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目的探讨Notch信号通路相关分子在脊椎生长过程中的差异表达及其对软骨细胞分化的调控作用。方法采用RT-PCR检测各不同发育年龄段小鼠椎间盘Notch信号分子mRNA的表达情况,筛选调控脊椎正常生长发育的重要信号分子,并采用免疫组化法进行验证。用重组腺病毒介导Dll-1、Jag-1、骨形态发生蛋白-2(bone morphogeneticprotein-2,BMP-2)在髓核(nucleus pulposus,NP)细胞中过表达,RT-PCR检测软骨细胞特征性标志物蛋白聚糖(aggrecan,ACAN)和Ⅱ型胶原(collagen typeⅡ,COL2A1)的表达,甲苯胺蓝染色检测软骨细胞基质分泌情况。结果小鼠脊椎生长发育过程中,Notch信号分子mRNA的表达总体呈下降趋势,其中Dll-1、Dll-3、Jag-1下降趋势尤其显著;Dll-1和Jag-1广泛表达于软骨细胞胞膜,随着小鼠年龄的增大,二者在椎间盘中的表达显著下降;BMP-2过表达可促进NP细胞成软骨分化,ACAN和COL2A1表达升高,甲苯胺蓝染色增强;Dll-1、Jag-1过表达可显著抑制BMP-2诱导的成软骨分化效应。结论 Notch信号分子,尤其是Dll-1、Jag-1对正常脊椎生长及椎间盘软骨细胞的成熟分化具有明显的负性调节作用。
Objective To investigate the differential expression of Notch signaling pathway related molecules in the process of spinal cord growth and its role in the regulation of chondrocyte differentiation. Methods RT-PCR was used to detect the mRNA expression of Notch in each disc of different developmental age. The important signal molecules that regulate the normal growth and development of the spine were screened and verified by immunohistochemistry. The recombinant adenovirus-mediated Dll-1, Jag-1, BMP-2 were overexpressed in nucleus pulposus (NP) cells and the expression of chondrocytes The expression of characteristic markers aggrecan (ACAN) and collagen typeⅡ (COL2A1) were detected by MTT assay. The secretion of cartilage matrix was detected by toluidine blue staining. Results The expression of Notch signaling molecule mRNA in the spine was decreased in general, especially in Dll-1, Dll-3 and Jag-1. Dll-1 and Jag-1 were widely expressed in chondrocytes Membrane, as the mice age, the expression of both in the disc decreased significantly; BMP-2 overexpression can promote the chondrogenic differentiation of NP cells, ACAN and COL2A1 expression, toluidine blue staining enhanced; Dll-1 Jag-1 overexpression significantly inhibited BMP-2-induced chondrogenic differentiation. Conclusion Notch signaling molecules, especially Dll-1 and Jag-1, have obvious negative regulation on normal spine growth and maturation of disc chondrocytes.