NOX4调控神经细胞增殖在神经管缺损发生中的作用  

Role of NOX4 regulating nerve cell proliferation in the development of neural tube defects

作　　者：付佳琳 黄琬淇 黄天楚[1] 顾卉[1] 袁正伟[1] Fu Jialin;Huang Wanqi;Huang Tianchu;Gu Hui;Yuan Zhengwei(The Key Laboratory of Pediatric Congenital Malformations,Affiliated Shengjing Hospital,China Medical University,Shenyang 110004,China)

机构地区：[1]中国医科大学附属盛京医院卫生部小儿先天畸形重点实验室,沈阳110004

出　　处：《中华小儿外科杂志》2023年第3期252-258,共7页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金(81771595);国家重点研发计划(2016YFC1000505)。

摘　　要：目的探究烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nicotinamide adenine dinucleotide phosphate oxidase 4,NOX4)通过调控细胞增殖对小鼠神经管缺损(neural tube defects,NTDs)形成的作用。方法选用体重18~20 g的健康C57BL/6J小鼠,雌鼠30只,雄鼠15只,雌雄小鼠按2∶1比例于晚上合笼、过夜,次日清晨观察小鼠阴道栓,有明显阴道栓确认已交配,当日记为孕0.5 d(E0.5)。选取明确受孕的18只小鼠,按随机数字表法随机分为小鼠NTDs组(9只)和对照组(9只)。孕8.5 d(E8.5)时,将小鼠NTDs组利用全反式维甲酸(all trans retinoid acid,ATRA)按70 mg/kg的浓度灌胃处理,对照组采用橄榄油灌胃处理。在孕9.5 d(E9.5)收集胎鼠。体式显微镜下观察胚胎神经管发育情况并拍照记录形态。通过免疫荧光染色法观察神经管形态,蛋白质印迹法(Western blot)检测NOX4和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在神经管的表达情况。构建NOX4过表达质粒(oe-NOX4)和对照(NC)以及NOX4沉默质粒(sh-NOX42^(#)、sh-NOX43^(#))和对照(sh-NC),分别转染至C17.2细胞系中,通过Western blot检测NOX4及增殖相关蛋白PCNA的表达情况;通过细胞计数试剂盒(cell counting kit-8,CCK8)检测转染质粒24 h、48 h、72 h、96 h后细胞生长情况,进一步明确NOX4对细胞增殖的调控作用。结果体式显微镜下观察E9.5胎鼠神经管发育情况,NTDs组存在明显神经管畸形。免疫荧光染色结果也显示NTDs组存在明显的神经管闭合不全,对照组神经管发育正常,且NTDs组神经管中NOX4表达略高于对照组,而PCNA的表达低于对照组。Western blot结果显示,NTDs组胚胎中NOX4表达(6.24±2.70)高于对照组(1.00±0.51),PCNA的表达(0.75±0.09)低于对照组(1.00±0.12),两组间的差异有统计学意义(P<0.05)。在C17.2神经干细胞中转染NOX4过表达质粒(oe-NOX4)和沉默质粒(sh-NOX42^(#)、sh-NOX43^(#)),利用Western blot检测NOX4表达水平的变化,结果显示,转染oe-NOX4质粒�Objective To explore the effect of NOX4 regulating cell proliferation on the formation of neural tube defects(NTDs)in mice.Methods A total of 45 adult C57BL/6J mice weighing 18-20g were selected.The ratio of female-to-male was 2:1.The animals were paired overnight and pregnancy was established by a presence of vaginal plug the next morning and noon of that day designated as embryonic day 0.5(E0.5).Eighteen pregnant mice were randomized into two groups of NTD(n=9)and control(n=9).At E8.5,pregnant mice received all trans retinoid acid(ATRA,70 mg/kg body weight)or control treatment(olive oil alone).Fetuses were collected at E9.5.Under a dissecting microscope,embryos were harvested for imaging.Neural tube morphology was observed after immunofluorescent stain and the expressions of NOX4 and PCNA in neural tube were detected by Western blot.NOX4 over-expression plasmid(oe-NOX4)and control(NC),NOX4 silencing plasmid(sh-NOX4 2# ,sh-NOX4 3#)and control(sh-NC)were constructed for transfecting C17.2 murine neural stem cell for examining the expressions of NOX4 and proliferation-related protein PCNA by Western blot.Counting kit-8(CCK8)was employed for detecting the effect of NOX4 on cell proliferation after transfecting plasmid for 24/48/72/96h.Results The development of neural tube of E9.5 embryo was observed under a dissecting microscope.NTD group exhibited obvious neural tube defects.Immunofluorescent stain results revealed apparent defects in neural tube in NTD group.There were a higher level of NOX4 and a lower level of PCNA in NTD group as compared with control group.Similarly,Western blot indicated that the expression of NOX4 were higher in NTD group than control group[(6.24±2.70)vs(1.00±0.51)]and the expression of PCNA declined as compared with control group[(0.75±0.09)vs(1.00±0.12)].Inter-group difference was statistically significant(P<0.05).NOX4 over-expression plasmid(oe-NOX4)and silencing plasmid(sh-NOX4 2# ,sh-NOX4 3#)were transfected into C17.2 neural stem cells,the results show that the expression of NOX4

关 键 词：神经管缺损 烟酰胺腺嘌呤二核苷酸磷酸氧化酶4 细胞增殖 

分 类 号：R741[医药卫生—神经病学与精神病学]