骨折后CHI3L1的表达变化及其功能研究

Research on CHI3L1 Expression Changes in Bone Fracture and Its Function

目的:研究在骨折过程中几丁质酶3样蛋白1(CHI3L1)的表达变化情况和CHI3L1蛋白可能具有的生物学功能。方法:构建了小鼠的骨折模型,同时用TNF-α在体外刺激成骨细胞,通过real time RT-PCR和Western blot检测CHI3L1表达变化情况。用抗TNF-α抗体中和TNF-α活性,验证TNF-α对CHI3L1表达的特异性诱导作用。用Bayl 1-7082抑制NF(?)B活化,考察NF(?)B活化对TNF-α的诱导作用是否是必须的。通过RT-PCR检测核结合因子a1(core binding factor a1,cbfa1)、骨钙素(osteocalcin,OCN)的表达,同时测定碱性磷酸酶(ALP)活性,以检测成骨细胞分化状况。结果:在骨折炎症反应期和TNF-α刺激的成骨细胞中,CHI3L1表达明显升高。用抗TNF-α抗体或者Bayl 1-7082都能抑制TNF-α对CHI3L1的诱导作用。说明TNF-α首先活化NF(?)B,再由后者刺激CHI3L1表达。转染表达CHI3L1的载体后,成骨细胞的ALP活性增强,cbfa1、OCN的表达也明显升高。结论:TNF-α通过激活NF(?)B促进CHI3L1表达;在体外实验中,CHI3L1具有促进成骨细胞分化的活性。CHI3L1在骨折组织中很可能受到TNF-α等细胞因子诱导表达。在体内它可能通过刺激骨分化促进骨折愈合。

Objectives： To investigate the changes of CHI3L1 expression levels in the process of bone fracture repair and explore the possible functions of CHI3L1 in the osteoblasts differentiation. Methods： The mouse model of bone fracture was generated and osteoblastic MC3T3-E1 cells were stimulated by TNF-α in vitro,then changes of CHI3L1 expression levels were examined by quantitative real time RT-PCR and western blot. The stimulating effect of TNF-α on CHI3L1 expression was confirmed by neutralizing the activity of TNF-α with anti-TNF-α antibody. The role ofNFκB activation in the induction process was investigated by using Bayl 1-7082, which was a specific inhibitor of NFκB activation. In order to examine the differentiation of osteoblasts, the expression levels of cbfa 1 and OCN were analyzed by semi-quantitative RT-PCR and ALP activity was measured. Results： CHI3L1 was up-regulated in the hemostasis and inflammatory phrase of bone fracture repair and TNF-α-stimulated osteoblasts showed CHI3L1 levels elevated on both RNA and protein levels. Neutralization of TNF-α activity or inhibition of NFκB activation prevented the up-regulation of CHI3L1 induced by TNF-α levels in osteoblasts. Therefore, TNF-α activated NFκB first, then the latter one mediated the elevation of CHI3L1 expression. Cells transfected with expression vectors of CHI3L1 showed ALP activity elevated. Cbfal and OCN expression were also induced compared with the cells transfected with empty vectors. Conclusion： TNF-α induces CHI3L1 expression in osteoblastsvia NFκB and CHI3L1 could induce osteoblasts differentiation in vitro. It is likely that CHI3L1 expression is stimulated in bone fracture tissues by cytokines produced by osteoblasts as well as other cells around the wound site and it might accelerate the healing of bone fracture through induction of osteoblasts differentiation in vivo