摘要
本研究探讨Notch信号通路在人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)体外增殖及向神经细胞分化过程中的作用。采集健康自愿者骨髓,体外培养获得hMSCs,取第3代hMSCs,在诱导剂(β-ME,DMSO,BHA)作用下向神经细胞分化。诱导后用免疫细胞化学鉴定神经元特异性烯醇化酶(neuron-specific enolase,NSE)和尼氏体的表达以确定诱导效果;用流式细胞术检测细胞生长周期时相的变化。在诱导前后,用免疫荧光和RT-PCR方法检测Notch通路中Notch1受体蛋白、配体Jagged1(JAG1)、调节蛋白活化相关物早老素1(presenilin1,PS1)、靶基因hairy and enhancer of split1 (HES1)信号分子表达的变化。结果显示:诱导前,处于G0/G1期的hMSCs占58.5%,S+G2/M期的细胞占41.5%;诱导后,G0/G1期细胞比例升高,而S+G2/M期细胞比例下降,NSE阳性细胞率达(77±0.35)%,细胞质中可见深蓝色的块状或颗粒状尼氏体。免疫荧光显示,诱导前后hMSCs内Notch1和JAG1均呈阳性表达,但RT-PCR检测发现诱导后Notch1、JAG1、PS1和HES1mRNA表达量较诱导前明显降低(均P<0.05)。结果表明,诱导hMSCs向神经细胞分化能抑制Notch信号分子表达,低水平的Notch信号激活可能有利于神经细胞的分化。
The Notch signaling pathway has been implicated in the regulation of cell-fate decisions such as differentiation of embryo stem cells and neural stem cells into neurons. We cultured human mesenchymal stem cells （hMSCs） in vitro and induced hMSCs to differentiate into neural cells by β-mercaptoethanol （β-ME）, DMSO and 3-tert-butyl-4-hydroxyanisole （BHA）. Immunocytochemistry was utilized to detect neuron-specific enolase （NSE） and Nissl body, and flow cytometry was used to determine cell growth phases. The expressions of signal molecules involved in the Notch pathway such as Notchl, Jagged 1 （JAG 1）, presenilin 1 （PS 1） and hairy and enhancer of split 1 filES 1 ） were observed by RT-PCR and immunofluorescent techniques. The results were as follows： （1） Before induction, the percentage of hMSCs at G0/G1 was 58.5%, and the percentage at S＋G2/M was 41.5%. After induction, the percentage of hMSCs at G0/G1 increased to 73.1%, 76.2% and 78.1%, respectively on days 2, 4 and 6, and the percentage at S＋G2/M decreased to 26.8%, 24.8% and 21.9%, respectively; The percentage of NSE-positive cells reached （77±0.35）%; Nissl＇s staining was positive in cytoplasm. （2） Notchl and JAG1 were both expressed in hMSCs before and after induction, but the mRNA expressions of both Notchl and JAG1, detected by RT-PCR, decreased obviously after induction（P〈0.05）. Notchl mRNA/β-actin was 1.157, 0.815, 0.756 and 0.570, and JAG1 mRNAJ β-actin was 0.437, 0.350, 0.314 and 0.362, respectively, on days 0, 2, 4 and 6 after induction. The Notch pathway activation participantPSI mRNA and Notch pathway target gene HES1 mRNA also decreased apparently after induction （P〈0.05）, and their mRNA/β-actin was 0.990, 0.449, 0.441, 0.454 and 0.370, 0.256, 0.266, 0.240 on days 0, 2, 4 and 6, respectively. These observations indicate that the expressions of Notch signal molecules were suppressed when hMSCs were induced to differentiate into neural cells. Based on these findings, we propose that low level of Notch signalling activation may contribute to neural cell differentiation.
出处
《生理学报》
CAS
CSCD
北大核心
2007年第3期267-272,共6页
Acta Physiologica Sinica
基金
the Science and Technology Foundation of Henan Province(No.0424410085)
the Natural Science Foundation of Henan Province (No.0611043000)
