GADD45与mir-21在燃煤污染型砷中毒基因病理性低甲基化中作用和机制及银杏制剂干预研究

Guizhou is characteristic endemic areas of coal-burning arsenism in China.But arsenic as a confirmed human carcinogen, so far there is not a widely recognized and consistent with the biological effect of arsenic carcinogenesis.Gene methylation level decreased is one of the important epigenetic changes in disease development.Gadd45 and miR-21 may play an important role in gene hypomethylation.Our study will point at characteristic endemic areas of coal-burning arsenism in Guizhou, using the present situation and intervention study of Ginkgo biloba and environmental and population epidemiology and molecular toxicology methods. Our study will focus on the methylation status of sensitive gene and genome of human exposuring to arsenic and the expression situation of Gadd45 and miR-21, and analyse the relationship between the methylation status and the expression situation told above and the occurrence and development of coal-burning arsenism as well. At the same time,we will construct the cell model of arseniasis and intervent with Ginkgo biloba extract and miR-21 mimetic or disruptors.Our study at the cellular level may supplement and verify the results of population study. Through analysising the relationship between Gadd45 and miR-21 and gene hypomethylation and coal-burning arsenism at population-based studies and cell research level,our study may provide a scientific basis for elucidating the molecular mechanism of coal-burning arsenism and early diagnosis and high-risk groups screening and preventive intervention.Meanwhile, we also try to find a natural, economic and effective drug for the prevention and treatment of arsenism.

贵州是我国特有的燃煤型地砷病重病区。但砷作为确认的人类致癌物，迄今还没有一个被广泛认可且符合砷生物学效应的致癌机制。基因甲基化水平降低是疾病发生发展中重要的表观遗传学改变之一。Gadd45与miR-21在基因低甲基化发生中可能扮演重要角色。本课题旨在以贵州燃煤型砷中毒病区为现场，通过现况与银杏制剂干预研究，采用环境和人群流行病学与分子毒理学方法，侧重研究砷暴露人群基因组、敏感基因甲基化状况及Gaddd45与miR-21表达情况，并分析其与燃煤型砷中毒发生发展的关系。同时，构建砷中毒细胞模型进行银杏提取物和miR-21模拟物或干扰物干预，在细胞水平对人群研究结果进行补充和验证。通过人群和细胞研究，分析Gadd45及miR-21与基因低甲基化及燃煤型砷中毒的关系，为阐明燃煤型砷中毒分子机理及早期诊断、高危人群的筛查和干预性防治提供科学依据,同时探索性寻找一种天然、经济、有效的砷中毒防治药物。

贵州曾是典型燃煤型砷中毒病区。经政府不懈努力，在砷中毒的病因防控上取得显著成就，现已无新发病人，但现患病人的治疗尚待解决。由于砷致病机制还不完全清楚，导致现患病人的治疗停留在对症治疗，缺乏特异性药物。DNA异常甲基化是砷中毒发病机制之一，Gadd45与miR-21在基因低甲基化发生中可能扮演重要角色。本课题开展了贵州省燃煤污染型砷中毒病区人群现况和干预研究，并在构建了砷中毒细胞模型的基础上进行银杏提取物、siRNA干预，在细胞水平对人群研究结果进行补充和深化，①探讨了砷暴露对全基因组、特异基因启动子区域甲基化及细胞周期和凋亡的影响。②探讨了Gadd45在砷致全基因组、特异基因启动子区域甲基化及细胞周期和凋亡改变中的作用。③探讨了Gadd45在银杏叶制剂改善砷致全基因组、特异基因启动子区域甲基化降低及细胞周期和凋亡改变中的作用。④探讨了miR-21-PTEN-ERK通路对Gadd45的调控作用。结果发现，①砷暴露可致全基因组、H-ras、C-myc动子区域甲基化率降低、G2/M期细胞阻滞增加、凋亡率增加，Gadd45、H-ras、C-myc基因表达增加；②抑制Gadd45表达后，砷暴露导致的上述毒效应可得到有效的改善。③银杏制剂可减少砷致干细胞毒性，改善砷中毒人群肝功能，其机制与其降低Gadd45表达，进而改善砷暴露所致上述毒效应有关。④砷暴露可致miR-21表达增加，ERK的表达降低，PTEN的表达先降低后升高，这与Gadd45表达趋势不一致。上述结果提示，砷暴露可致全基因组、H-ras、C-myc动子区域甲基化率降低、G2/M期细胞阻滞增加、凋亡率增加，这与Gadd45表达增加有关，而miR-21-PTEN-ERK通路对Gadd45的表达无直接调控作用；银杏制剂可减少砷致肝细胞毒性，改善砷中毒人群肝功能，其机制与其降低Gadd45表达，进而改善砷暴露所致上述毒效应有关。本研究为阐明燃煤污染型砷中毒对人体损害乃至癌变的分子机理及早期诊断、高危人群的筛查和干预性防治提供科学依据,亦为银杏制剂用于砷中毒防治提供了科学依据。