摘要
探讨神经营养因子-3（neurotrophin-3，NT-3）对大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）增殖及凋亡的影响及可能机制。方法将NT-3过表达及干扰慢病毒转染的BMSCs细胞与神经元细胞共培养，分为过表达对照组、NT-3转染组、抑制对照转染组、shRNA-NT-3转染组（NT-3沉默组）4组，MTT检测共培养24 h、48 h、72 h细胞增殖情况；流式细胞术检测48 h细胞周期变化及凋亡情况；实时荧光定量PCR检测C/EBPβ mRNA表达情况；Western blot检测C/EBPβ蛋白表达情况。结果MTT结果显示，与过表达对照组比较，48 h及72 h时间点NT-3转染组BMSCs细胞增殖能力（0.650±0.042，0.826±0.074）显著增强（P〈0.05），与抑制对照转染组比较，48 h及72 h时间点NT-3沉默组BMSCs细胞增殖能力（0.426±0.062，0.516±0.088）显著降低（P〈0.05）；流式细胞术检测48 h细胞周期及凋亡结果显示，NT-3转染组BMSCs细胞G1期比例降低，G2及S期升高，凋亡减少；NT-3沉默组BMSCs细胞G1期比例升高，G2及S期降低，凋亡增加；荧光定量PCR及Western blot结果表明C/EBPβ mRNA及蛋白水平在NT-3转染组BMSCs细胞中显著上调，而在NT-3沉默组BMSCs细胞中显著降低（P〈0.05）。结论NT-3可能通过促进C/EBPβ表达影响其下游相关靶基因的表达，从而抑制BMSCs细胞增殖分化过程中的细胞凋亡。
ObjectiveTo investigate the effect of neurotrophin-3 （NT-3） on the proliferation and apoptosis of bone marrow mesenchymal stem cells （BMSCs） in rats and possible mechanisms.MethodsThe NT-3 overexpression and lentiviral transfection of BMSCs were co-cultured with neuronal cells respectively and then they were divided into overexpression control group, NT-3 transfection group and shRNA-NT-3 transfection group （NT-3 silencing group）. MTT assay was used to detect the cell culture for 24 h, 48 h and 72 h. Cell cycle and apoptosis were detected by flow cytometry for 48 h. Real-time quantitative PCR was used to detect the expression of C/EBPβmRNA.The expression of C/EBPβprotein was detected by Western blot.ResultsMTT results showed that the proliferation ability of BMSCs in the NT-3 overexpression group was significantly higher than that in the control group （0.650±0.042, 0.826±0.074） at 48 h and 72 h （P〈0.05）. Compared with the control group （P〈0.05）, the cell cycle and apoptosis of BMSCs in NT-3 silencing group were significantly decreased at 48 h and 72 h （P〈0.05）. The results of 48 h cell cycle and apoptosis showed that the percentage of G1 phase in BMSCs was decreased, G2 and S were increased and the apoptosis was decreased. The percentage of G1 phase in G2-S phase and the increase of apoptosis were increased in NT-3 silencing group. The results of Western Blot showed that C/EBPβ mRNA and protein levels were significantly up-regulated in BMSCs of NT-3 overexpression group and significantly decreased in NT-3 silencing group （P〈0.05）.ConclusionNT-3 may promote the expression of C/EBP beta and affect the expression of its downstream target genes, which can inhibit the apoptosis of BMSCs cells.
出处
《中华行为医学与脑科学杂志》
CAS
CSCD
北大核心
2018年第1期12-16,共5页
Chinese Journal of Behavioral Medicine and Brain Science
基金
山东省科学技术发展计划项目 （2012GSF11838）
