【作者】 李鹤；

【导师】 邹志华；

【作者基本信息】 吉林大学 ， 生物学， 2024， 硕士

【摘要】 骨关节炎(osteoarthritis,OA)是一种发病率非常高、严重影响人类尤其是老年人健康的疾病,但是目前还没有能改变疾病进程的骨关节炎治疗药物(disease-modifying osteoarthritis drugs)。由于缺乏血管和淋巴系统,软骨组织中的营养物质和氧气供应有限,废物和代谢产物不能及时清除,使软骨细胞的生存变得脆弱。一旦软骨组织,特别是关节软骨受到损伤后,其再生修复能力很差。在骨性关节炎软骨组织中,软骨细胞的分解(catabolic)与合成(anabolic)代谢平衡失调,导致进行性软骨基质胶原和蛋白多糖降解、软骨细胞凋亡,因此,促进软骨细胞再生和软骨基质合成是骨关节炎领域的研究热点。成纤维细胞生长因子(Fibroblast growth factor,FGF)家族成员FGF18通过其受体FGFR3c和FGFR2c而在骨骼和软骨发育与再生过程中发挥重要作用。FGF18激活的FGFR2c促进长骨发育过程中的硬骨形成,而被FGF18激活的FGFR3c则促进软骨细胞增殖和软骨形成。FGF18在骨骼和软骨发育和形成中的双重作用使得研究者们注意到了其在骨关节炎中的潜在应用价值。因此,重组人FGF18(Sprifermin)被用于骨关节炎治疗的研究,已在多种动物模型中均表现出了显著的促软骨再生的活性。目前,Sprifermin的研究已经进展至临床Ⅲ期,在多项临床数据中展现出关节软骨的结构改善、关节软骨的厚度增加的显著疗效,但其缓解疼痛症状的疗效有限,且其促硬骨形成的活性可能引起骨刺生长。本研究旨在设计和筛选促软骨增殖的活性更高、硬骨形成的作用更低的新型FGF18类似分子,以促进FGF18类药物在骨关节炎等疾病治疗中的应用。FGF18与FGF8和FGF17一起组成一个FGF小家族,三者在氨基酸序列和生物活性方面享有很高同源性,其中FGF18与FGF17的同源性更高。为此,本研究根据FGF18和FGF17的蛋白结构域的构造及其与活性之间的关系,设计了3个新型FGF18类似分子(FGF18-3,FGF18-4,FGF18-5),并在大肠杆菌原核表达系统中成功诱导表达。经过包涵体的变性溶解、稀释复性及三步纯化得到纯度较高的目的蛋白。接下来,为研究这些新型FGF18类似分子对各种FGF受体(FGFR)的作用特异性和活性强度,我们构建了只表达单个FGFR的7种Baf3单克隆细胞系,包括FGFR1b/Baf3,FGFR1c/Baf3,FGFR2b/Baf3,FGFR2c/Baf3,FGFR3b/Baf3,FGFR3c/Baf3和FGFR4/Baf3细胞系。由于每种FGFR的细胞质段都采自FGFR1,因此当某种FGFR被FGF激活时,表达这种FGFR的Baf3细胞会快速增殖,从而反映该FGF分子对这种FGFR的活性。实验结果显示,3种新型FGF18类似分子均能刺激FGFR2c/Baf3,FGFR3c/Baf3和FGFR4/Baf3细胞增殖,而对FGFR1b/Baf3,FGFR1c/Baf3,FGFR2b/Baf3,FGFR3b/Baf3细胞则几乎没有活性,表明3种新型FGF18类似分子具有与野生型FGF18和FGF17相同的受体特异性。通过对半数有效剂量(EC50)进行仔细测定发现,相较于FGF18,FGF18-4对FGFR3c/Baf3细胞的EC50低了约4倍,而对FGFR2c/Baf3细胞的EC50高了3倍。这些结果表明,与FGF18相比,FGF18-4的促软骨增生的活性可能更高、而硬骨形成的作用会更低。为此,我们专注于FGF18-4的体内外活性检测,及其促进软骨再生能力的验证。在体外,1μg/mL FGF18-4显著提高了人关节软骨细胞的增殖,其促有丝分裂的效果与FGF18相近。此外,q PCR结果表明FGF18-4显著上调了蛋白多糖的成分Aggrecan和Ⅱ型胶原蛋白的表达,FGF18-4处理的人关节软骨细胞团番红-O着色较深,从而验证了FGF18-4促进软骨基质的合成和分泌的活性。为验证和比较FGF18-4在体内促进软骨修复的活性,我们用雄性SD大鼠构建了膝关节软骨划痕损伤模型。关节腔注射给药和切片番红-O染色结果显示,FGF18-4治疗后在划痕处再生出了成熟的透明软骨,且其疗效比FGF18有较大提高。综上所述,本研究设计并表达纯化得到了3种新型FGF18类似分子,经FGFR3c/Baf3细胞系筛选后,我们确定了FGF18-4为本研究的重点关注对象。活性研究结果显示,FGF18-4在体内外均展现出了显著促软骨再生作用。本研究为骨关节炎的治疗提供了一种新的候选药物分子。更多还原

【Abstract】 Osteoarthritis(OA)is a highly prevalent disease that severely affects human health,especially among the elderly,but currently there is no disease-modifying osteoarthritis drugs(DMOADs)available.Due to the lack of blood and lymphatic systems,the supply of nutrients and oxygen in cartilage tissue is limited,and waste and metabolic products cannot be cleared in a timely manner,making chondrocytes vulnerable.Once cartilage tissue,particularly articular cartilage,is damaged,its regenerative capacity is poor.In osteoarthritic cartilage tissue,the balance between catabolic and anabolic metabolism of chondrocytes is disrupted,leading to progressive degradation of cartilage matrix collagen and proteoglycans and chondrocyte apoptosis.Therefore,promoting chondrocyte regeneration and cartilage matrix synthesis is a hot topic in the field of osteoarthritis research.Fibroblast growth factor(FGF)family member FGF18 plays a significant role in bone and cartilage development and regeneration through its receptors FGFR3 c and FGFR2 c.FGF18-activated FGFR2 c promotes the formation of hard bone during long bone development,while FGF18-activated FGFR3 c promotes chondrocyte proliferation and cartilage formation.The dual roles of FGF18 in bone and cartilage development and formation has attracted researchers’ attention to its potential value in osteoarthritis.Therefore,recombinant human FGF18(Sprifermin)has been studied for the treatment of osteoarthritis and has shown significant chondrogenic activity in various animal models.Currently,Sprifermin research has progressed to phase III clinical trials,showing significant therapeutic effects in multiple clinical studies,such as structural improvement of articular cartilage and increased thickness of articular cartilage.However,its efficacy in relieving pain symptoms is limited,and its activity in promoting hard bone formation may cause formation of osteophyte.Therefore,this study aims to design and screen new FGF18-like molecules with higher activity in promoting cartilage proliferation and lower activity in hard bone formation,to promote the application of FGF18 drugs in the treatment of diseases such as osteoarthritis.FGF18,along with FGF8 and FGF17,forms a small FGF family,with high homology in amino acid sequence and biological activity,among which FGF18 and FGF17 have even higher homology.Therefore,based on the construction of protein structure domains of FGF18 and FGF17 and their relationship with activity,this study designed three new FGF18-like molecules(FGF18-3,FGF18-4,FGF18-5)and successfully induced their expression in the Escherichia coli prokaryotic expression system.High-purity target proteins were obtained through inclusion body denaturation,dilution refolding,and three-step purification.Next,to research the specificity and intensity of activity of these new FGF18-like molecules on various FGF receptors(FGFRs),we constructed seven Baf3 monoclonal cell lines that only express a single FGFR,including FGFR1b/Baf3,FGFR1c/Baf3,FGFR2b/Baf3,FGFR2c/Baf3,FGFR3b/Baf3,FGFR3c/Baf3 and FGFR4/Baf3 cell lines.Since the cytoplasmic segments of each FGFR were collected from FGFR1,when a certain FGFR is activated by FGF,Baf3 cells expressing this FGFR will proliferate rapidly,reflecting the activity of the FGF molecule on this FGFR.The experimental results showed that all three new FGF18-like molecules could stimulate the proliferation of FGFR2c/Baf3,FGFR3c/Baf3,and FGFR4/Baf3 cells,while they had almost no activity on FGFR1b/Baf3,FGFR1c/Baf3,FGFR2b/Baf3 and FGFR3b/Baf3 cells,indicating that the three new FGF18-like molecules have the same receptor specificity as wild-type FGF18 and FGF17.Careful determination of the half-maximal effective concentration(EC50)revealed that compared to FGF18,FGF18-4 had about four times lower EC50 for FGFR3c/Baf3 cells and three times higher EC50 for FGFR2c/Baf3 cells.These results suggest that FGF18-4 may have higher activity in promoting cartilage proliferation and lower activity in hard bone formation compared to FGF18.Therefore,we focused on the in vitro and in vivo activity testing of FGF18-4,as well as the validation of its ability to promote cartilage regeneration.In vitro,1 μg/m L FGF18-4 significantly increased the proliferation of human articular chondrocytes,with a mitogenic effect similar to that of FGF18.Additionally,qPCR results showed that FGF18-4 significantly upregulated the expression of proteoglycan components Aggrecan and type II collagen,and the Alcian blue-O staining of FGF18-4-treated human articular chondrocytes was darker,thereby verifying the activity of FGF18-4 in promoting the synthesis and secretion of cartilage matrix.To validate and compare the activity of FGF18-4 in promoting cartilage repair in vivo,we established a knee joint cartilage scratch injury model in male SD rats.Articular cavity injection and Alcian blue-O staining results showed that after FGF18-4 treatment,mature hyaline cartilage was regenerated at the scratch site,and its therapeutic effect was significantly improved compared to FGF18.In summary,this study designed and expressed three new FGF18-like molecules,and after screening with FGFR3c/Baf3 cell lines,we identified FGF18-4 as the focus of this study.Activity studies showed that FGF18-4 exhibited significant chondrogenic activity both in vitro and in vivo.This study provides a new candidate drug molecule for the treatment of osteoarthritis.更多还原