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-NOX4 2 #、sh-NOX4 3 #)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-NOX4 2 #、sh-NOX4 3 #)，利用Western blot检测NOX4表达水平的变化，结果显示，转染oe-NOX4质粒后，NOX4的表达(1.26±0.13)显著高于转染NC组(1.00±0.02)，而转染oe-NOX4质粒后增殖相关蛋白PCNA的表达(0.71±0.10)较NC组(1.00±0.12)显著降低，差异有统计学意义( P<0.05)；相反，转染sh-NOX4 2 #、sh-NOX4 3 #质粒后，NOX4的表达为(0.85±0.06)、(0.71±0.06)，显著低于转染sh-NC质粒组(1.00±0.04)，PCNA的表达(1.61±0.17)、(1.43±0.17)较sh-NC组(1.00±0.17)明显增加，差异有统计学意义( P<0.05)。利用CCK8实验探究NOX4对细胞增殖能力的影响，结果提示，过表达NOX4，24 h后与NC组相比，吸光度为(0.48±0.01)比(0.48±0.02)，48 h为(0.82±0.04)比(1.12±0.03)，72 h为(1.07±0.10)比(2.10±0.10)，96 h为(2.53±0.20)比(3.01±0.43)，细胞增殖能力呈下降趋势，差异有统计学意义( P<0.05)；而与sh-NC相比，当转染sh-NOX4 2 #、sh-NOX4 3 #，24 h吸光度分别为(0.32±0.02)、(0.31±0.02)、(0.30±0.01)，48 h为(0.77±0.02)、(0.64±0.04)、(0.37±0.04)，72 h为(1.94±0.05)、( 1.69±0.03)、(1.24±0.08)，96 h为(2.33±0.11)、( 2.11±0.05)、(2.01±0.01)，细胞增殖能力有显著提高，差异有统计学意义( P<0.05)。 结论:NTDs中异常高表达的NOX4能够通过降低增殖相关蛋白PCNA的表达，抑制细胞增殖，导致小鼠神经管闭合异常。

abstractsObjective: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 was significantly higher in oe-NOX4 group than NC group[(1.26±0.13) vs (1.00±0.02)]and the expression of proliferation-related protein PCNA was lower than NC group[(0.71±0.10) vs (1.00±0.12)]. The difference was statistically significant ( P<0.05); On the contrary, after transfecting sh-NOX4 2# and sh-NOX4 3# plasmids, the expression of NOX4 (0.85±0.06, 0.71±0.06) dropped markedly than sh-NC group (1.00±0.04) and the level of PCNA (1.61±0.17, 1.43±0.17) was significantly higher than sh-NC group (1.00±0.17). The difference was statistically significant ( P<0.05). CCK8 experiment was performed for examining the effect of NOX4 on cell proliferation.The results showed that absorbance of 24 h was (0.48±0.01, 0.48±0.02) in oe-NOX4 group as compared with NC group; 48h (0.82±0.04, 1.12±0.03), 72 h (1.07±0.10, 2.10±0.10), 96 h (2.53±0.20, 3.01±0.43), cell proliferation capability showed a downward trend and the difference was statistically significant ( P<0.05). It implied that NOX4 could suppress C17.2 cell proliferation.On the contrary, when C17.2 cells were transfected with sh-NOX4 2#, sh-NOX4 3# and sh-NC, absorbance was (0.32±0.02, 0.31±0.02, 0.30±0.01) at 24 h, 48 h (0.77±0.02, 0.64±0.04, 0.37±0.04), 72 h (1.94±0.05, 1.69±0.03, 1.24±0.08), 96 h (2.33±0.11, 2.11±0.05, 2.01±0.01), cell proliferation capability significantly improved and the difference was statistically significant ( P<0.05). It implied that silencing NOX4 could promote cell proliferation. Conclusions:Abnormally up-regulated NOX4 in NTDs may suppress cell proliferation through down-regulation of proliferation-related protein PCNA, resulting in a failure of neural tube closure in mice.