小鼠肠神经前体细胞培养、转染和功能研究  

Culturing, transfection and function study of routine enteric neural progenitor cells

作　　者：朱中贤 杜春霞 谢华[1] 李红星[1] 唐维兵[1] 

机构地区：[1]南京医科大学附属儿童医院外科,210008

出　　处：《中华小儿外科杂志》2018年第1期64-70,共7页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金项目（81370473,81570467）;江苏省“青蓝工程”项目

摘　　要：目的建立小鼠肠神经前体细胞体外培养和纯化的方法，寻找合适的肠神经前体细胞转染方式进行增殖分化等功能实验，为研究肠神经系统发育及疾病奠定基础。方法取胎龄16.5 d（E16.5）的ICR小鼠胚胎肠道，经胶原酶消化后制成单细胞悬液，接种到神经前体细胞培养基中，多次换液后进行免疫学鉴定。纯度达到95%的神经前体细胞使用lipo2000，RNAimax，慢病毒等三种方式进行mmu-miR-346转染，通过实时荧光定量PCR检测神经前体细胞内mmu-miR-346的表达来比较这三种方式的转染效率。使用EdU法检测肠神经前体细胞的增殖情况，通过分化培养基诱导检测肠神经前体细胞的分化情况。结果培养第2天出现神经球，经过4次全换液，免疫学鉴定显示（96.85±0.5357）%细胞为Nestin/p75双阳性。lipo2000，RNAimax，慢病毒这三种转染方式中仅慢病毒方法的转染效率最高，能显著提升mmu-miR-346表达水平（差异倍数：117.45）。EdU阳性率与TuJ1阳性率分别从阴性对照组的（17.87±1.13）%和（24.87±0.6）%降至转染组的（6.73±0.43）%与（11.90±0.85）%，两组在增殖与分化中的差异均具有统计学意义（P〈0.0001；P〈0.001）。结论通过分离消化E16.5小鼠肠道可以建立小鼠肠神经前体细胞培养和纯化的方法，慢病毒方法在肠神经前体细胞的转染效率最高，能成功进行增殖与分化实验，可以为肠神经系统发育和疾病的研究奠定基础。ObjectiveTo establish and improve the method for culturing and purifying enteric neural progenitor cells （ENPCs） derived from murine embryos and seek optimal transfection for cell proliferation and differentiation experiments for further studies of the development and diseases of enteric nervous system.MethodsGuts were collected from E16.5 mouse embryos and treated with collagenase.Single cell suspension was plated on medium and immunocytochemical identification performed after several exchanges of medium.Once ENPCs purity reached over 95%, mmu-miR-346 was transfected into ENPCs by lipo2000, RNAimax and lentivirus （LV） respectively.For assessing transfection efficiency, the gene expression of mmu-miR-346 was detected by real-time polymerase chain reaction （PCR）. Ethynyldeoxyuridine （EdU） assay was performed for examining the effect of mmu-miR-346 on the proliferation of ENPCs.The differentiation of ENPCs was detected by the induction of differentiation medium.ResultsNeurospheres formed at Day 2.After 4 full exchanges of medium, immunocytochemical identification indicated （96.85±0.5357）% cells expressed Nestin and p75.The overexpression efficiency of LV was the highest among three methods.EdU showed mmu-miR-346 decreased the ratio of EdU（＋ ） cells from （17.87±1.13）% to （6.73±0.43）%.TuJ1 immunofluorescence showed that mmu-miR-346 decreased the ratio of TuJ1（＋ ） cells from （24.87±0.6）% to （11.90±0.85）%.Significant differences existed in cell proliferation and differentiation between transfected and control groups.ConclusionsA primary method of culturing and purifying murin ENPCs may be improved by isolating and dissociating E16.5 murine gut.And an efficient lentivirus transfection is successfully selected for cell proliferation and differentiation assays.Thus it lays a foundation for understanding the development and diseases of enteric nervous system.

关 键 词：先天性巨结肠 肠神经系统 转染 

分 类 号：R725.7[医药卫生—儿科]