摘要
目的观察先天性巨结肠症(hirschsprung disease,HSCR)患者携带的SEMA3C/3D基因错义突变编码蛋白对原代培养的肠神经嵴细胞(enteric neural crest cell,ENCC)分化、成熟和迁移的影响。方法分别用野生型(wild type,WT)和突变型SEMA3C/3D质粒瞬时转染HEK293T细胞获取上清液,用含0.5 nmol/ml相应蛋白的上清液处理ENCC 72 h,分别用神经元、胶质细胞和未成熟神经元特异性标志物(Peripherin、GFAP、MASH1)多重免疫荧光染色法检测处理后细胞的分化倾向和分化后神经元的成熟速度,用Transwell迁移实验检测细胞迁移能力的变化。结果与WT相比,突变型SEMA3C(S329G、V337M)处理组的ENCC分化为神经元和成熟神经元的能力均下降(WT,S329G,V337M组的Peripherin/GFAP比值分别为0.95±0.34,0.53±0.35,0.28±0.07;Peripherin/MASH1比值分别为1.14±0.17,0.95±0.38,0.60±0.19),其中以V337M突变型SEMA3C处理组的下降最为明显(P<0.01),而野生型和突变型SEMA3C/3D对神经球的迁移均没有明显影响。结论突变型SEMA3C可以抑制ENCC分化为神经元/成熟神经元,从而影响肠神经系统的正常发育,这可能为HSCR的发病机制研究提供新的线索。
Objective To explore the effect of SEMA3C/3D mutations in Hirschsprung disease patients on the differentiation,maturation and migration of the enteric neural crest cells（ ENCC）. Methods HEK293 T cells were transfected with wild-type（ WT） and mutant SEMA3C/3D-AP plasmids to collect the fusion protein supernatant. The ENCCs were treated with wild-type and mutant supernatants containing 0. 5 nmol/ml of corresponding fusion protein for 72 h. Differentiation,maturation and migration abilities of the ENCC were determined by multiple-immunofluorescence（ Peripherin,GFAP,MASH1） and Transwell migration assay. Results Compared with WT group,the ability of ENCC to differentiate into neurons/mature neurons both decreased in mutant SEMA 3C groups,especially in V337 M SEMA 3C group（ P〈0. 01）. The Peripherin/GFAP ratios of WT,S329 G and V337 M were 0. 95 ± 0. 34,0. 53 ± 0. 35 and0. 28 ± 0. 07,respectively; and the Peripherin/MASH1 ratios of WT,S329 G and V337 M were 1. 14 ± 0. 17,0. 95 ± 0. 38 and 0. 60 ±0. 19,respectively. There was no statistically significant difference in migration capacity of ENCC between WT group and mutant SEMA3C/3D group. Conclusion Mutant SEMA3 C may affect the development of the enteric nervous system by inhibiting the ENCC to differentiate into neurons/mature neurons,which may provide a new clue for the pathogenesis of HSCR.
出处
《山西医科大学学报》
CAS
2017年第8期817-822,共6页
Journal of Shanxi Medical University
基金
国家自然科学基金青年科学基金资助项目(81300266)
北京市自然科学基金面上项目(7142029)
北京市优秀人才培养个人项目(2013D003034000007)
北京市科技新星基金资助项目(Z151100000315091)
