摘要
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 differential expression of Notch signaling pathway-related molecules during spine development as well as its regulatory effect on differentiation of chondrocytes.Methods The mRNA expressions of Notch signaling molecules in intervertebral discs of mice at various stages of development were determined by RT-PCR,based on which the most important signaling molecules regulating normal development of spine were screened and verified by immunohistochemical assay.Dll-1,Jag-1 and bone morphogenetic protein-2（BMP-2）were over-expressed in nucleus pulposus（NP）cells in mediation of recombinant adenovirus,and the expression of characteristic markers of chondrocytes,aggrecan（ACAN）and collagen typeⅡ（COL2A1）were determined by RT-PCR,while the secretion of cartilage oligomeric matrix by toluidine blue staining.Results In general,the expression of Notch signal molecules,especially Dll-1,Dll-3 and Jag-1,showed a decreasing tendency during the development of mouse spine.Dll-1 and Jag-1 were widely expressed on the membrane of chondrocytes,of which the expression levels in intervertebral discs decreased with the increasing age of mice.BMP-2 overexpression promoted the differentiation of NP cells into chondrocytes with increased expression of ACAN and COL2A1 and enhanced toluidine blue staining.However,the overexpressions of Dll-1 and Jag-1 significantly inhibited the chondrogenic differentiation induced by BMP-2.Conclusion Notch signaling molecules,especially Dll-1 and Jag-1,showed significantly negative regulatory effect on normal spine development and differentiation of intrevertebral disc chondrocytes.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第8期1094-1099,共6页
Chinese Journal of Biologicals
基金
国家自然科学基金(81100309
81172545
81001197)
关键词
NOTCH信号通路
椎间盘
软骨细胞
骨形态发生蛋白-2
重组腺病毒
Notch signaling pathway
Intervertebral disc
Chondrocytes
Bone morphogenetic protein-2（BMP-2）
Recombinant adenovirus
