【作者】 钟招明；

【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】 研究背景黄韧带骨化（Ossification of ligamentum flavum,OLF）是一种发生在脊柱韧带的特殊类型异位骨化形式。OLF主要累及亚洲黄种人,尤其是日本人。男女发病率文献报道不一,约为2-4:1,随年龄增长而发病率增高,年龄大于65岁的亚洲人群发病率高达20%。OLF可发生在颈,胸和腰椎,但好发生于下胸椎和胸腰段,导致椎管狭窄,常引起脊髓压迫受损,表现出一系列的神经功能障碍。目前,OLF的临床治疗局限于椎管减压手术,但是其诊断延误、OLF的多发性和术后骨化复发是造成患者脊髓功能恢复差的重要原因。近年来,OLF逐渐引起国内外学术界的关注与重视。国内、外文献有关OLF病因学研究的报道逐渐增加,虽然目前认为遗传因素、生长因子、力学因素、内分泌与代谢异常等因素与OLF发病密切相关。但其具体发病机制仍不清楚。组织学研究证实骨化黄韧带组织表现为韧带肥厚,弹力纤维减少,胶原纤维大量增生、肿胀,在骨化与非骨化韧带组织间的交界区（骨化移行区）可见大量的软骨样细胞。因此,这提示OLF属于异位软骨内成骨病变。免疫组织化学研究发现某些成骨诱导因子（如骨形态发生蛋白（BMPs）和转化生长因子（TGF）-β）和骨形态发生蛋白受体（BMPRs）在骨化移行区的未成熟的软骨样细胞中阳性表达。体内、外实验进一步证实BMP-2诱导OLF形成的作用。提示这些成骨诱导因子可通过旁分泌或自分泌的形式与黄韧带细胞胞膜上的受体结合并刺激细胞成骨分化,在黄韧带的软骨内骨化过程发挥重要作用。我们的前期研究表明体外分离培养的下胸椎黄韧带骨化患者非骨化区域的黄韧带细胞呈现典型的成骨细胞表型特征。近年来,体外研究表明牵张应力和瘦素等致病因素可诱导人黄韧带细胞向成骨细胞分化。因此,各种致病因素诱导黄韧带细胞向成骨细胞分化可能是OLF发病的细胞学基础。有丝分裂原活化蛋白激酶（MAPK）家族成员由多种同功酶组成,其中ERK1/2,p38和JNK发现较早,生物学功能研究较为透彻。MAPK信号转导途径是细胞外信号引起细胞核内反应的通道之一,可被多种刺激因素激活,通过三级酶促级联反应,最终磷酸化靶蛋白,激活转录因子,调节特定的基因表达,参与细胞的增殖、分化及凋亡等多种生理过程。许多研究证实MAPK信号通路参与成骨细胞增殖分化过程。最近,体外研究表明牵张应力和瘦素通过激活MAPK信号通路诱导黄韧带细胞向成骨细胞分化。因此,MAPK信号通路在OLF的发病过程中可能发挥重要作用。生长分化因子-5（GDF-5）又称为BMP-14或软骨衍生形态发生蛋白-1（CDMP-1）,是TGF-β超家族中的新成员。其编码基因定位于20q11.2染色体,由两个外显子（exon）和一内含子（intron）组成,全长488 kb,其编码多肽共含有501个氨基酸,由位于N端的信号肽、中间的前体肽以及C端的成熟肽构成。翻译后的前体蛋白裂解得到成熟肽,由其发挥生物活性作用。成熟的GDF-5二聚体分子量为25KD,而单体分子为13.6KD。不同种属来源的GDF-5分子具有高度同源性。它与骨软骨发育,肌腱/韧带损伤修复、神经营养等密切相关。GDF-5像其它TGF-β超家族的成员（如BMPs）一样,以同源二聚体的形式与跨膜Ser-Thr激酶受体结合,通过胞浆内Smad信号途径和非Smad信号途径（如MAPK）将其信号传导到核内,调节特定基因的表达。最近,MAPK信号通路在GDF-5生物效应中的作用引起一些学者的重视。体外研究证实GDF-5可诱导人脐静脉平滑肌细胞的ERK磷酸化和小鼠软骨细胞系ATDC5的p38和ERK磷酸化。体内研究表明人重组GDF-5（rhGDF-5）可促进骨缺损修复和脊柱后外侧融合;将含rhGDF-5的基质植入大鼠皮下或肌肉内,可诱导异位骨形成。体外研究报道GDF-5能诱导多能间充质细胞C2C12、脂肪干细胞、骨髓基质细胞和骨膜细胞等多种细胞向成骨细胞分化。因此,GDF-5可诱导多种细胞向成骨细胞分化和异位骨形成。组织病理学研究表明GDF-5在OLF的黄韧带组织中呈阳性表达,而在无骨化的对照样本中无表达。这一研究结果提示GDF-5可能是OLF的主要致病因子之一,但其具体机制不清楚。因此,研究GDF-5在OLF发病过程中分子机制,以其作为切入点进一步阐明OLF的发病机制,将为临床OLF的预防、早期干预与治疗提供新思路。目的1.探讨人黄韧带细胞的分离与体外培养方法,建立黄韧带细胞系。2.探讨rhGDF-5诱导人黄韧带细胞成骨分化的作用。3.探讨rhGDF-5对人黄韧带细胞的MAPK信号通路激活的作用,并观察信号阻断剂对rhGDF-5诱导黄韧带细胞成骨分化的影响。方法1.人黄韧带细胞的分离与培养采集胸腰椎骨折患者后路减压术中和腰椎间盘突出症患者后路开窗髓核摘除术中的黄韧带标本。黄韧带标本用PBS液清洗后,剪成大小约0.5 mm的碎块,并用0.2%Ⅰ型胶原酶消化液消化90 min。将胶原酶预消化组织块均匀贴附在6孔细胞培养板或10 ml培养皿内,置于37℃、5%CO₂、饱和湿度的孵箱中静置8 h后,沿孔板侧壁小心加入含10%胎牛血清（FBS）的DMEM培养液继续培养。培养5-7d后首次更换培养液,随后每隔3天更换。原代细胞从组织块迁出、生长至80%融合时,用含0.25%胰蛋白酶和0.02%EDTA的细胞消化液消化并传代培养。倒置相差显微镜下观察细胞从组织块迁出时间,原代和传代细胞的形念和生长状态。四甲基偶氮唑盐（MTT）比色法分析传1、3、5代细胞（P1、3、5）的增殖特性,绘制细胞生长曲线,计算细胞群体倍增时间,并比较细胞传5代以内的生物学特性的变化。对传3代细胞进行波形蛋白和Ⅰ型胶原的免疫荧光化学染色。2.rhGDF-5诱导人黄韧带细胞成骨分化的作用（1）黄韧带细胞碱性磷酸酶（ALP）活性检测人黄韧带细胞与100ng/ml rhGDF-5分别共孵育0,4,7,10,14 d或分别与0,10,50,100,500ng/ml rhGDF-5共孵育10 d后,收集细胞,提取细胞总蛋白,检测ALP活性。（2）黄韧带细胞骨钙素mRNA表达的检测人黄韧带细胞与100ng/ml rhGDF-5分别共孵育0,4,7,10,14 d或分别与0,10,50,100,500ng/ml rhGDF-5共孵育10 d后,收集细胞,提取细胞总RNA,反转录多聚酶链式反应（RT-PCR）检测黄韧带细胞的骨钙素mRNA农达,以β-acin为内对照,分析骨钙素mRNA相对表达的变化。（3）黄韧带细胞骨钙素蛋白表达的检测人黄韧带细胞与100ng/ml rhGDF-5分别共孵育0,4,7,10,14d或分别与0,10,50,100,500ng/ml rhGDF-5共孵育10d后,收集细胞,提取细胞总蛋白,免疫印迹法（Western blot）检测黄韧带细胞的骨钙素蛋白表达,以β-acin为内对照,分析骨钙素蛋白相对表达的变化。（4）黄韧带细胞骨钙素定位表达的检测人黄韧带细胞接种于置有盖玻片的6孔细胞培养板内,加入含10.0%FBS的DMEM培养基培养。细胞生长达80%融合后,加入100ng/ml rhGDF-5继续培养10d。免疫荧光化学染色检测骨钙素的定位表达,细胞爬片依次用1:100稀释的山羊抗骨钙素多克隆抗体和1:50稀释的FITC荧光标记的兔抗山羊IgG孵育,共聚焦显微镜下观察。（5）茜素红矿化结节染色人黄韧带细胞接种于6孔细胞培养板内,加入含10.0%FBS的DMEM培养。细胞达融合后,加入100ng/ml rhGDF-5或者对照培养基继续培养28d。茜素红染色检测矿化结节形成。3.rhGDF-5诱导人黄韧带细胞成骨分化的信号转导机制（1）MAPKs磷酸化活性的检测人黄韧带细胞与100 ng/ml rhGDF-5分别共孵育0、5、15、30、60、120、240min后收集细胞;在阻断实验中,黄韧带细胞与20gM的信号阻断剂U0126、SB203580或SP600125预孵育60min,将含阻断剂的培养基吸出,无血清培养基清洗一次。再加入含100ng/ml rhGDF-5的培养基,60分钟后收集细胞,提取细胞总蛋白。Western blot检测MAPK（ERK1/2、p38、JNK）总蛋白及磷酸化蛋白含量,以总蛋白为对照,分析磷酸化蛋白含量相对值的变化。（2）MAPKs信号阻断剂对rhGDF-5诱导人黄韧带细胞成骨分化的影响人黄韧带细胞与20μM U0126、SB203580或SP600125和100ng/ml rhGDF-5共孵育10d后收集细胞,提取细胞总蛋白,检测ALP活性,Western blot检测骨钙素蛋白表达。结果1.人黄韧带细胞的分离与培养（1）倒置相差显微镜观察在培养第10-14d后,部分黄韧带组织块周围开始有细胞迁出,细胞呈多种形态,主要为梭形和多角形,细胞接近融合时,呈放射状生长;传代细胞主要呈长梭形,少许呈扇形或多角形,接近融合时呈涡流状生长;连续传代培养至第5代,细胞仍保持良好细胞形态。（2）传代细胞增值、生长曲线和细胞群体倍增时间的变化传代细胞P1、3、5按2.0×10³/ml的密度接种后,生长迅速,0-3d为生长潜伏期;3-5d,细胞增生极为活跃,呈指数递增,为对数增生期;此后细胞增长逐渐减缓,进入平台期,细胞生长曲线呈S形。MTT测量P1、3、5代细胞增值无统计学差异（F＝0.188,P＝0.831）;细胞生长曲线呈S形;细胞群体倍增时间无统计学差异（F＝0.405,P＝0.675）。（3）波形蛋白和Ⅰ型胶原的免疫荧光染色所有细胞的波形蛋白和Ⅰ型胶原免疫荧光化学染色均呈阳性。2.rhGDF-5诱导人黄韧带细胞向成骨细胞分化的影响（1）rhGDF-5诱导黄韧带细胞增加ALP活性100ng/ml rhGDF-5与人黄韧带细胞共孵育0,4,7,10,14d后,细胞ALP活性随干预时间延长而升高,呈时间依赖效应（F＝273.905,P＝0.002）。同时,rhGDF-5又以剂量依赖的方式增加黄韧带细胞的ALP活性（F＝64.091,P＝0.004）。（2）rhGDF-5诱导黄韧带细胞表达骨钙素mRNA100ng/ml rhGDF-5与人黄韧带细胞共孵育0,4,7,10,14d后,细胞骨钙素mRNA表达随干预时间延长而升高,呈时间依赖效应（F＝46.573,P＝0.018）。同时,rhGDF-5又以剂量依赖的方式增加黄韧带细胞的骨钙素mRNA表达（F＝70.300,P＝0.002）。（3）rhGDF-5诱导黄韧带细胞表达骨钙素蛋白100ng/ml rhGDF-5与人黄韧带细胞共孵育0,4,7,10,14d后,细胞骨钙素蛋白表达随干预时间的延长而升高,呈时间依赖效应（F＝287.754,P＝0.001）。同时,rhGDF-5又以剂量依赖的方式增加黄韧带细胞的骨钙素蛋白表达（F＝115.087,P＝0.004）。人黄韧带细胞与100 ng/ml rhGDF-5共培养10d,细胞免疫荧光化学染色可见黄韧带细胞胞质中均有骨钙素表达,而对照组细胞未见骨钙素表达。（4）rhGDF-5诱导黄韧带细胞矿化结节形成融合黄韧带细胞与100ng/ml rhGDF-5共培养28d。茜素红染色结果表明rhGDF-5组可见橘红色钙化结节,而对照组未见明显矿化结节形成。3.rhGDF-5激活人黄韧带细胞的ERK1/2和p38信号通路,阻断剂U0126和SB203580抑制rhGDF-5诱导的成骨分化效应（1）rhGDF-5对人黄韧带细胞的ERK1/2、p38、JNK磷酸化的影响黄韧带细胞与100ng/ml rhGDF-5共培养0、5、15、30、60、120、240min,rhGDF-5以时间依赖的方式增强ERK1/2和p38磷酸化活性（F＝75.940,P＝0.008;F＝19.537,P＝0.030）,而JNK磷酸化活性在各时间点无统计学差异（F＝0.250,P＝0.688）。ERK1/2特异性阻断剂U0126（20gM）和p38特异性阻断剂SB203580（20gM）可分别抑制ERK1/2和p38磷酸化。（2）ERK1/2阻断剂U0126和p38阻断剂SB203580对rhGDF-5的诱导人黄韧带细胞向成骨细胞分化效应的影响黄韧带细胞与100ng/ml rhGDF-5和U0126或SB203580共培养10d,U0126和SB203580可明显抑制rhGDF-5诱导的ALP活性增加（t＝14.470,P＝0.001;t＝20.430,P＝0.001）和骨钙素蛋白表达（t＝3.431,P＝0.027;t＝3.559,P＝0.024）。结论1.胶原酶预消化组织块培养法能成功分离人黄韧带细胞。原代培养的黄韧带细胞主要呈纤维细胞样表型,在细胞传5代以内,其生物学特性稳定。2.GDF-5体外诱导人黄韧带细胞增加ALP活性、表达骨钙素、形成矿化结节,通过激活细胞内ERK1/2、p38 MAPK信号途径诱导人黄韧带细胞成骨分化。更多还原

【Abstract】 BackgroudOssification of ligamentum flavum（OLF） is characterized by a heterotopic bone formation in the spinal ligamentum flavum that is normally composed of fibrous tissues.It has been reported more often in Asian populations,particularly in Japanese subjects.The incidence rate is as high as 20%in Asian populations older than 65 years and the ratio of male to female subjects reported in the literature varies about 2-4:1.OLF can be seen in the cervical,thoracic and lumbar region,but it occur significantly more frequently in the lower thoracic region or the thoracic-lumbar junction.The ossified ligament protrudes into the spinal canal and compresses the spinal cord,resulting in serious neurological damages.Therefore,OLF is now appreciated as an important cause of thoracic myeloradiculopathy.Although en bloc laminectomy has been commonly used for treating the condition,the outcome of surgical treatment of thoracic OLF is not always satisfactory.Moreover,the delays of diagnosis,multilevel ossification and postoperative recurrence of ossification in the adjacent segment have a negative effect on functional recovery of the compressed spinal cord.There has been growing concern regarding OLF over the past two decades.Although numerous studies showed that systemic and local factors, including the genetic factors,mechanical stress,growth factors,cytokines,and the endocrine/metabolic abnormalities,were responsible for the pathogenesis of OLK its precise pathogenesis has not been conclusively established.Histological studies in the OLF samples demonstrated that the ossific ligamentum flavum showed loss of elastic fibers and increase of collagen fibers,and the numerous fibrocartilaginous cells were observed within and around the ossification fronts,which supported this theory that the developmental mode of OLF was endochondral ossification.Immunohistochemical studies have also documented that osteogenesis cytokines,such as bone morphogenetic proteins（BMPs）, transforming growth factor（TGF）-β,and the receptors of BMP（BMPRs） were localized around the ossification front.These causative factors could play a role in initiation of cellular events that ligamentum flavum（HLF） cells differentiate into chondrocytes and osteoblasts,which further supported by the subsequent studies.In our previous study,the OLF cells,which were isolated from the ligamentum flavum tissues from OLF patients during surgery,showed phenotypic characterization of osteoblasts.In the past decade,several in vitro studies showed that some factors,such as mechanical stress and leptin,induces osteogenic differentiation in HLF cells. Therefore,the osteogenic differentiation of HLF cells may be is a key event in the genesis and development of OLF.Mitogen-activated protein kinases（MAPK）,including a variety of isoenzyme, are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment..The ERK1/2,p38 and JNK of them were found earlier, and their biological functions were extensively investigated.They regulate diverse processes ranging from proliferation and differentiation to apoptosis.Increasing evidence showed that the MAPK pathways are involved in the proliferation and differentiation of osteoblast.It was recently demonstrated that mechanical stress and leptin which are known as the cause of OLF,induced osteogenic differentiation in HLF cells through the activation of MAPK pathways.Thus,the activation of MAPK pathways may be associated with the genesis and development of OLF.Growth/differentiation factor-5（GDF-5）,known as BMP-14 or cartilage-derived morphogenetic protein-1（CDMP-1）,is a new member of the TGF-beta superfamily, and is also a subfamily of the highly conserved group of bone morphogenetic protein （BMP） signaling molecules.GDF-5 gene,a 488 kb fragment,locates on chromosome 20q11.2,and consists of only two coding exons and one intron.It encodes polypeptide with a total of 501 amino acids,consists of the N-terminus signal peptides,the precursor peptide and C-terminal mature peptides.The post-translational precursor protein split into mature peptides,which play a role in biological activity. The molecular weight of mature GDF-5 dimer is of 25KD,and the monomer molecules weight is of 13.6KD.GDF-5 is known to play crucial roles in skeletal, tendon and ligament morphogenesis.Like other members of the TGF-beta superfamily,GDF-5 transduces its effects through binding to serine/threonine kinase receptors,then activates Smad-dependent and Smad-independent signaling and modulates gene transcription in the nucleus. Recently,some researchers have considerable concerns on the effect of MAPK signaling pathway in the biological activation of GDF-5.in vitro studies have showed that GDF-5 could induce the phosphorylation of ERK in human umbilical vein smooth muscle cells,and induce the phosphorylation of ERK and p38 MAPK in a mouse chondrogenic cell line,ATDC5.The in vivo studies have been demonstrated that GDF-5 promotes the closure of osteochondral defects in a minipig model by enchondral ossification,and induces posterolateral lumbar fusion in a New Zealand white rabbit model.Other studies also showed that the matrices loaded with rhGDF-5 induced ectopic cartilaginous and osseous tissue when implanted in subcutaneous or intramuscular site of rats.Several studies have been demonstrated that it could induce in vitro the osteogenic differentiation in many cell types,such as mesenchymal cell C2C12,fat-derived stromal cells,marrow mesenchymal stem cells and periosteum-derived cells.Histopathological study found that GDF-5 was detected in spindle-shaped cells and chondrocytes in the OLF tissues,but no in the cells in non-ossified sites.The results suggested that GDF-5 may be involved in the progression of OLF.However,it precise pathogenesis is still unclear.Therefore,the identification of the molecular mechanisms of GDF-5-induced OLF has implications for clarifying further the pathogenesis of OLF,and may offer some new information in the prevention,early intervention and treatment of this disease.Objectives1.To explore the method of the isolation and culture of HLF cells in vitro,and establish this cell lines.2.To investigate whether GDF-5 induces osteogenic differentiation in HLF cells.3.To investigate the effect of GDF-5 on the phosphorylation of MAPKs,and investigate the effect of MAPK inhibitors on GDF-5-induced osteogenic differentiation in HLF cells.Methods1.Isolation and culture of HLF cells in vitro.Tissue samples of ligamentum flavum were harvested during surgery from patients with thoracolumbar burst fractures or from patients with lumbar disc herniation.The samples were washed with phosphate-buffered saline（PBS）,after which any surrounding tissues attached to the specimen were carefully removed.The collected ligaments were minced into pieces of approximately 0.5 mm³ and washed twice with PBS.The minced tissue was digested at 37℃for 90 min with 0.2% collagenase type I in serumless containing DMEM.Collagenase-treated ligament chips were washed with DMEM and then placed in 6-well plates or 10 ml dishes in DMEM supplemented with 10%fetal bovine serum（FBS）,100 U/ml penicillin and 100 pg/ml streptomycin,and incubated in a humidified atmosphere of 95%air and 5%CO₂ at 37℃.The medium was firstly changed at day 5-7 after explant culture and then replaced at three-day intervals.The outgrown cells grown to confluence were subcultured using trypsinization with 0.2%trypsin/0.02%ethylenediamine tetraacetic acid（EDTA）.Under an inverted phase microscope,the cultures were examined for cell outgrowth and Cell morphology and growth status.MTT colorimetric method was used to analysis the proliferation of HLF cells at the first,third and fifth passage.Cell growth curve was then drawed and cell population doubling time was calculated.We compared the biological characteristics of the HLF cells within 5 generation. Immunofluorescence staining was used to detect the expression of vimentin and collagen type I.2.To investigate whether GDF-5 induces osteogenic differentiation in HLF cells（1） Alkaline phosphatase（ALP） activity assayThe OLF cells were exposed to 100ng/mlrhGDF-5 for 0,4,7,10,14 days or 0,10, 50,100,500ng/ml rhGDF-5 for 10 days,respectively.Cells were collected and their total protein was then prepared with RIPA lysis buffer.The ALP activity was examined using the commercial reagents.（2） Determination of the expression of osteocalcin mRNA in HLF cellsThe OLF cells were exposed to 100ng/mlrhGDF-5 for 0,4,7,10,14 days or 0, 10,50,100,500ng/ml rhGDF-5 for 10 days,respectively.Cells were collected and Total RNA was extracted using the TRI Reagent ^? according to the manufacturer’s instruction.The expression of osteocalcin mRNA was analyzed by reverse transcriptase-polymerase chain reaction（RT-PCR）.Relative expression of osteocalcin was calculated usingβ-actin mRNA expression as an internal control.（3） Determination of the expression of osteocalcin protein in HLF cellsThe OLF cells were exposed to 100ng/ml rhGDF-5 for 0,4,7,10,14 days or 0, 10,50,100,500ng/ml rhGDF-5 for 10 days,respectively.Cells were collected and their total protein was then prepared with RIPA lysis buffer.The expression of osteocalcin protein was analyzed by Western blot.（4） Determination of the location expression of osteocalcin protein in HLF cellsHLF cells were planted on glass coverslips in 6-well plates,and cultured in the DMEM medium containing 10.0%FBS.Cells grown to 80%confluence were treated with or without 100ng/mlrhGDF-5 for 10 days.Immunofluorescence staining was used to detect the location expression of osteocalcin.The coverslips were treated by 1:100 dilution of goat polyclonal anti-osteocalcin antibody and subsequent 1:50 dilution of FITC fluorescent-labeled goat,and observed under the confocal microscope.（5） Alizarin red stainingHLF cells were planted in 6-well plates,and cultured in the DMEM medium containing 10.0%FBS.Cells at confluence were treated with or without 100ng/ml rhGDF-5 for 28 days.Mineralized nodule formation was examined by the alizarin red staining.3.The signal transduction mechanism of rhGDF-5-induced osteogenic differentiation in HLF cells.（1） Determination of the phosphorylation of MAPKsThe OLF cells were exposed to 100ng/ml rhGDF-5 for 0,5,15,30,60,120,240 min,and collected for protein isolation.Inhibition experiments were performed by 1 h pretreatment with the MAPKs inhibitors U0126,SB203580 and SP600125 before rhGDF-5 stimulation.The total protein and phosphorylated protein of MAPKs （ERK1/2,p38 and JNK） was analyzed by Western blot.（2）Effect of MAPKs inhibitors on rhGDF-5-induced osteogenic differentiation in HLF cells.The OLF cells were exposed to 100ng/ml rhGDF-5 for 10 days with or without 20μM U0126,SB203580 and SP600125,respectively.The ALP activity and osteocalcin protein were examined.Results1.Isolation and culture of HLF cells in vitro.（1） Inverted phase microscopeHLF cells were successfully isolated using a collagenase-pretreated explant method.Within 14 days after explants,outgrowth of cells was observed from ligament tissue explants and became monolayer.Morphologically,HLF cell lines varied widely in appearance,and ranged from thin,spindle-shaped cells to polygonal cells or oval-shaped cells.The primary cells showed radial growth at sub-confluence. The passage cells arranged mainly spindle-shaped,some were fan-shaped or polygonal.They showed vortex-like growth at sub-confluence.When HLF cells subculture continuously to the fifth passage,they still maintain a good morphology.（2） The proliferation,growth curve and cell population doubling time in the passage cells.HLF cells were planted at a density of 2×10⁴ cells per well in 96-well plates. They grow rapidly and showed S-shaped growth curve,0-3 days for the incubation phase of cell growth;3-5 days for proliferative phase;thereafter cell growth entering the plateau.In the MTT analyses,the proliferation of the P1,P3 and P5 cells were no significant difference（F＝0.188,P＝0.831）.The cell population doubling times were no significant difference（F＝0.405,P＝0.675）.（3） Immunofluorescence stainingImmunofluorescence staining showed that the vimentin and collagen type I expressed in the all HLF cells.2.rhGDF-5 induces osteogenic differentiation in HLF cells.（1） rhGDF-5 induces increase of ALP activity in HLF cellsAfter the stimulation of HLF cells with 100ng/ml rhGDF-5 for 0,4,7,10,14 days or 0,10,50,100,500ng/ml rhGDF-5 for 10 days,rhGDF-5 induces increase of ALP activity in a time- dependent（F＝273.905,P＝0.02） and dose -dependent manner（F＝64.091,P＝0.004）.（2） rhGDF-5 induces expression of osteocalcin mRNA in HLF cellsRT-PCR measurement showed that rhGDF-5 treatment resulted in a time-dependent（F＝46.573,P＝0.018） and dose-dependent（F＝70.300,P＝0.002） increasing expression of osteocalcin mRNA.（3） rhGDF-5 induces expression of osteocalcin protein in HLF cellsWestern blot measurement showed that rhGDF-5 treatment resulted in a time-dependent（F＝287.754,P＝0.001） and dose-dependent（F＝115.087, P＝0.004） increasing expression of osteocalcin protein.Immunofluorescence staining showed that the expression of osteocalcin was located in the cytoplasmic in the rhGDF-5 group cells,but not in the control group cells.（4） rhGDF-5 induces mineralized nodule formation in HLF cellsAlizarin red staining showed that the mineralized nodule formation was seen in rhGDF-5 treated cells,but not in the control cells.3.rhGDF-5 stimulated the phosphorylation of ERK1/2 and p38 MAPK,their inhibitors U0126 and SB203580 inhibited the rhGDF-5-induced osteogenic differentiation in HLF cells.After the stimulation of HLF cells with 100ng/ml rhGDF-5 for 0,5,15,30, 60,120,240min,Western blot analyses showed that rhGDF-5 treatment resulted in increasing the phosphorylation of ERK1/2 and p38 MAPK in time-dependent manner （F＝75.940,P＝0.008;F＝19.537,P＝0.030）,but not affected the the phosphorylation of JNK（F＝0.250,P＝0.688）.The pretreatment of ERK1/2 inhibitor U0126 and p38 inhibitor SB203580 could inhibit the phosphorylation of ERK1/2 and p38, respectively.ERK1/2 inhibitor U0126 and p38 inhibitor SB203580 could inhibit the increasing ALP activity and osteocalcin protein induced by rhGDF-5.Conclusions1.The HLF cells can be successfully isolate by collagenase-predigested explant culture.HLF cells in primary culture showed fibroblast-like phenotype.It is stabile that the biological characteristics of cells within 5 generations2.rhGDF-5 can in vitro induce osteogenic differentiation in HLF cells through the ERK1/2 and p38MAPK pathways.更多还原