同型半胱氨酸致神经管畸形小鼠模型的建立及其作用机制

Establishment of a mouse model of neural tube defect caused by homocysteine and action mechanism

目的建立同型半胱氨酸(homocysteine,Hcy)致神经管畸形(neural tube defect,NTD)的小鼠模型,并探讨其作用机制。方法以单独使用同型半胱氨酸硫代内酯(homocysteine thiolactone,HTL)及联合胱硫醚-β-合成酶(cystathionine-β-synthase,CBS)抑制剂氨基氧乙酸(aminooxyacetic acid,AOAA)建立小鼠NTD模型,其中单独使用HTL的剂量为100、300、500、700 mg/(kg·d),联用组中HTL的剂量为400、500、600 mg/(kg·d),AOAA剂量均为30 mg/(kg·d),于小鼠孕6.5~10.5 d连续给药,均经腹腔注射。选择最佳剂量建立NTD小鼠模型(NTD组),同时设正常对照组。ELISA法检测孕鼠血液中Hcy水平及胚胎脑组织中S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)和S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)水平,Click-i T EdU成像试剂盒检测小鼠胚胎神经上皮细胞的增殖情况,Western blot法检测胚胎脑组织中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达水平。结果建立NTD的小鼠模型最佳方案为500 mg/(kg·d)HTL+30 mg/(kg·d)AOAA,该条件下NTD发生率最高,死亡率较低。与正常对照组比较,NTD组Hcy、SAH和SAM含量均显著升高(P<0.01),SAM/SAH比值明显下降(P<0.01);胚胎神经上皮细胞增殖受到显著抑制(P<0.05);PCNA的表达水平显著下降(P<0.05)。结论联合使用HTL与AOAA成功建立了小鼠的NTD模型,抑制细胞增殖可能是导致NTD发生的重要机制。

Objective To establish a mouse model of neural tube defect(NTD)caused by homocysteine(Hcy)and investigate the relevant mechanism.Methods Mouse model of NTD was established with homocysteine thiolactone(HTL)alone or in combination with aminooxyacetic acid(AOAA),an inhibitor of cystathionine-β-synthase(CBS).The dosages of HTL for administration alone were 100,300,500 and 700 mg/(kg·d)respectively.However,the dosages of HTL for combined administration were 400,500 and 600 mg/(kg·d)respectively,while that of AOAA was 30 mg/(kg·d).All the mice were injected i.p.daily on days 6.5~10.5 of pregnancy.The dosages were optimized to establish the NTD model,serving normal mice as control.The levels of Hcy in blood of pregnant mice as well as the S-adenosylhomocysteine(SAH)and S-adenosylmethionine(SAM)in embryonic brain tissues were determined by ELISA,while the proliferation of mouse neuroepithelial cells by Click-i T EdU imaging kit,and the expression level of proliferating cell nuclear antigen(PCNA)in embryonic brain tissue by Western blot.Results The optimal protocol for establishment of mouse model of NTD was 500 mg/(kg·d)HTL+30 m/(kg·d)AOAA,with the highest incidence of NTD and low mortality.Compared with those in normal control group,the Hcy,SAH and SAM levels in NTD group increased significantly(P<0.01),while the SAM/SAH ratio decreased significantly(P<0.01),the proliferation of embryonic neuroepithelial cells was inhibited significantly(P<0.05),and the expression level of PCNA decreased significantly(P<0.05).Conclusion Mouse model of NTD was successfully established by the combination of HTL and AOAA.The inhibition of cell proliferation may be an important mechanism of incidence of NTD.