑制作用,探讨砷性皮肤损伤的分子机制。方法：1.以原代培养的人皮肤成纤维细胞为研究对象,用噻唑蓝(MTT)还原法检测不同浓度的亚砷酸钠、DMA(5价)、MMA(3价)(0 umol/1、0.5μmol/1、1μmol/15μmol/1、10μmol/1、15μmol/1)对人皮肤成纤维细胞体外增殖抑制作用。2.流式细胞仪检测不同浓度的亚砷酸钠、砷酸钠、DMA(5价)、MMA(3价)(0μmol/l、0.5μmol/l、5μmol/l、10μmol/l)处理人皮肤成纤维细胞后细胞周期分布情况。3.透射电镜观察不同浓度的亚砷酸钠对皮肤成纤维细胞超微结构的影响。4.半定量逆转录RT-PCR技术检测不同浓度的亚砷酸钠、砷酸钠、DMA(5价)、MMA(3价)处理人皮肤成纤维细胞后DNMT1、DNMT3A、DNMT3B、As3MT的mRNA表达。结果：1.MTT还原法检测结果显示：与对照组比较,人皮肤成纤维细胞经不同浓度的亚砷酸钠、DMA(5价)、MMA(3价)诱导后,终浓度为0.5μmol/l、1μmol/l、5μmol/l的亚砷酸钠、DMA(5价)、MMA(3价)吸光度值均升高,存在剂量-效应关系(P<0.05),终浓度为10μmol/l、15μmol/l的亚砷酸钠、DMA(5价)、MMA(3价)吸光度值均降低,存在剂量-效应关系(P<0.05)；荧光倒置显微镜下观察原代培养皮肤成纤维细胞至第13代生长缓慢；正常皮肤成纤维细胞生长曲线可观察出,96小时皮肤成纤维细胞生长速度较快趋势。2.透射电镜观察0.5μmol/l亚砷酸钠作用皮肤成纤维细胞,细胞表面微绒毛多,细而长,细胞核大,核心不规则,核仁明显,细胞质里的细胞器数量少,细胞里部分线粒体空泡变；10Lmol/l亚砷酸钠作用皮肤成纤维细胞显示细胞表面微绒毛减少较5μmol/l浓度组明显,溶酶体较5Lmol/1浓度组增多,内质网扩张,未见核仁,线粒体空泡变较明显。3.流式细胞仪检测细胞周期分布结果显示,低剂量亚砷酸钠、砷酸钠、DMA(5价)、MMA(3价)处理组细胞G1期细胞相对于对照组所占百分数减少,未出现G1期阻滞；高、中剂量亚砷酸钠、砷酸钠、DMA(5价)、MMA(3价)处理组细胞G1期细胞所占百分数增多,细胞周期阻滞在G1期。4. DNMT3A、DNMT1、DNMT3B的mRNA表达量随着亚砷酸钠、MMA药物作用剂量的增加表达量有下降趋势,且随着砷酸钠、DMA药物作用剂量的增加,mRNA表达量有增加趋势,除低剂量亚砷酸钠处理组对DNMT1的mRNA表达与对照组比较无统计学意义外,其它趋势均有统计学意义且低于对照组mRNA表达量(P<0.05)；As3MT的mRNA表达量随着亚砷酸钠、MMA药物作用剂量的增加表达量有增加趋势,且随着砷酸钠、DMA药物作用剂量的增加,mRNA表达量有下降趋势。除低剂量亚砷酸钠、高剂量砷酸钠作用于皮肤成纤维细胞对细胞As3MT的mRNA表达量与对照组相比差异无统计学意义(P>0.05)外,其它趋势均有统学意义(P<0.05)。结论：低浓度亚砷酸钠、DMA(5价)、MMA(3价)刺激人皮肤成纤维细胞增殖,高浓度具有细胞毒性；高、中剂量的亚砷酸钠、砷酸钠、DMA(5价)、MMA(3价)使皮肤成纤维细胞阻滞于G1期,低剂量未出现G1期阻滞；电镜观察显示随着亚砷酸钠药物浓度增加皮肤成纤维细胞内线粒体空泡变明显,细胞表面微绒毛逐渐减少,核仁边集→核仁成份有分离→核仁消失；不同浓度亚砷酸钠、砷酸钠、DMA(5价)、MMA(3价)影响DNA甲基转移酶相关基因(DNMT3A、DNMT1 DNMT3B、As3MT)的表达,这在砷性皮肤损伤的分子机制研究中有重要意义。
Objective:To observe different effects of iAsⅢ、iAsⅤ、DMAⅤ、MMAⅢof the different doses impact on the proliferation and inhibition in human skin fibroblasts and possible molecular mechanism in the skin lesions of arsenic. Methods:1.The different effects of iAsⅢ、DMAⅤ、MMAⅢof the different doses impact on the proliferation and inhibition in human skin fibroblasts was tested by MTT reduction assay.2-The cell cycle distribution was detected by flow cytometry when cells were treated with or without different concentrations of iAsⅢ、iAsⅤ、DMAⅤ、MMAⅢin human skin fibroblasts.3.Cell structure examined by electron microscopy.4. Semi-quantitative RT-PCR method was applied to detect the mRNA expression level of DNMT1,3A and 3B、As3MT. Results: 1. The results of MTT assay showed that with different concentration of iAsⅢ、DMAⅤ、MMAⅢ(0.5-5μmol/L), the skin fibroblasts were markedly stimulated, and a dose-effect relationship was observed; F13 skin fibroblasts grew slowly, the fast growing phase of skin fibroblasts was at the 96 h.2. The different doses of sodium arsenic can injury the normal microstructure of skin fibroblasts. After be treated with 0.5μmol/l sodium arsenite,Cell morphology appeared several changes:cell surface microvillus change more,thin and long, cell nucleus is big, core is irregular, nucleoli is obviously, There are small number of organelle in the cytoplasm, there are vacuoles in the part of mitochondria. After be treated with 10μmol/l sodium arsenite, Cell morphology appeared several changes:cell surface microvillus showed significantly reduced than 5μmol/l sodium arsenite. Lissome have significantly increased than 5μmol/l sodium arsenite. There are a lot of vacuoles in the mitochondria. Nucleolus has disappeared.3.High or middle doses of iAsⅢ、iAⅤ、DMAⅤ、MMAⅢcould increase cell percentage in G1 phase, That was showed G1 phase of cell cycle was being stilled.4. The result of RT-PCR showed that the gene expression of DNA methyltransferase were not in the same levels among different groups on the skin fibroblasts. Conclusion:The proliferation of the skin fibroblasts is regulated in a double-phase manner by inorganic arsenic and its metabolites. Low concentration induces stimulation effect, but high concentration inhibits the cell proliferation. High or middle doses of iAsⅢ、iAsⅤ、DMAⅤ、MMAⅢincreased cell percentage in G1 phase, blocked G1 phase. The different doses of sodium arsenic can injury the normal structure of skin fibroblasts. It plays a vital role in the skin lesions of arsenic.
Objective:To observe different effects of iAsⅢ、iAsⅤ、DMAⅤ、MMAⅢof the different doses impact on the proliferation and inhibition in human skin fibroblasts and possible molecular mechanism in the skin lesions of arsenic. Methods:1.The different effects of iAsⅢ、DMAⅤ、MMAⅢof the different doses impact on the proliferation and inhibition in human skin fibroblasts was tested by MTT reduction assay.2-The cell cycle distribution was detected by flow cytometry when cells were treated with or without different concentrations of iAsⅢ、iAsⅤ、DMAⅤ、MMAⅢin human skin fibroblasts.3.Cell structure examined by electron microscopy.4. Semi-quantitative RT-PCR method was applied to detect the mRNA expression level of DNMT1,3A and 3B、As3MT. Results: 1. The results of MTT assay showed that with different concentration of iAsⅢ、DMAⅤ、MMAⅢ(0.5-5μmol/L), the skin fibroblasts were markedly stimulated, and a dose-effect relationship was observed; F13 skin fibroblasts grew slowly, the fast growing phase of skin fibroblasts was at the 96 h.2. The different doses of sodium arsenic can injury the normal microstructure of skin fibroblasts. After be treated with 0.5μmol/l sodium arsenite,Cell morphology appeared several changes:cell surface microvillus change more,thin and long, cell nucleus is big, core is irregular, nucleoli is obviously, There are small number of organelle in the cytoplasm, there are vacuoles in the part of mitochondria. After be treated with 10μmol/l sodium arsenite, Cell morphology appeared several changes:cell surface microvillus showed significantly reduced than 5μmol/l sodium arsenite. Lissome have significantly increased than 5μmol/l sodium arsenite. There are a lot of vacuoles in the mitochondria. Nucleolus has disappeared.3.High or middle doses of iAsⅢ、iAⅤ、DMAⅤ、MMAⅢcould increase cell percentage in G1 phase, That was showed G1 phase of cell cycle was being stilled.4. The result of RT-PCR showed that the gene expression of DNA methyltransferase were not in the same levels among different groups on the skin fibroblasts. Conclusion:The proliferation of the skin fibroblasts is regulated in a double-phase manner by inorganic arsenic and its metabolites. Low concentration induces stimulation effect, but high concentration inhibits the cell proliferation. High or middle doses of iAsⅢ、iAsⅤ、DMAⅤ、MMAⅢincreased cell percentage in G1 phase, blocked G1 phase. The different doses of sodium arsenic can injury the normal structure of skin fibroblasts. It plays a vital role in the skin lesions of arsenic.
引文
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[2]Vahter.M.Mechanisms of arsenicbiotrans formation[J].Toxicology,2002,118-182:211-217.
[3]Yamanaka K,Mizoi M,Tachikawa M,et al.Oxidative DNA damage following exposure to dimethylarsinous iodide:the formation of cisthymine glycol[J]. Toxicol Lett,2003, 143(2):145-153.
[4]Naranmandura H, Suzuki N, Suzuki KT. Trivalent arsenicals are bound to proteins during reductive methylation [J]. Chem Res Toxicol,2006,19:1010-1018.
[5]Hayakawa T, Kovyahi Y, Cui X, et al. A new metabolic pathway of arsenite: arsenic-glutathione complexes are substrates for human arsenic methyltransferase Cyt19[J].Arch Toxicol,2005,79:183-191
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[16]Sakural T,Kaise T,Matsubara C.Inorganic and methylatedarsenic compounds induce cell death in murine macrophagesvia different mechanisms[J]. Chem Res Toxicol,1998, 11(4):273-281.
[17]Mass MT, Wang L. Arsenis alter, cytosine Methylation patterns of the promoter of the tumor suppressor gene p53 in human lung cells:a model for a mechanism of carcion-genesis [J]. Mutate Res,1997,386:263-277.
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