【作者】 徐俊昌；

【导师】 陈建庭；

【作者基本信息】 南方医科大学 ， 骨外科学， 2009， 博士

【摘要】 研究背景异位骨化(heterotopic ossification,HO)是指在正常情况下没有骨组织的软组织内形成的新生骨,它不同于高钙血症、营养不良等代谢性疾病所致的局部钙化,它形成的是成熟的板层新生骨。根据形成原因,HO可以分为四种类型:创伤后HO、神经源性HO、遗传性HO及烧伤、血友病、镰刀细胞型贫血、脊髓灰质炎、破伤风、多发硬化引起的HO,其中以创伤后HO最为常见。它常发生于肌肉骨骼创伤、关节脱位、髋、膝、肩、肘关节置换和骨折内固定等外科手术后。HO对骨科医生一直是个挑战,特别在创伤和关节置换领域,它可以导致疼痛、关节活动范围减小,甚至关节强直需要外科手术干预。然而,对HO的发病机制至今仍然缺乏足够的认识,Chalmers等提出了HO形成必需的三个条件:①成骨的前体细胞;②成骨诱导物;③允许成骨的组织环境,并认为HO的形成与否取决于局部和全身多种刺激成骨和抑制成骨因素相互作用的结果。诸多学者认为HO的形成起源于软组织内的多潜能干细胞,在特定的成骨诱导因子作用下分化为成骨细胞。这些因子有骨形态发生蛋白(BMPs)、转化生长因子(Transforming growth factor-β,TGF-β)、碱性成纤维生长因子(Basic fibroblast growth factor,bFGF)、胰岛素样生长因长(Insulin-like growthfactor,IGF)等,此外还有一些没有确定的骨调节生物活性因子。基于对肥胖基因的研究,1994年美国Howard Hughes医学研究所首次通过ob/ob小鼠基因定位克隆发现了ob基因表达产物——瘦素(Leptin,LEP)。LEP是一个由白色脂肪组织分泌、167个氨基酸组成的多肽,分子量为16kU,作为一个骨调节因子,LEP和其受体广泛存在机体各种组织中,暗示其可能参与了多种代谢的调节。它不仅是调节摄食和能量平衡的一个细胞因子,还具有活化巨噬细胞、调节炎症反应、维持内环境稳定、促进造血和血管生成、促进胶原合成、促进细胞修复、增强免疫功能,在创伤、应激、感染及组织修复过程中发挥一定的保护机制。临床上对LEP在肥胖症、心血管疾患、Ⅱ型糖尿病、肾脏病、乳腺癌、脂肪肝、不育症、骨质疏松症和骨关节炎发病机制中的作用研究较多,但LEP是否参与了HO的形成仍不清楚,对这方面的研究,国内外罕见报道。近年来,随着人工关节的广泛应用及骨折切开复位内固定术的普及,HO的发病率显著增高,其发病机制不明,缺乏理想的预防和治疗方法,致残率很高,因此,其病因和发病机制受到广泛关注,对这一课题进行广泛深入的研究将具有深远地意义。第一章Leptin在跟腱切断术诱导异位骨化模型大鼠骨化组织中的表达目的通过跟腱切断法建立大鼠HO模型,并初步研究其形成机制;测定模型大鼠血清LEP、骨碱性磷酸酶(bone alkaline phosphatase,BALP)水平,并分析二者之间的相关性;采用逆转录-聚合酶链反应(Reverse transcrip tion poly-merasechain reaction,RT-PCR)和免疫组化法分别检测大鼠跟腱和周围软组织中LEPmRNA和蛋白的表达情况。方法36只雄性Sprague-Dawley(SD)大鼠随机分为3组(n=12):单纯切开皮肤组(Sham)、跟腱半切组(Partial Achilles’tenotomy,PAT)、跟腱全切断组(Achilles’tenotomy,AT),于术后5、10周分别行跟腱X线和组织学检查,观察异位骨形成情况;采集术前和术后(5、10 w)的血液样品用于测定LEP和BALP;同时,采用RT-PCR和免疫组化法分别检测大鼠跟腱和周围软组织中LEP mRNA(5、10w)和蛋白(10w)表达情况。结果本实验通过跟腱切断法成功建立大鼠HO模型,Sham和PAT组5、10周均未发生HO;AT组:术后5周,3只大鼠X线平片可见异位骨,3只组织学检查发现有软骨形成;术后10周,6只大鼠均出现HO,这种异位骨具有骨小梁和骨髓结构,是通过软骨化骨形成。比较三组血清LEP和BALP水平,Sham组和PAT组在术前、术后5、10w 3个时间点差异无统计学意义(P＞0.05);AT组在术后5、10 w高于其它两组和术前(P＜0.05)。此外,术后血清LEP和BALP呈正相关(R=0.890,P＜0.05)。Sham组跟腱组织中无LEP mRNA和蛋白表达,在PAT组有弱表达,而在AT组则有较强的表达。结论跟腱切断术能有效诱导大鼠HO,该方法简单、有效、易行,结果稳定,这种异位骨是通过软骨化骨形成;HO模型大鼠血清LEP增加,且和BALP呈正相关,且在HO跟腱分离组织中有较强的LEP和LEP mRNA的表达。基于以上这些证据,我们认为LEP可能参与了HO的形成,其机制为:促进各类细胞的增殖/分化、胶原合成、细胞外基质的矿化和血管化来诱导骨形成和成熟。第二章Leptin对rhBMP-2诱导裸鼠异位成骨效能的影响目的研究LEP对基因重组骨形态发生蛋白-2(Recombinant human bonemorphogenetic protein-2,rhBMP-2)诱导裸鼠异位成骨效能的影响,探讨LEP和rhBMP-2对异位成骨的联合作用,并分析其机制,为临床应用提供理论依据。方法以胶原(Collagen,COL)材料作为载体,与不同的生长因子进行复合,根据复合生长因子的有无和不同分为A组:COL;B组:COL+LEP;C组:COL+rhBMP-2;D组:COL+rhBMP-2+LEP。在裸鼠(n=12)皮下分别植入这四种材料(采用自身左右配对设计,双侧腋窝、腹股沟),术后4、8周标本取材后采用放射学、骨密度(bone mineral density,BMD)、碱性磷酸活性测定(alkalinephosphatase,ALP)、组织学观察及成骨半定量分析评分等方法对各组成骨效能进行研究。结果术后4-8周,大体观察可见B、C、D组裸鼠材料植入区有半圆形包块形成,触之较韧,包块逐渐增大变硬,以D组最大;X线平片,A组裸鼠材料植入处无骨显影,B、C、D组均有显影,以D组的骨痂大而明显;术后4、8周,X线阻射值、BMD、ALP活性测定和成骨半定量分析评分,分组与时间交互效应统计值分别为F=5.637,P=0.003;F=1.835,P=0.156;F=4.447,P=0.009;F=3.386,P=0.047。同一时间点不同组间各检测值比较差异均有统计学意义(P均＜0.05),同一组不同时间点之间差异具有统计学意义(A组除外),8w水平高于4w(P＜0.05);组织学观察,A组可见部分未吸收材料,材料周围可见纤维组织增生,未见新骨形成;B、C、D组,可见部分未吸收材料和明显的骨、软骨形成,以D组新生骨组织最为成熟。结论以COL为载体复合rhBMP-2和LEP的材料具有高效骨诱导活性,LEP可明显增强rhBMP-2的骨诱导活性,其机制可能是通过促进新血管生成和多种细胞的增殖与分化。该材料具有控制释放、完全降解、生物相容性好等优点,随着研究的不断深入,在骨科领域有着广阔的应用前景。第三章Leptin对hBMSCs生物学行为干预的研究目的研究LEP对体外培养的人骨髓基质干细胞(human bone marrow stromalcells,hBMSCs)增殖与成骨分化的作用,筛选刺激增殖与成骨分化的最佳诱导浓度,并对机制进行初步探讨。方法抽取健康成人骨髓,采用全骨髓培养法,多次消化以纯化hBMSCs,取第三代细胞用于试验。采用流式细胞仪检测与干细胞相关的细胞表面标志物,确定细胞类型;同时在体外对细胞进行向成骨细胞、软骨细胞及脂肪细胞诱导分化,并分别以茜素红染色、阿利辛蓝染色及油红O染色进行定性鉴定。诱导增殖、成骨分化实验分为6组,待细胞贴壁后各组分别加入400 ng/ml LEP、200 ng/mlLEP、100 ng/ml LEP、50ng/ml LEP和(或)100ng/mL BMP(阳性对照)、普通培养基(Common nutrient medium,CNM,阴性对照)各2mL进行培养,于培养后第2、3、4、5、6d MTT法测定细胞增殖情况并绘制细胞增殖曲线,筛选LEP刺激增殖的最佳浓度;于培养第7d行ALP染色、21d行矿化结节茜素红染色,倒置相差显微镜观察各组染色结果;于培养后7、14、2ld,检测各组ALP活性、骨钙素(osteocalcin,OCN)水平,筛选LEP促成骨分化的最佳浓度;200ng/mLLEP、100ng/mL BMP和CNM组细胞培养7d后,采用RT-PCR方法检测成骨相关基因:核心结合因子(Core-binding factorα1,Cbfα1)、ALP、Ⅰ型胶原(CollagenⅠ,COLⅡ)、OCN mRNA的表达情况。结果培养的第三代细胞表面抗原检测显示:表达CD105、CD166和CD29,不表达造血和内皮标志CD34、CD45和CD14。在体外分别诱导出其向成骨、成软骨和成脂肪细胞分化。各组hBMSCs细胞增殖情况,分组与时间之间存存交互效应(F=46.983,P=0.000),同一组内不同时间点间OD值比较差异均有统计学意义(P均＜0.05),除在第2d各组间OD值比较差异无统计学意义(P＞0.05)外,其余各时间点各组间OD值比较差异均有统计学意义(P＜0.05)。hMSCs细胞生长曲线显示各浓度LEP组细胞增殖较快,生长曲线呈“S”形,细胞接种经过约3d的潜伏期后进入对数生长期,在第5d进入平台期,与CNM组比较差异具有统计学意义(P＜0.05),以200ng/mL LEP组最为显著。诱导培养7d后ALP染色显示,400 ng/ml LEP组,200 ng/ml LEP组,100 ng/ml LEP组,50ng/ml LEP组和100ng/mL BMP组细胞胞浆均呈蓝色阳性着色,CNM组呈弱阳性。诱导培养21d后矿化结节茜素红染色结果显示,400 ng/ml LEP组,200 ng/ml LEP组,100 ng/ml LEP组,50ng/ml LEP组和100ng/mL BMP组细胞染色阳性,CNM组呈阴性。诱导培养后第7、14、21天,200ng/mL LEP组ALP表达量与其他各组比较,差异均有统计学意义(P＜0.05)。诱导培养后第7、14天,200ng/mL LEP组OCN表达量与400ng/mL、100ng/mL、50ng/mL LEP组比较,差异无统计学意义(P＞0.05),但高于CNM组,低于100ng/mL BMP组(P＜0.05);诱导培养后第2l天,200ng/mL LEP组OCN表达量与其他各组比较,差异均有统计学意义(P＜0.05),200 ng/mL LEP为最佳诱导增殖、分化浓度。200ng/ml LEP组和100ng/mL BMP组和CNM组细胞培养7d后,CNM组Cbfα1、ALP、COLⅠ、OCN mRNA均不表达,200ng/ml LEP组和100ng/mLBMP组各产物mRNA均有表达,但200ng/ml LEP组表达量低于100ng/mLBMP组。结论我们采用全骨髓培养法成功地从健康成人骨髓中分离、培养出hBMSCs,本实验成功地建立了一整套有关hBMSCs分离、培养和鉴定的较简便的方法,在体外经分离传代培养后细胞成分较为单一,具有多向分化潜能,符合间充质干细胞的基本功能特征;LEP可促进hBMSCs的增殖和成骨分化,以200ng/ml为最佳诱导浓度。其促hBMSCs成骨分化的机制为促进了成骨相关基因Cbfα1、ALP、COLⅠ、OCN mRNA的表达。更多还原

【Abstract】 BackgroundHO is characterized as formation of bone in tissues other than skeletal system, which is usually seen in the cases of musculoskeletal trauma,spinal cord or central nervous system damage,joint dislocations and surgical procedures such as hip,knee, shoulder,or elbow arthroplasty and internal fixation after fractures.HO continues to pose a substantial challenge to the orthopedic surgeon,particularly in the fields of trauma and arthroplasty,it may cause pain,decrease motion;in severe cases, complete joint ankylosis could happen whereby surgical intervention follows. However,the exact etiology of HO remains unclearly,it is complicate with multiple etiologies.It requires osteoinductive factors,inducible osteoprogenitors,and a favorable microenvironment.The process of HO may include appropriate differentiation of pluripotent mesenchymal cells that generally exist in soft tissues into bone-forming cells under the influence of various osteoinductive factors. Numerous previous studies concerning inductive factors have been performed to understand HO etiology,which concentrated in bone morphogenetic proteins(BMPs), transforming growth factor-beta(TGF-β),basic fibroblast growth factor(bFGF), insulin-like growth factor(IGF)and so on,there are still some signaling factors thought to be involved.LEP is a polypeptide hormone comprised of 167 amino acids and secreted primarily by white adipose tissue.It is a main hormone not only regulating the steady state of energy,but also has such physiological functions as activating macrophage,regulating inflammatory,maintaining homeostasis,promoting homeostasis and angiogenesis,promoting cytothesis,enhancing immunological function,which have protective effect in trauma,stress,infection and reparative progress of tissue.LEP and its receptor have been revealed a bone-conditioning factor, spread widely in a variety of body tissue,suggesting that LEP may be involved in many aspects of metabolic regulation.Recently,the role of LEP is worth more attention.To our knowledge,current research have focused on LEP in the pathogenesis of osteoporosis and osteoarthritis,and whether LEP involved in the formation of HO is yet to know.Research in this area is rarely reported both at home and abroad.Accompanying with the increasing in open reduction and internal fixation of fracture,the incidence of HO gradually increased,therefore,systemic further researches on it may be of great importance.Part oneExpression of Leptin in heterotopic ossification-isolated tissue of rats with Achilles’ tenotomyObjective(1)To explore the way for estimating animal model of HO induced by Achilles’ tenotomy on Sprague-Dawley(SD)rats and to investigate the possible mechanism for its formation;(2)To detect the changes of serum LEP levels in model rats;(3)To determine whether LEP mRNA and protein are expressed in HO-isolated tissue.MethodsAll animal’s procedures were performed in accordance with the guidelines of the Southern Medical University Animal Care and Use Committee.36 SD male rats were divided into 3 groups(n=12).In sham group,incision and suturation were merely performed on the left leg;Partial tenotomy were performed on the left Achilles tendons in PAT group;In AT group,tenotomy were performed on the left Achilles tendons to build up animal model of HO.X-ray and histological examination were made at 5,10 weeks after operation respectively.Preoperative and post-operative (5,10 w)bloody sample were harvested for assaying LEP,Meanwhile,the expression of LEP mRNA(5,10 w)and protein(10 w)in Achilles tendons and the surrounding tissue were examined respectively by using RT-PCR and immunohistochemical methods.ResultsIn this study,we have successfully established HO model through Achilles’ tenotomy,no HO occurred in the Sham and PAT groups at 5,10 weeks after operation;In AT group,3 rats showed cartilage and bone formation on X-rays while the remaining 3 rats showed chondrification in histological examination at 5 weeks after operation;At 10 weeks,all specimens of AT group presented cartilage and bone formation with trabecular bone,this HO occured through a process of endochondral ossification.Compared with serum LEP and bone alkaline phosphatase(BALP) levels in 3 groups,there was no significant difference between sham and PAT on 3 time points(P＞0.05),those of AT on weeks 5,10 were significantly higher than those in others and pre-operative(P＜0.05).Furthermore,positive correlations were found between post-operative LEP and BALP(R=0.890,P＜.05);There were no LEP mRNA and protein expression in sham and a moderate expression in PAT of Achilles tendons and surrounding tissue,whereas there was a strong expression in AT.ConclusionsTenotomy is a simple,effective and feasible method to induce HO,and this heterotopic ossification occured through a process of endochondral ossification.The serum LEP levels in models markedly increased,positive correlations were found between LEP and BALP.Furthermore,there were strong expression of LEP and its mRNA in HO-isolated tissue.Therefore,based on these evidences,we conclude that LEP at least partly plays a role in the formation of HO,its action mechanisms are related to induce bone formation and maturation through a series of cellular events, including:proliferation/differentiation of many kinds of cells,collagen synthesis, mineralization and vascularization of the extracellular matrix. Part twoExperimental investigations for the effect of Leptin on the ectopic osteogenesis induced by rhBMP-2 in nude miceObjectiveTo study the effect and mechanism of LEP on the ectopic osteogenesis induced by rhBMP-2 with collagen as a carrier in nude mice,so as to provide the theoretical basis for clinical application in the future.MethodsThe composite of material(group A:COL,group B:COL+LEP,group C:COL +rhBMP-2,group D:COL+rhBMP-2+LEP)were subcutaneouly implanted into the back of nude mice(n=12)respectively.The effect of ectopic bone formation was evaluated by radiography,dual-energyⅩabsorptionmetry(DEXA),biochemical examination of alkaline phosphatase(ALP),histological observation and semiquantitative evaluation in 4 and 8 weeks after operation.ResultsMacroscopical observation at 4-8 weeks after operation,there are semi-circular masses in the operative areas in groups B,C,and D.The masses are tenacious, increasing and stiffening gradually,the masses in group D are the biggest by macroscopical observation and radiographs.At 4,8 weeks after operation,there were significant difference between all groups of the X-ray’ s score,BMD,the activity of ALP,histological semi-quantitative evaluation in same timepoint(P＜0.05);There were significant difference between in each group at different timepoint(except group A),those at 8 w significantly higher than those at 4w(P＜0.05).In histology, COL were partly absorbed and no bone and cartilage formation were observed at 4,8 weeks postoperatively in groups A;however,COL were mostly absorbed,and a large quantity of cartilages and bones formed in groups B,C and D,especially,the new bony tissue in group D were maturest.ConclusionsThe sustainedly released material with COL as a carrier compounded with rhBMP-2 and LEP has a very good osteoinductive activity,and LEP can enhance the activity of rhBMP-2 obviously in inducing bone formation.Its mechanisms are probably related to promote the formation of new-vessels and proliferation/ differentiation of many kinds of cells.It has broad prospect in tissue engineering for the advantages of controlled release,completely degradable and the good biological compatibility.Part threeThe study of intervention by Leptin on biological behaviour with human bone marrow stromal cellsObjectiveTo study the effect and mechanism of LEP on promoting proliferation and osteoblastic differentiation of hBMSCs in vitro,and so as to screen the best concentration of LEP on proliferation and osteoblastic differentiation.Methods:Bone marrow was aspirated from the healthy adult human and cultured -expanded in vitro by the method of total marrow.The hBMSCs were purified by discarding suspended cells through exchanging medium.For identifying hBMSCs, cells surface antigens,including CD105,CD166,CD29,CD34,CD45 and CD14 were detected by flow cytometry,and hBMSCs were induced with the special inducer, then its trilineage differential potential into osteoblast,chondrocyte and adipocyte were observed by using Alizarin red staining,Alcian staining and Oil Red O staining under microscope.The third passage cells were used for next experiment.The hBMSCs were cultured in vitro,after treated with 400 ng/ml LEP,200 ng/ml LEP, 100 ng/ml LEP,50ng/ml LEP,100 ng/ml BMP and common nutrient medium(CNM) respectively,MTT were assayed at day 2,3,4,5 and 6 after being treated to establish a growth curve of cells cultivated,and so as to screen the optimal concentration of LEP on promoting proliferation;Staining of ALP(NBT/BCIP)at 7 days and mineralized nodules(chinalizarin)at 21 days in hBMSCs were performed respectively.Meanwhile,ALP and OCN activitives of hBMSCs in supernatant was determined at 7,14 and 21 days respectively to ensure the best concentration of LEP on osteoblastic differentiation.After been cultured 7 days by common nutrient medium,200 ng/ml LEP and 100 ng/ml BMP medium,the mRNA level of osteoblastic related gene(cbfα1,ALP,COL I,OCN)in hBMSCs were evaluated by RT-PCR.ResultsThe result of flow cytometry showed that the third passage hBMSCs highly expressed CD105,CD166 and CD29,and were negative for any hematopoietic and endothelial markers(CD34,CD45 and CD14).Functionally,they could differentiate into the osteoblast,adipocyte and chondrocyte by induced respectively with the special inducer.The cell proliferation curves of hBMSCs showed that the cells in different groups of LEP multiplied rapidly and entered the logarithmic phase after 3 days latent phase,then entered the plateau at 5 days,and there were significance compared to the CNM group(P＜0.05),the group of 200ng/mL LEP is the most significant.Positive staining of ALP in groups of 400ng/mL LEP,200ng/mL LEP, l00ng/mL LEP,50ng/mL LEP,l00ng/mL BMP and lower levels of positive staining in group of CNM were found at 7 days;The red-stained mineralized nodules in groups of 400ng/mL LEP,200ng/mL LEP,l00ng/mL LEP,50ng/mL LEP, l00ng/mL BMP and negative staining in group of CNM were observed at 21 days; Compared with the ALP and OCN activity of hBMSCs cells in 6 groups,its in group of 200ng/mL LEP were lower significantly than those in group of l00ng/mL BMP (P＜0.05),but significant higer than those in others at 7,14 and 21 days(P＜0.05), therefore,the optimal concentrations was 200 ng/ml LEP.At 7 days after culture, group CNM witnessed no expression of Cbfal,ALP,Col I and OCN mRNA,while group l00ng/mL LEP witnessed expression of all those indexes,but was less than that of group l00ng/mL BMP.Conclusion We successfully isolated and cultured the hBMSCs from the bone marrow of healthy adult human in vitro by the method of total marrow.And then we established a set of convenient methods about isolation,culture and identification for the hBMSCs.The cultured cells have only composed of undifferentiated hBMSC and great potentiality of proliferation and osteogenic,chondrogenic and adipogenic differentiation in vitro.So they have the properties of stem cells.Our results demonstrate that LEP can stimulats proliferation and osteoblastic differentiation of hBMSCs in vitro,and 200ng/ml LEP is the optimal concentrations.LEP leads to the high expression of osteoblastic related gene(Cbfal,ALP,COL I,OCN mRNA)in hBMSCs,which may be parts of the mechanisms underlying the anabolic osteogenetic effect of LEP.更多还原