低氧环境和生长分化因子-5联合诱导促进人骨髓基质干细胞分化成软骨细胞的研究

目的 探讨低氧环境下生长分化因子-5 (GDF-5)诱导人骨髓基质干细胞(hBMSCs)“自组装”形成工程化软骨的可行性和有效性。方法 分离培养hBMSCs,流式细胞学方法鉴定。将hBMSCs用含100 ng/mL GDF-5软骨诱导液(CM)分别在低氧（A组）和正常氧（B组）环境下诱导培养3周,RT-PCR检测Ⅱ型胶原和Aggrecan表达情况。将A、B两组hBMSCs消化后,按一定密度接种于2％琼脂糖包被的24孔板,在不同氧浓度下CM继续诱导3周,免疫组化法检测组织Ⅰ、Ⅱ型胶原表达,甲苯胺蓝染色检测葡萄糖胺聚糖(GAG)表达。结果 hBMSCs呈梭形漩涡状生长,高表达CD44、CD29,不表达CD45分子。诱导5d后A组细胞较B组明显增殖,诱导10dA组细胞体积较B组小。含GDF-5的CM诱导hBMSCs 3周后,Ⅱ型胶原和Aggrecan mRNA表达阳性,A组Ⅱ型胶原和Aggrecan表达量较B组均明显增加,差异有统计学意义(P＜0.05)。GDF-5诱导hBMSCs可“自组装”形成一定形状大小类软骨样组织,A组Ⅱ型胶原表达较B组增加,A组Ⅰ型胶原表达量下降,B组表达阳性;A组甲苯胺蓝染色异染加深。结论 低氧促进GDF-5诱导hBMSCs向软骨分化,低氧环境下GDF-5诱导软骨分化的hBMSCs“自组装”形成的工程化软骨更具有软骨表型。     

Structural and molecular analyses of bone bridge formation within the growth plate injury site and cartilage degeneration at the adjacent uninjured area

Injury to the growth plate is common and yet the injured cartilage is often repaired with undesirable bony tissue, leading to bone growth defects in children. Using a rat tibial growth plate injury model, our previous studies have shown sequential inflammatory, fibrogenic, osteogenic and bone maturation responses involved in the bony repair. However, it remains unclear whether there is progressive accumulation of bone within the injury site and any potential degenerative changes at the adjacent non-injured area of the growth plate. This study examined effects of growth plate injury on the structure, composition and some cellular and molecular changes at the injury site and adjacent uninjured area. Micro-CT analysis revealed that while the bone volume within the injury site at day 14 was small, the bone bridge was considerably larger at the injury site by 60 days post-injury. Interestingly, formation of bone bridges in the adjacent uninjured area was detected in 60% of injured animals at day 60. Immunohistochemical analyses revealed reduced chondrocyte proliferation (PCNA labelling) but increased apoptosis (nick translation labelling) in the adjacent uninjured area. RT-PCR analysis on adjacent uninjured growth plate tissue found increased expression of osteocalcin at day 60, differential expression of apoptosis-regulatory genes and alterations in genes associated with chondrocyte proliferation/differentiation, including Sox9 and IGF-I. Therefore, this study has demonstrated progressive changes in the structure/composition of the injury site and adjacent uninjured area and identified cellular and molecular alterations or degeneration in adjacent uninjured growth plate in response to injury.     

低氧诱导因子对骨形成影响的研究进展

氧不仅是一种非常重要的底物,而且是一类控制特殊基因程序表达的调节信号。细胞对缺氧的适应性反应的关键介质是低氧诱导转录因子家族(HIFs)。有研究证实低氧环境下,低氧诱导因子1α(HIF-1α)对软骨细胞的生存和分化起重要作用,并能促进骨形成。在此总结了近期相关研究,对HIFs在骨形成中的作用加以阐述,旨在说明HIFs在成骨过程中的重要作用。     

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.     

周期性张应变力对幼年和成年及骨关节炎软骨细胞产生糖胺多糖的影响

目的观察周期性张应变力（cyclic tensile strain,CTS）对体外培养的幼年、成年及骨性关节炎（Osteoarthritis,OA）兔软骨细胞产生糖胺多糖（Glycosaminoglycans,GAG）的影响。方法取2月龄雄性新西兰大白兔3只作为幼年组,5月龄雄性新西兰大白兔10只,随机分2组（即成年组与OA组）,每组5只。OA组采用前交叉韧带切断术（ACLT）制作OA动物模型,成年组仅行双膝关节切开,但不行ACLT。成年组及OA组动物于术后20周空气栓塞处死,取左侧膝关节标本分别做大体评分及Mankins评分观察OA造模情况。将三组兔膝软骨细胞进行消化分离及体外培养。将每个组原代软骨细胞均分别培养于2个BioFlex6孔培养板上,随机分为对照组细胞和加载组细胞,每组6个样本,同置于培养箱内,加载组每天CTS（sin10%,0.5Hz,6h/次）作用6h,对照组不予特殊处理。加载组与对照组在首次加载后24h、48h与72h分别吸取培养细胞上清液,阿尔新蓝染色沉淀法测定上清液GAG含量,比较各组GAG分泌的变化。结果①实验组动物关节软骨明显退变,大体评分及Mankin＇s评分均明显高于对照组（P〈0.05）;②与对照各组细胞相比,幼年、成年及OA组细胞进行加载后其GAG的分泌水平随时间均升高（P〈0.05）;且各细胞在CTS干预前后GAG分泌随时间变化趋势存在统计学差异（P〈0.05）。结论 Flexercell-4000力学加载系统可对体外培养的软骨细胞施加精确的力学负荷加载,并从刺激各类软骨细胞产生蛋白多糖。与正常细胞相比,OA软骨细胞对力学刺激的响应特点发生了较明显的变化。     

Celecoxib inhibits nitric oxide production in chondrocytes of ligament-damaged osteoarthritic rat joints

The purpose of this study was to demonstrate the direct effects of celecoxib, one of the selective cyclo-oxygenase (COX)-2 inhibitors, on nitric oxide (NO) and prostaglandinE2 (PGE2) synthesis in cultured osteoarthritic chondrocyte comparing with those of indomethacin. Articular chondrocytes were isolated from rat osteoarthritic knee joint with damaged anterior cruciate ligament and also from the sham knee joint. Chondrocytes were preincubated with or without IL-1 alpha, and were exposed to celecoxib, indomethacin (non-selective COX inhibitor), or nothing. The amounts of NO and PGE2 in culture supernatants of chondrocytes were measured by EIA or the Griess reaction. In a series of experiments preincubated with or without IL-1 alpha and exposed to nothing, PGE2 and NO levels were significantly higher in osteoarthritic chondrocytes than in sham chondrocytes. Celecoxib and indomethacin inhibited the increase of PGE2 in osteoarthritic chondrocytes. Celecoxib inhibited and indomethacin did not inhibit the increase of NO levels in osteoarthritic chondrocytes.     

周期性单轴牵张应力对大鼠前软骨干细胞相关增殖基因的影响

目的观察周期性单轴牵张应力对大鼠前软骨干细胞增殖相关基因的影响。方法采用四点弯曲应力仪分别以2000μstrain和4000μstrain的牵张力刺激前软骨干细胞1h,并设空白对照组,荧光定量PCR检测相关增殖基因PCNA和CyclinD1的表达情况,并用免疫荧光检测力学激前后Ki67阳性细胞的表达情况。结果2000μstrain刺激组细胞表达PCNA以及CyclinDl较对照组高（P〈0．05）,免疫荧光检测提示2000μstrain刺激组的细胞核内Ki67表达较对照组明显增多;而4000μstrain刺激组细胞PCNA的表达与对照组相比明显降低（P〈O．05）,而CyclinD1降低不明显（P〉0．05）。结论适宜的周期性单轴牵张力能引起大鼠前软骨干细胞相关增殖基因的表达增加。     

二氮嗪对软骨细胞氧化损伤的影响

目的:探讨二氮嗪对双氧水诱导的软骨细胞氧化损伤的影响。方法:取SD乳鼠膝关节软骨细胞进行原代培养,将对数生长期的软骨细胞分成5组,分别为阴性对照组(A组),双氧水组(B组),H2O2+0.1μmol·L-1二氮嗪组(C组),H2O2+1.0μmol·L-1二氮嗪组(D组),H2O2+10μmol·L-1二氮嗪组(E组);A组细胞不做特殊处理,B组用0.3 mmol·L-1双氧水在37℃恒温箱内孵育4 h,C、D、E、F组预先分别用0.1μmol·L-1DZ,1.0μmol·L-1DZ,10μmol·L-1DZ,100μmol·L-1在37℃的恒温箱孵育30分钟,PBS洗涤细胞,再用0.3 mmol·L-1双氧水在37℃恒温箱内孵育4小时。分别检测ABCDE各组细胞的细胞活性,ROS的量,细胞凋亡情况。结果:①MTT法检测各组细胞活性,细胞活性率从大至小依次为D组C组E组F组B组;②用活性氧探针DCFH-DA检测各组细胞中的ROS的量,ROS产量从多至少依次为B组E组D组C组A组;③流式细胞仪检测各组细胞凋亡情况,各组细胞凋亡率由大至小依次为B组E组D组C组。④Western bolt检测各组软骨细胞中HIF-1α蛋白的表达,各组软骨细胞中HIF-1α蛋白表达量依次为C组D组E组A组B组。结论:二氮嗪可降低双氧水诱导的软骨细胞的凋亡率,同时也降低双氧水诱导的软骨细胞ROS的产生,并诱导软骨细胞产生HIF-1α蛋白,可能存在二氮嗪诱导HIF-1α的表达,引起下游相关基因的转录,总体提高软骨细胞对氧化损伤的耐受力,阻碍自由基诱导的软骨细胞的凋亡。     

IL-1β对小鼠软骨细胞中 c-myc 蛋白表达的影响

目的：通过观察不同浓度IL-1β对软骨细胞c-myc蛋白表达的影响,探讨IL-1β在骨关节炎软骨损伤中的作用及机制并寻找有效治疗骨关节炎的方法。方法选用20只C57BL/6小鼠,体外进行关节软骨细胞的分离及原代培养;将原代培养的软骨细胞分为4组：A 组为空白对照组,采用含10% FBS 的RPMI-1640常规培养基培养;B、C、D组为IL-1β处理组,分别用含有1、10和100 ng/ml IL-1β的常规培养基培养,12 h后进行实验分析。采用Western blot和流式细胞仪检测法,观察各组c-myc蛋白表达情况及细胞凋亡情况。结果原代培养细胞24 h后开始贴壁,呈圆形或多角形,传至第4代、第5代细胞体积变大,逐渐成为梭形,甲苯胺蓝染色为软骨细胞内见蓝紫色异染颗粒。与对照组相比较,不同浓度IL-1β处理组软骨细胞 c-myc 蛋白表达水平逐渐增高,呈剂量依赖性（P＜0.05）;与对照组相比较,不同浓度 IL-1β处理组软骨细胞凋亡率逐渐增高,呈剂量依赖性（P＜0.05）。结论实验体外分离小鼠关节软骨,可成功获得高纯度,且传至3代以内活性率超过90%的软骨细胞;IL-1β可以按照剂量依赖方式上调软骨细胞c-myc蛋白的表达,同时增加软骨细胞凋亡率,说明IL-1β可能通过c-myc蛋白诱导软骨细胞凋亡。     

RNA干扰环氧合酶2对人腰椎退变终板软骨细胞生长增殖及凋亡的影响

目的 观察沉默环氧合酶2(COX-2)基因对人腰椎退变软骨终板细胞增殖、凋亡的影响.方法 根据COX-2mRNA的序列,分别设计合成4对不同的且能干扰COX-2基因表达的siRNA并筛选出最高效抑制序列,构建以COX-2基因为靶点的RNA干扰质粒并将其转染腰椎退变软骨终板细胞.分为空白对照组(不予任何处理)、阴性对照siRNA组(30 nmol/L阴性siRNA)、siRNA1组(15 nmol/L COX-2 siRNA)和siRNA2组(30 nmol/L COX-2 siRNA).siRNA转染48 h后,采用荧光定量PCR法检测COX-2基因表达;WST-8检测细胞增殖情况;实时荧光定量聚合酶链反应(qPCR)法检测细胞凋亡相关基因survivin、bcl-x1、bax基因表达的情况.结果 构建的4组重组体插入片段的碱基序列完全正确.转染COX-2siRNA 48 h后人腰椎退变软骨细胞中COX-2mRNA表达量在siRNA1组和siRNA2组分别为(1.3±7.2)%和(35.4±3.6)%,均显著低于空白对照组(100±5.7)%和阴性对照组siRNA(83.4±2.8)%,P＜0.05.免疫印迹实验显示COX-2蛋白表达量明显下降,以siRNA2组表达量最低(P＜0.05＝.WST-8法检测显示4组细胞存活率分别为(100.0±8.3)%、(84.9±4.2)%、(52.5±6.7)%、(48.9±5.4)%,siRNA1组和siRNA2组与其他各组相比,差异有统计学意义(P＜0.05＝.实时荧光定量聚合酶链反应(qPCR)检测显示,随着转染浓度的递增,退变软骨细胞中survivin、bcl-2基因表达较对照组显著降低,bax基因表达则较对照组逐渐升高.结论 RNA干扰COX-2基因能明显抑制人腰椎终板软骨细胞COX-2的表达,抑制细胞生长增殖,并可通过下调抗凋亡基因survivin和bcl-2的表达,上调促凋亡基因bax的表达,从而诱导退变软骨终板细胞凋亡.

Abstract：

Objective To investigate the influence of siRNA-COX-2 gene upon the growth inhibition and apoptosis of cartilage endplate chondrocytes and provide new methods and evidence for siRNA in gene therapy of cartilage endplate chondrocytes. Methods According to the sequence of COX-2 mRNA,COX-2 siRNA was designed, synthesized, cloned into the GFP reporter pcDNA6. 2GW/EmGFPmiR vector and transfected into Hep cell line. The integrity of inset fragment was detected by colony PCR ( polymerase chain reaction) and sequencing analysis. The cultured cartilage endplate chondrocytes were divided into 4 groups: control group (untreated), negative siRNA group (treatment with 30 nmol/L negative control siRNA), siRNA1 group (treatment with 15 nmol/L COX-2 siRNA) and siRNA2 group (treatment with 30 nmol/L COX-2 siRNA). The biological activity of recombinants was identified with the interference efficiency of COX-2 siRNA recombinant by real-time PCR and Western blot. And the effects of COX-2 inhibitor on the growth of chondrocytes were detected by WST-8 and the mRNA expressions of survivin, bcl-2 and bax genes measured by real-time PCR. Results The sequences of inset fragment in 4 siRNA expressing recombinants were correct After COX-2 transfection, the expression of COX-2 mRNA in chondrocytes was 51.3% ±7. 2%in the siRNA1 group and 35.4% ±3.6% in the siRNA2 group. Western blot showed that the expression of COX-2 protein decreased, especially in siRNA2 group (P ＜ 0.05 ). And the cell survival rate was 100.0% ±8.3% in the control group, 84.9% ±4.2% in the negative control siRNA group, 52.5% ±6. 7% in the siRNA1 group and 48.9% ± 5.4% in the siRNA2 group ( P ＜ 0. 05 ). Meanwhile, the expressions of mRNA of survivin and bcl-2 decreased while the expression of bax mRNA increased in degenerative cartilage endplate chondrocytes transfected with COX-2 siRNA ( P ＜ 0. 05 ). Conclusion COX-2-targeting siRNA inhibits the expression of COX-2, suppresses the proliferation of chondrocytes and induces the cell apoptosis. These effects may be attributable to the up-regulation of survivin and bcl-2 and the downregulation of bax.