Notch1(NICD)过表达真核载体构建及对大鼠BMSCs增殖分化的影响  

Construction of Notch1(NICD) Eukaryotic Expression Vector and Its Influence on the Proliferation and Differentiation of Rat Bone Marrow Mesenchymal Stem Cells in vitro

作　　者：杜红阳 李东宁 付海燕 包翠芬 秦书俭 

机构地区：[1]医学院附属第一医院皮肤科,辽宁121001 [2]重症医学科,121001 [3]医学院解剖学教研室,辽宁121001

出　　处：《天津医药》2014年第9期883-888,共6页Tianjin Medical Journal

基　　金：国家自然科学基金面上项目(31170930);辽宁省科技厅自然科学基金计划项目(2013022059);辽宁医学院青年科技启动基金项目(Y2011Z010)

摘　　要：目的探讨过表达Notch1(NICD)基因对大鼠骨髓间充质干细胞(BMSCs)增殖分化的影响。方法构建Notch1(NICD)过表达真核载体,实验分正常对照组(CON组,不做转染)、阳性对照组(空载体组,将质粒pEGFP-N1转染BMSCs)和转染组(将重组质粒pEGFP-N1-NICD转染BMSCs)。转染48 h后观察细胞一般状态,Real-timePCR和Western blotting检测神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和Notch1基因和蛋白表达,流式检测转染后细胞凋亡、细胞周期,MTT检测细胞增殖情况。结果重组质粒pEGFP-N1-NICD编码序列与设计完全一致。转染组和空载体组BMSCs均可表达绿色荧光。转染组GFAP、Notch1的基因和蛋白表达均高于CON组和空载体组(均P<0.05),空载体组与CON组差异无统计学意义;各组间NSE的基因和蛋白表达差异无统计学意义。转染组存活细胞比率低于CON组和空载体组,早期细胞凋亡比率、晚期细胞凋亡比率均高于CON组和空载体组;空载体组晚期细胞凋亡比率高于CON组。转染组细胞处于G1/G0期的比例高于CON组和空载体组,处于S期和G2/M期的比例低于CON组和空载体组(均P<0.05)。除转染1 d时各组MTT A490值差异无统计学意义外,其余时点转染组MTT A490值均低于CON组和空载体组(均P<0.05)。结论高表达Notch1(NICD)基因可能在一定程度上诱导BMSCs凋亡、抑制其增殖,且表现诱导向神经胶质样细胞分化。Objective To investigate the effect of construct the Notch1 （NICD） eukaryotic expression vector on the proliferation and differentiation of rat bone marrow mesenchymal stem cells （BMSCs） in vitro. Methods Rat BMSCs were experimented as the object. NICD eukaryotic expression vector was constructed. pEGFP-N1-NICD expressing plasmids were used to transfect BMSCs. The study included control group （CON group）, empty vector group （VEC group） and the trans-fection group （TRA group）. After 48-hour transfection, BMSCs were observed for general morphology. The protein expres-sions of NSE, GFAP and Notch1 were detected by real-time PCR and Western blotting assay respectively. The apoptosis, cy-cle distribution and cell proliferation were evaluated by flow cytometry and MTT assay. Results The DNA sequencing con-firmed that the pEGFP-N1-NICD recombinant plasmid was successfully constructed, and both VEC group and TRA group expressed green fluorescence after 48-hour transfection. The relative expression levels of Notch1 and GFAP mRNA and pro-tein were significantly higher in TRA group than those in VEC group and CON group （P＆lt;0.05）, and there was no significant difference between VEC group and CON group. After 48-hour transfection, the ratio of living cells was significantly lower in TRA group than that of CON group and VEC group, and the early apoptotic rate and late apoptotic rate were significantly higher in TRA group than those of CON group and VEC group （P＆lt;0.05）. The late apoptotic rate was significantly higher in VEC group than that of CON group. The proportion of G1/G0 cells was significantly higher in TRA group than that of CON group and VEC group, but S and G2/M cells were significantly lower （P＆lt;0.05）. The value of growth curve was gradually de-creased in TRA group than that of CON group and VEC group （P＆lt;0.05）. Conclusion The high expression of NICD gene might induce apoptosis of BMSCs, inhibit the proliferation in part, and induce into glial-like cell differentia

关 键 词：细胞增殖 细胞分化 转染 神经胶质原纤维酸性蛋白质 磷酸丙酮酸水合酶 NOTCH信号通路 骨髓间充质干细胞 诱导 Notch1(NICD) 

分 类 号：R349.5[医药卫生—基础医学]