摘要
目的探讨地黄多糖诱导大鼠骨髓间充质干细胞(BMSCs)分化为神经样细胞的作用,及其对Notch1蛋白表达的影响。方法以大鼠BMSCs为研究对象,取第3代细胞,分为对照组、β-巯基乙醇诱导组(5mmol/L BME)和地黄多糖诱导组(0.2 mg/mL)进行诱导实验,应用流式细胞仪检测诱导前后细胞生长周期改变情况,免疫细胞化学染色法检测神经标志物:神经巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和Notch1蛋白的表达,Western blot检测Notch蛋白胞内片段(NICD)的表达情况。结果流式细胞仪检测示地黄多糖诱导后G0/G1期细胞比例明显升高,而S+G2/M期的细胞比例明显下降。免疫细胞化学染色显示,对照组不表达神经细胞标志物,各诱导组诱导后产生特异性神经细胞标志物表达。其中地黄多糖组nestin及NSE阳性细胞率高于β-巯基乙醇诱导组(P<0.05);GFAP阳性细胞率低于β-巯基乙醇诱导组,差异有统计学意义(P<0.05);免疫细胞化学染色示地黄多糖诱导组诱导前为Notch1蛋白强阳性或阳性染色,并随诱导时间延长阳性细胞逐渐减少。Western blot结果显示地黄多糖诱导组诱导分化24 h后细胞内NICD含量逐渐下降,到第5天时,低于诱导前水平,且仍明显低于对照组。结论地黄多糖可能通过抑制Notch1蛋白表达而诱导BMSCs向神经样细胞分化,且主要向神经元样细胞方向分化。
Objective To investigate the effect of Rehmannia glutinosa polysaccharide （RGP） on the Notchl protein expression in inducing differentiation of rat bone marrow mesenchymal stem ceils （BMSCs） into neuron -like cells in vitro. Methods The third passage cells of rat BMSCs were divided into 3 groups ： control group, β - mereaptoethanol （5 mmoL/L, BME） induction group and RGP （0. ：2 mg/mL） induction group. Fiow eytometry was applied for testing the growth cycle changes before and after induction. Immunoeytochemetry was used to detect neural specific marks such as nestin, NSE, GFAP and Notch 1 protein. Western blot was constructed for assessment of intracellular domin of Notch （NICD） protein expression. Results Obvious increase of G0/G1 phase cells and reduction in S ＋ Gz/M ceils were revealed after RGP induction according to flow cytometry. Neural cell markers were expressed in induction groups, but not in control group. There were significantly higher positive rates of nestin and NSE in RGP induction group than BME induction group （ P 〈 0. 05 ） and higher positive rate of GFAP in the latter group （ P 〈 0. 05 ）. The all cells were Notch 1 protein positive in RGP induction group, which were reduced gradually over time, according to immunoeytochemistry. Western blot results show that the contents of NICD was up - regulated 24 h after RGP induction and decreased gradually, and even fell below the baseline level and significantly lower than control group at 5th day （ P 〈 0. 05 ）. Conclusion RGP inhibits Notchl protein expression thus induces BMSCs differentiation into neuron -like cell.
出处
《广东医学》
CAS
CSCD
北大核心
2012年第9期1202-1206,共5页
Guangdong Medical Journal
基金
国家自然科学基金资助项目(编号:31170930)
辽宁省教育厅科研项目(编号:LT2010061)
辽宁医学院青年科技启动基金项目(编号:Y2010Z011
Y2011Z010)
关键词
地黄多糖
骨髓间充质干细胞
NOTCH1蛋白
诱导
神经样细胞
Rehmannia Glutinosa Polysaceharide
bone marrow mesenchymal stem cells
Notchl protein
induc- tion
neuron - like cells
