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为进一步阐明三氧化二砷的发育毒性和致畸作用机制,本实验用非放射性原位杂交等技术研究了砷诱导畸胎发生过程中p16和iNOS基因表达的变化。腹腔注射砷剂量依次为0、1、4和10mg/kg。结果发现砷对SD大鼠胚胎的发育毒性与剂量正比关系,10mg/kg时,发育正常胚胎仅为16.7％。原位杂交结果显示在对照和1mg/kg砷组可见p16mRNA阳性表达信号,但iNOSmRNA表达阴性;在4和10mg/kg组p16mRNA阳性杂交信号未检出,而iNOSmRNA大量表达。p16蛋白缺失与胚胎细胞分裂周期有关;iNOS过表达可导致内源性NO产量增高,进而对胚胎造成氧化性损伤。扫描电镜下,可见≥4mg/kg砷对胚胎表皮细胞、卵黄囊胎盘间皮和内皮细胞表面的微绒毛及细胞膜均有明显的损害作用。
In order to further elucidate the developmental toxicity and teratogenic mechanism of arsenic trioxide, we used non-radioactive in situ hybridization to study the changes of p16 and iNOS gene expression in the process of arsenic-induced teratogenicity. Intraperitoneal arsenic doses were 0, 1, 4 and 10 mg / kg. The results showed that the developmental toxicity of arsenic to SD rat embryos was proportional to the dose, and at 10 mg / kg, only 16.7% of normal embryos were developed. In situ hybridization results showed that p16 mRNA positive expression signal was found in the control and 1 mg / kg arsenic group, but iNOS mRNA expression was negative. The p16 mRNA positive hybridization signal was not detected in 4 and 10 mg / kg group, but iNOS mRNA was abundantly expressed. Deletion of p16 protein is associated with the cycle of embryonic cell division; overexpression of iNOS results in increased endogenous NO production and oxidative damage to embryos. Scanning electron microscopy, we can see ≥ 4mg / kg of arsenic on embryonic epidermal cells, yolk sac placental and endothelial cells on the surface of microvilli and cell membrane were significantly damaged.