Mex3c参与调控小鼠胚胎神经管发育PAX3、Nestin表达的作用

Role of Mex3c in regulating the expression of PAX3 and Nestin in mouse embryonic neural tube development

目的探讨秀丽隐杆线虫肌肉表达物3(caenorhabditis elegans muscle excess,Mex3c)参与调控小鼠胚胎神经管发育中的配对盒基因(paired box 3,PAX3)、巢蛋白(Nestin)的表达及其对于神经管发育的影响。方法选取Mex3c+/-和正常野生型小鼠各18只和正常野生型雄鼠6只(用于繁殖)。在母鼠孕10.5 d、12.5 d、14.5 d时,两组各取6只母鼠获取小鼠胚胎并记录总胚胎数、吸收胎数、存活胎数。免疫组织化学染色及蛋白质印迹法检测小鼠胚胎Nestin、PAX3表达,免疫荧光染色检测PAX3,Tunel染色检测细胞凋亡,HE染色观察形态学变化。总胚胎数、吸收胎数、存活胎数为计数资料采用卡方检测;测定的Nestin蛋白、PAX3蛋白、Mex3c蛋白、细胞凋亡数据检测符合正态分布,进行单因素方差分析及S-N-K两两比较。结果免疫组织化学染色检测母鼠下丘脑Mex3c蛋白表达,Mex3c组(16.52±1.60)较对照组(26.05±2.00)低,且差异有统计学意义(P<0.0001)。各组不同时期吸收胎及总胚胎数差异无统计学意义(P>0.05),表明缺陷Mex3c基因不会导致子代胚胎发育畸形。HE染色结果显示,与对照组相比,Mex3c组神经细胞较小、密度较低。Tunel染色结果显示,Mex3c组孕10.5 d、12.5 d、14.5 d小鼠胚胎神经管内凋亡细胞表达阳性率分别为(12.20±1.30)%、(11.20±0.84)%和(10.00±0.71)%,与对照组的(4.83±1.17)%、(5.17±0.75)%和(7.17±0.75)%比较,差异均有统计学意义(P均<0.01)。免疫组织化学染色结果显示,Nestin蛋白表达在神经纤维,PAX3蛋白表达在细胞核。免疫荧光实验检测PAX3蛋白结果显示,Mex3c组孕12.5 d和孕14.5 d小鼠胚胎PAX3蛋白表达阳性率分别为(9.20±1.30)%和(8.80±1.30)%,与对照组(5.83±0.98)%和(3.67±0.82)%比较,差异均有统计学意义(P<0.01)。蛋白质印迹法结果显示,Mex3c组孕10.5 d、12.5 d、14.5 d时小鼠胚胎Nestin蛋白表达水平分别为0.06±0.02、0.57±0.09和0.69±0.04,与对照组0.79±0.07、0.39±0.14和0.27±0.07比较,Mex3c组表达呈上升趋势而对照组呈下降趋势,组间不同时间点比较,差异均有统计学意义(P均<0.05或0.001)。Mex3c组孕10.5 d、12.5 d、14.5 d时小鼠胚胎PAX3蛋白表达水平分别为0.17±0.02、0.63±0.09和0.72±0.03,与对照组0.03±0.01、0.46±0.08和0.45±0.06比较,Mex3c组表达呈上升趋势而对照组呈下降趋势,组间不同时间点比较,差异均有统计学意义(P均<0.01或0.001)。结论Mex3c参与调控小鼠胚胎神经管发育中PAX3、Nestin蛋白表达并抑制神经管中神经细胞发育及成熟和促使神经管内细胞凋亡。

Objective To explore the role of caenorhabditis elegans muscle excess(Mex3c)in regulating the expression of paired box 3(PAX3)and nestin in neural tube development and its influence on neural tube development of murine embryo.Methods Mex3c+/-(n=18)and wild-type mice(n=18)were selected along with 6 normal wild-type male mice(for reproduction).From each of two groups,6 female mice were employed for obtaining murine embryos and recording the counts of total embryos and absorbed fetuses at pregnancy days 10.5/12.5/14.5.Immunohistochemical staining and Western blot were employed for detecting the expressions of nestin and PAX3.Immunofluorescent staining was utilized for detecting PAX3 and TUNEL staining for detecting cell apoptosis and hematoxylin-eosin(HE)staining for observing morphological changes.Results The results of immunohistochemical staining showed that Mex3c group(16.52 of total embryos)and control group(26.05±2.00)had statistically significant difference(P<0.0001).No difference in the counts of absorbed fetuses and total embryos indicated that defective Mex3c gene caused abnormal embryonic development of offspring.HE staining results indicated that,as compared with control group,nerve cells were smaller with a lower density of Mex3c group.TUNEL staining results:the expression levels of apoptotic cells were(12.20±1.30),(11.20±0.84),and(10.00±0.71)in Mex3c group at days 10.5/12.5/14.5 and(4.83±1.17),(5.17±0.75)and(7.17±0.75)in control group.The differences were statistically significant(P<0.01).The results of immunohistochemical staining indicated that nestin protein was expressed in nerve fibers and PAX3 protein in nucleus.Immunofluorescent test:the positive rate of PAX3 protein expression at days 12.5/14.5 were(9.20±1.30)%and(8.80±1.30)%in Mex3c group and(5.83±0.98)%and(3.67±0.82)%in control group.The differences were statistically significant(P<0.01).Western blot revealed that the nestin protein expression levels at days 10.5/12.5/14.5 were(0.06±0.02),(0.57±0.09)and(0.69±0.04)in Mex3c group and(0.79±0.07),(0.39±0.14)and(0.27±0.07)in control group.The expression of Mex3c group showed an upward trend while control group sloped downward.And the inter-group differences were statistically significant at different timepoints(P<0.01,or 0.001).The expression levels of PAX3 protein at days 10.5/12.5/14.5 were 0.17±0.02,0.63±0.09 and 0.72±0.03 of PAX3 protein in Mex3c group versus 0.03±0.01,0.46±0.08 and 0.45±0.06 of PAX3 protein in control group(P<0.01 or 0.001).Conclusions Mex3c is involved in regulating the expressions of PAX3 and nestin protein in the development of murine embryonic neural tube through suppressing the development and maturation of neural cells and promoting cell apoptosis on neural tube.