文章摘要
王 莉,董艳婷,丁晓琛,牛玉虎,牛 勃.RTX诱导神经管畸形动物模型的建立及机制研究[J].南京医科大学学报,2016,(4):420~425
RTX诱导神经管畸形动物模型的建立及机制研究
Establishment of animal model of neural tube defects induced by RTX and the mechanisms study
投稿时间:2015-08-28  
DOI:10.7655/NYDXBNS20160408
中文关键词: 神经管畸形  胸苷酸合成酶  雷替曲塞  DNA损伤  细胞凋亡与增殖
英文关键词: neural tube defects  thymidylate synthase  raltitrexed  DNA damage  cell apoptosis and proliferation
基金项目:国家自然科学基金(81370312)
作者单位
王 莉 山西医科大学基础医学院,山西 太原 030001 
董艳婷 山西医科大学基础医学院,山西 太原 030001 
丁晓琛 山西医科大学基础医学院,山西 太原 030001 
牛玉虎 山西医科大学基础医学院,山西 太原 030001 
牛 勃 山西医科大学基础医学院,山西 太原 030001
首都儿科研究所儿童发育营养组学北京市重点实验室,北京 100020 
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中文摘要:
      目的:通过胸苷酸合成酶(thymidylate synthase,TS)特异性抑制剂——雷替曲塞(RTX)干预孕鼠,建立小鼠神经管畸形(neural tube defects,NTDs)模型,探讨细胞凋亡和增殖?DNA损伤在RTX诱导的NTDs发生中的作用。方法:将孕7.5 d C57BL/6J小鼠随机分为6组:1组为对照组,5组为RTX干预组。对照组腹腔注射0.9%NaCl,RTX干预组分别腹腔注射不同剂量的RTX(5.0?10.0?11.5?13.5?15.0 mg/kg),孕11.5 d取胚胎并观察其发育情况。苏木精-伊红(HE)染色观察神经管闭合情况,放射性法测定TS活性,高效液相色谱检测胸苷酸(dTMP)及尿苷酸(dUMP)含量,Western blot分析复制蛋白A2(RPA2)?γ-H2AX和caspase-3表达,免疫组化检测细胞增殖。结果:RTX注射剂量为11.5 mg/kg时,NTDs发生率最高(30.56%);RTX干预后,TS活性较对照组显著降低,dUMP含量显著升高,dTMP含量显著下降;RTX建立的NTDs模型中,RPA2和γ-H2AX磷酸化水平显著升高;caspase-3表达量显著增加,细胞增殖显著减少。结论:通过RTX干预孕鼠可建立小鼠NTDs模型,dTMP合成障碍导致DNA损伤?细胞凋亡与增殖失调可能是NTDs发生的重要机制。
英文摘要:
      Objective:To establish a murine model of neural tube defects (NTDs) by inhibition of thymidylate synthase (TS) via a specific inhibitor, raltitrexed (RTX). Based on the model, The role of DNA damage, cell apoptosis and proliferation in RTX-induced NTDs were investigated. Methods: The adult C57BL/6J pregnant of 7.5 d mice were randomly divided into six groups: one was for control and the other five were RTX-treated groups. NTDs were induced by intraperitoneally injection of various doses of RTX (5.0, 10.0, 11.5, 13.5, 15.0 mg/kg body weight) on gestational day 7.5. Control mice were injected with 0.9% NaCl at equal volume. Neural tube closure was examined by hematoxylin-eosin (H&E) staining on gestational day 11.5 and TS activity was measured by radioactive method following RTX treatment. Levels of thymidylate (dTMP) and uridylate (dUMP) were detected by high performance liquid chromatography (HPLC). Western-blotting was performed to analyze the expressions of replication protein A2 (RPA2), γ-H2AX and caspase-3 in NTDs embryos induced by RTX. Cell proliferation was analyzed by immunohistochemical method. Results: At 11.5 mg/kg bw, RTX induced the highest incidence of NTDs (30.56%). TS activity was significantly reduced by RTX treatment. Besides, levels of dUMP were increased associated with decreased dTMP levels after RTX treatment. Furthermore, phosphorylation of RPA2 and γ-H2AX were significantly increased in RTX-induced NTDs. The expression of caspase-3 was significantly increased and cell proliferation was significantly decreased. Conclusion: Murine model of NTDs was successfully established by inhibition of TS via RTX. DNA damage and imbalance between apoptosis and proliferation were important events caused by impairment of dTMP biosynthesis, which may be one of the key mechanisms underlying the development of NTDs.
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