5-碘代杀结核菌素对小鼠神经管的影响及其作用机制  

Effect of 5-iodotubercidin on neural tube of mice and relevant mechanism

作　　者：张晓敏[1] 李美宁[1] 王文卓 李丹丹 牛玉虎[1] 牛勃[1,2] ZHANG Xiao-min;LI Mei-ning;WANG Wen-zhuo;LI Dan-dan;NIU Yu-hu;NIU Bo(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China)

机构地区：[1]山西医科大学生物化学与分子生物学教研室,山西太原030001 [2]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

出　　处：《中国生物制品学杂志》2021年第6期682-687,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81741023);北京市自然科学基金(7182025)。

摘　　要：目的探讨腺苷激酶(adenosine kinase,ADK)抑制剂5-碘代杀结核菌素(5-iodotubercidin,ITU)对小鼠神经管的影响及其作用机制。方法将妊娠7.5 d的C57BL/6J小鼠随机分为ITU组(经腹腔分别注射2、3、4、5、6、7 mg/kg的ITU)和对照组(经腹腔注射含2%DMSO的生理盐水),妊娠11.5 d时,取胚胎,观察其发育情况。取对照组及神经管缺陷(neural tube defect,NTD)组(最佳剂量ITU)胚胎进行HE染色,观察小鼠神经管发育情况;ELISA法检测NTD组孕鼠血清中腺苷、AMP、S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)、S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)水平;N6-甲基腺苷(N6-methyladenosine,m6A)甲基化检测试剂盒检测两组小鼠胚胎脑组织中m6A含量;EdU及TUNEL法分别检测两组小鼠胚胎脑组织中上皮细胞增殖及凋亡情况;Western blot法检测m6A相关酶蛋白(METTL3、FTO、YTHDF2)、细胞增殖相关蛋白(PCNA)及细胞凋亡相关蛋白(caspase-3)的相对表达水平。结果对照组胚胎生长发育良好,未出现NTD;ITU组随着剂量的增加,NTD的发生率逐渐增加,剂量为5 mg/kg时,小鼠NTD的发生率最高(32.2%),确定该剂量为建立NTD模型的最佳ITU剂量。对照组胚胎神经管发育良好,管腔规则,神经管闭合完全;NTD组胚胎神经管管壁厚薄不一,管腔不规则,神经管未完全闭合。NTD组孕鼠血清中腺苷、SAM、SAH的含量于注射后8 h显著增加(P<0.01),AMP含量显著下降(P<0.05)。与对照组比较,NTD组小鼠胚胎脑组织m6A含量降低,METTL3的相对表达量明显减少(P<0.05),FTO、YTHDF2的相对表达量无明显变化(P>0.05);PCNA的相对表达量明显减少(P<0.05),caspase-3的相对表达量明显增加(P<0.05),表明小鼠胚胎脑组织中上皮细胞增殖减弱,细胞凋亡增加。结论ITU可诱导小鼠NTD的发生,其作用机制可能与m6A含量降低导致神经上皮细胞增殖和凋亡紊乱相关。Objective To investigate the effect of 5-iodotubercidin(ITU),an adenosine kinase(ADK)inhibitor,on the neural tube of mice as well as the relevant mechanism.Methods C57BL/6J mice 7.5 d after gestation were randomly divided into test the control groups.The mice in test groups were injected i.p.with ITU at dosages of 2,3,4,5,6 and 7 mg/kg respectively,while those in control groups with normal saline containing 2%DMSO,of which the embryos were taken out and observed for development 11.5 d after gestation.The embryos of mice injected with ITU at the optimal dosage and those in control group were observed for the development of neural tube by HE staining.The adenosine,AMP,S-adenosylhomocysteine(SAH)and S-adenosylmethionine(SAM)levels in sera of mice were determined by ELISA.The m6A content in mouse embryonic brain tissue was determined by m6 A methylation kit.The proliferation and apoptosis of epithelium in mouse embryonic brain tissue were determined by EDU and TUNEL methods respectively.The relative expression levels of m6A-assocaited enzyme proteins METTL3,FTO and YTHDF2,cell proliferation-associated protein PCNA and cell apoptosis-associated protein caspase-3 were determined by Western blot.Results The embryos in control group grew and developed well,while no neural tube defect(NTD)appeared.However,the incidence of NTD in test groups increased with the increasing dosage of ITU.When the dosage was 5 mg/kg,the incidence of NTD in mice was the highest(32.2%),thus the dosage was determined as the optimal dosage for establishment of mouse model of NTD.The neural tubes in control group developed normally,which were closed completely and the tube cavities were regular.However,the wall thicknesses of neural tubes in test groups were uneven,while the tube cavities were irregular,and the tubes were incompletely closed.The contents of adenosine,SAM and SAH in sera of mice increased significantly 8 h after ITU intervention(P<0.01),while the AMP content decreased significantly(P<0.05).Compared with those in control group,the m6

关 键 词：神经管畸形 腺苷激酶 5-碘代杀结核菌素 N6-甲基腺苷 细胞增殖 细胞凋亡 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]