论文部分内容阅读
目的:探讨母系表达基因3(MEG3)启动子甲基化对p53/p21信号通路的调控及其在先天性巨结肠(HSCR)发病机制中的作用。方法:检测30例HSCR患儿有神经节细胞段、无神经节细胞段p53 mRNA、蛋白表达和MEG3基因启动子甲基化情况。应用MEG3过表达和沉默质粒转染人神经母细胞瘤SK-N-BE(2)细胞株，建立MEG3过表达(MEG3-OE)组、MEG3抑制(MEG3-KD)组及空白对照(NC)组，使用氮杂胞苷(5-aza-CdR)调控各组后检测细胞株的增殖、凋亡情况及其p21 mRNA和蛋白的表达情况。结果:①无神经节细胞段与有神经节细胞段肠管组织p53 mRNA的相对表达量分别为10.56±0.37和2.24±0.3，p53蛋白的相对表达量分别为1 058.5±106.9和583.6±87.6，MEG3启动子甲基化率分别为(58.3±4.5)%和(33.3±1.3)%，两组比较，差异均有统计学意义(n t＝95.67，n P<0.05；n t＝18.82，n P<0.05；n χ2＝6.25，n P<0.05)。②加入氮杂胞苷共培养72 h时：MEG3-OE组较MEG3-KD组、NC组增殖能力明显增高(吸光度值分别为0.52±0.06、0.51±0.01、0.47±0.05)、凋亡率降低[共培养72 h时细胞凋亡率分别为(2.22±1.12)%、(4.08±0.57)%、(7.59±3.87)%]，且p21 mRNA及蛋白的相对表达量4.54±2.13和7.09±0.95均较另两组0.83±0.16、0.06±0.04和6.21±0.34、3.24±1.40显著升高，以上结果，各组间差异均具有统计学意义(n P均<0.05)。n 结论:HSCR中存在MEG3启动子高度甲基化，可能通过上调p53/p21信号通路介导神经嵴细胞增殖和凋亡，参与HSCR的发病。“,”Objective:To explore the regulatory mechanism of MEG3 promoter methylation for p53/p21 signaling pathway in the pathogenesis of HSCR.Methods:The expressions of p53 gene and methylation of MEG3 were detected in aganglionosis and ganglion cell tissue segment of 30 HSCR-confirmed cases. MEG3 overexpression and silencing plasmid were employed for transfecting SK-N-BE(2) cell line, overexpression of MEG3 and knockdown of MEG3. Blank groups were established. Methylation inhibitor azacytidine (5-aza-CdR) was added to each SK-N-BE(2) group. The proliferation, apoptosis and expression of p21 were detected for all groups.Results:The relative expression of p53 mRNA in aganglionosis segment and ganglion cell tissue was 10.56±0.37 & 2.24±0.3; the relative expression of p53 protein 1058.5±106.9 & 583.6±87.6; the incidence of MEG3 methylation (58.33±4.5)% & (33.30±1.3)%. Statistically significant differences existed in the above results ( n t=95.67, n P<0.05;n t=18.82, n P<0.05;n χ2=6.25, n P<0.05). After adding 5-aza-CdR: MEG3-OE group was higher than MEG3-KD and NC groups (72h absorbance value: 0.516±0.06 & 0.505±0.01 & 0.466±0.05); apoptotic rate was lower (2.22%±1.12% & 4.08%±0.57% & 7.59%±3.87%). The expression of p21 mRNA was (4.54±2.13 & 0.83±0.16 & 0.06±0.04) in MEG3-OE group and the relative expression of p21 protein (7.09±0.95 & 6.21±0.34 & 3.24±1.4); statistically significant difference existed in the above results (n P<0.05).n Conclusions:Highly methylated in HSCR, MEG3 promoter is involved in the pathogenesis of HSCR by up-regulating the p53/p21 signaling pathway for mediating the apoptosis of neural crest cells.