【作者】 李平；

【导师】 李保界；

【作者基本信息】 上海交通大学 ， 生物学， 2018， 博士

【摘要】 肝素结合表皮生长因子HB-EGF属于EGF超家族,它可以在蛋白酶的作用下形成可溶性形式,通过自分泌或旁分泌作用发挥功能。有报道称关节炎病人样本及关节手术造模鼠中HB-EGF表达升高,EGFR信号通路被激活。HB-EGF还可以通过调控成骨细胞相关基因表达促进破骨细胞的形成。本课题在前人研究基础上,使用Cre/lox P技术,构建了间质干细胞(MSC)、软骨细胞、破骨细胞中特异性过表达或敲除HB-EGF的突变体小鼠,系统研究HB-EGF在骨骼系统中的作用及潜在机制。我们发现在Dermo1标记的MSC中特异性敲除HB-EGF时,敲除鼠骨骼大小和关节软骨均无异常,而骨量及骨密度都有一定程度上升。相反,Dermo1标记的MSC中过表达HB-EGF,小鼠骨量及骨密度均显著下降,骨形成明显减弱且骨矿化不足。组织学研究发现,实验鼠关节严重畸形,关节膨大,关节软骨退化,关节软骨下有内生软骨瘤样结构。进一步分析还发现,生长板软骨增殖增加,关节软骨纤维化显著提高,并且Dermo1-HB-EGF小鼠脊柱软骨板也有软骨瘤样表型。与此同时,本研究发现MSC中过表达HB-EGF的小鼠中,破骨细胞的数量及分化并未受到影响。为了更全面的说明HB-EGF对骨代谢的作用,我们构建了在Ly M标记的破骨祖细胞中过表达HB-EGF的小鼠。实验鼠骨骼大小和生长板软骨均无异常,骨量及骨密度均无变化。实验鼠骨髓细胞体外破骨分化能力没有差别,这说明自分泌及旁分泌的HB-EGF对成骨-破骨之间的偶联及破骨细胞自身的调控都没有作用。通过分子生物学研究发现,HB-EGF通过激活EGFR信号下游的Erk及Akt信号通路促进MSC的增殖。另外,HB-EGF通过抑制BMP信号通路下游Smad1/5/8信号抑制MSC分化。EGFR信号通路的抑制剂AG1478对过表达小鼠软骨异常表型具有良好的挽救作用。综上所述,本研究首次发现并详细的分析了HB-EGF在小鼠骨骼发育及骨代谢中的作用。研究结果表明HB-EGF在MSC中无论敲除和过表达都会对成年之后骨代谢平衡产生影响,MSC中敲除HB-EGF不影响软骨的正常发育,相反HB-EGF的过表达却对关节及生长板的发育造成严重破坏,导致软骨发育缺陷和关节炎的发生。以上研究结果拓展了我们对关节炎的发生及内生软骨瘤的产生的认识,也为临床提供了新的检测指标和治疗策略。更多还原

【Abstract】 Heparin-binding EGF-like growth factor(HB-EGF)is a membraneanchored glycoprotein.It belongs to the EGF superfamily.It can form soluble HB-EGF under the action of proteases.In vitro studies have found that HB-EGF-EGFR signaling can promote MSC proliferation and inhibit its differentiation.It have also been reported that HB-EGF levels are elevated in osteoarthritic chondrocytes in arthritic patients and in ACLT or DMM-induced osteoarthritis,and in which EGFR signaling pathway is activated.HB-EGF can also promote the formation of osteoclasts by regulating the expression of genes,such as RANKL,OPG,M-CSF in osteoblasts.In this thesis,based on the previous studies.I used Cre/lox P technology to generate mutant mice that specifically over-express HB-EGF in MSCs,chondrocytes and osteoclasts.We study the role of HB-EGF and underlying pathological mechanisms in the skeleton systematically.We found that when HB-EGF was specifically knocked out in Dermo1-labeled mesenchymal cells,there was no abnormality in bone size and articular cartilage,but there was a moderate increase in bone mass and bone density.In contrast,overexpression of HB-EGF in Dermo1-labeled mesenchymal cells significantly decreased bone mass and bone density in mice,markedly decreased bone formation and bone mineralization.This shows that HBEGF expressed in stromal cells plays an important role in bone remodeling in mice.By H/E staining of the bone sections,we found that the mutant mice had distorted knee joints,increased femur width,degraded articular cartilage.The mutant mice also developed chondroma and fibrosis-like phenotypes,with the later stained positive for FSP1,a fibroblast marker.Ki67 staining revealed that HB-EGF overexpression led to an increase in the number of proliferating chondrocytes in the growth plate and at the articular cartilage surface,suggesting that HB-EGF promoted expansion of chondrocytes at these two locations.We also found chondroma in the spinal growth plate.At the same time,we found that overexpressing HB-EGF in MSC preduced no difference in osteoclastogenesis.To more fully demonstrate the effect of HB-EGF on bone resorption,we constructed mice that overexpress HB-EGF in Lyz-labeled osteoclasts.There was no abnormality in bone size and growth plate cartilage,no change in bone mass and bone density,and no difference in osteoclast differentiation ability.Further mechanism analysis revealed that HB-EGF promotes MSC proliferation by activating the Erk and Akt signaling pathways downstream of EGFR signaling.In addition,HB-EGF inhibits MSC differentiation by inhibiting the Smad1/5/8 signal downstream of the BMP signaling pathway.AG1478,an inhibitor of EGFR signaling pathway in mice,can well rescue the abnormal phenotype of cartilage.In summary,this study first analyzed in details the role of HB-EGF in bone development and bone metabolism in mice.The results of the study indicate that HB-EGF overexpression in stromal cells can influence bone growths,whereas overexpression of HB-EGF in osteoclasts has no effect on bone.Knocking out HB-EGF in stromal cells does not affect the normal development of cartilage.On the contrary,overexpression of HB-EGF may have a destructive effect on the development of joint and growth plate chondrocytes.These findings expand our understanding of the occurrence of arthritis and the production of endogenous chondromas,and provide new possible therapeutic strategies for clinical treatment.更多还原