NELL-1蛋白对小鼠ATDC5细胞软骨分化的调控机制和对人血管周细胞成骨分化作用的研究

发布时间：2018-03-19 18:21

本文选题：NELL-1　切入点：Nfatc2　出处：《浙江大学》2011年博士论文　论文类型：学位论文

【摘要】：NELL-1作为一种生长因子,对于骨骼的成正常发育以及骨和软骨细胞分化过程中胞外基质蛋白的产生都非常重要。本研究利用基因芯片和实时定量PCR技术筛选并鉴定出转录因子Nfatc2是NELL-1的一个初级响应基因。并且研究重组蛋白NELL-1对于ATDC5细胞系以及小鼠原代软骨细胞的作用。Runx2是一种骨软骨特异性的转录因子,利用腺病毒载体过量表达野生型和显性负性突变(DN)的、Runx2,发现Runx2有可能在NELL-1和Nfatc2之间起着调节作用。NELL-1可以同时在转录和蛋白质水平上诱导Nfatc2基因的表达并维持较短暂的时间,而这种诱导作用在DN-Runx2转导的细胞中被显著抑制,这表明Runx2对于Nfatc2的诱导是必要的。细胞分化实验揭示出NELL-1可抑制ATDC-5的细胞分化,尽管细胞增殖并未受到影响,但是软骨分化的特异性基因的表达降低,并且软骨节的形成与蛋白聚糖的积累也受到抑制。而利用siRNA技术敲除Nfatc2可以使得以上抑制效果得以恢复。为了进一步阐明在体内Nell-1、Runx2以及Nfatc2之间的关系,选取新生的和胚胎不同发育阶段的小鼠,利用免疫组织化学技术检测Nell-1、Runx2以及Nfatc2在野生型和Nell-1缺陷型小鼠的股骨中的分布和表达情况。结果表明在新生小鼠的股骨中这三种蛋白在野生型小鼠的软骨膜中共表达,而在Nell-1缺陷型小鼠的软骨膜中染色却很微弱或者完全不存在。因此在NELL-1调节的体内的或体外的骨软骨细胞的分化过程中,Nfact2有可能起着非常重要的作用。据我们了解,这将是首次证实Nfatc2是NELL-1的初级响应基因。此外,本论文还初次探索了人胎儿胰腺血管周细胞在体外培养条件下的成骨分化潜能,结果显示NELL-1可显著提高人血管周细胞的体外增殖并促进成骨分化和血管发生作用。
[Abstract]:NELL-1 as a growth factor, It is very important for the normal development of bone and the production of extracellular matrix protein during the differentiation of bone and chondrocytes. In this study, the transcription factor Nfatc2 was screened and identified as a kind of NELL-1 by gene chip and real-time quantitative PCR. To study the effect of recombinant protein NELL-1 on ATDC5 cell line and mouse primary chondrocytes. Runx2 is a osteochondral specific transcription factor. Using adenovirus vector to overexpression wild-type and dominant negative mutant DN2, it was found that Runx2 might play a regulatory role between NELL-1 and Nfatc2. NELL-1 could induce the expression of Nfatc2 gene at both transcriptional and protein levels and maintain a relatively short period of time. This induction was significantly inhibited in DN-Runx2 transduced cells, which indicated that Runx2 was necessary for the induction of Nfatc2. Cell differentiation experiments revealed that NELL-1 could inhibit the differentiation of ATDC-5 cells, although cell proliferation was not affected. But the expression of specific genes for cartilage differentiation decreased, Moreover, the formation of chondrocytes and the accumulation of proteoglycan were also inhibited. Knockout of Nfatc2 by siRNA technique could restore the inhibitory effect. In order to further elucidate the relationship between Nell-1 Runx2 and Nfatc2 in vivo, New born and embryonic mice at different stages of development were selected. Immunohistochemical technique was used to detect the distribution and expression of Nell-1G Runx2 and Nfatc2 in femur of wild and Nell-1 deficient mice. The results showed that these three proteins were co-expressed in the chondrocytes of wild type mice in newborn mice. However, the staining in the chondrocytes of Nell-1 deficient mice is weak or not present at all. Therefore, Nfact2 may play a very important role in the differentiation of osteochondrocytes regulated by NELL-1 in vivo or in vitro. As far as we know, Nfact2 may play a very important role in the differentiation of osteochondrocytes in vivo and in vitro. It will be the first time to confirm that Nfatc2 is the primary response gene of NELL-1. In addition, we also explored the osteogenic differentiation potential of human fetal pancreatic perivascular cells in vitro. The results showed that NELL-1 could significantly increase the proliferation of human pericytes in vitro and promote osteogenic differentiation and angiogenesis.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

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