转化生长因子β1诱导永生化人前软骨干细胞向髓核样细胞分化的实验研究

  目的 研究转化生长因子β1 (TGF-β1) 诱导永生化人前软骨干细胞 (IPSCs) 向髓核样细胞分化的可行性。方法 将体外培养的IPSCs 接种于温敏型壳聚糖水凝胶 (C/GP) 三维支架上,加入含TGF-β1的诱导培养基,低氧条件下进行诱导培养,观察三维支架上IPSCs的生长及分化情况。7 d后行Alcian Blue染色,分析细胞外基质糖胺聚糖（GAG）合成情况,并收集细胞,提取RNA。RT-PCR检测诱导前后髓核样细胞标志基因Ⅱ型胶原（Collagen Ⅱ）和蛋白聚糖（Aggrecan）的表达情况,Western Blot 检测诱导前后细胞内β-catenin蛋白表达水平的变化。结果 IPSCs在三维支架上生长状态良好,诱导7 d 后,Alcian Blue染色表明,细胞外基质GAG的合成明显增多,实验组（诱导培养基）明显多于对照组（普通培养基）。RT-PCR证实Ⅱ型胶原和Aggrecan的基因表达水平明显增高,实验组明显高于对照组;Western Blot证实细胞内β-catenin蛋白表达水平明显上调,实验组明显高于对照组。结论 IPSCs经TGF-β1诱导可在体外定向分化为髓核样细胞,诱导分化后的细胞具有良好的分泌功能,能够有效地分泌细胞外基质成分。TGF-β1可能通过上调细胞内β-catenin的表达诱导IPSCs向髓核样细胞分化。     

转化生长因子-β1诱导骨髓间充质干细胞向髓核样细胞转化的研究

目的 探讨转化生长因子-β1(TGF-β1)诱导兔骨髓间充质干细胞(BMSCs)向髓核样细胞分化的可能性.方法 密度梯度法分离培养兔BMSCs,取BMSCs(P3).实验组:含TGF-β1的诱导培养基,对照组:不含TGF-β1诱导培养基,体外培养两周.阿尔新蓝法(Alcian blue)检测培养基内葡糖胺聚糖(GAG)含量.培养第14天,实时定量聚合酶链反应(Real-time PCR)检测髓核样细胞Ⅱ型胶原(CollagenⅡ)及聚集蛋白多糖(Aggrecan) mRNA基因表达.免疫组织化学法测定CollagenⅡ的含量变化.结果 GAG检测结果显示,第7、10、13天实验组GAG含量均显著高于对照组(P＜0.01).实验组CollagenⅡ免疫组织化学染色阳性,表达量较对照组高.Real-time PCR结果证实:实验组CollagenⅡ和Aggrecan mRNA表达水平较对照组显著增高(P＜0.01).结论 TGF-β1能明显增加BMSCs向髓核样细胞分化的诱导生物活性,促进BMSCs向髓核样细胞定向分化.     

兔髓核细胞体外培养和重组人转化生长因子-β1对其代谢影响的研究

目的：探讨体外单层和立体培养兔髓核细胞时的变化及重组人转化生长因子-β1（rhTGF-β1，10ng／ml）对其代谢的影响。方法：体外培养兔髓核细胞，分为3组。A组，单层培养组；B组，Ⅱ型胶原支架立体培养组；C组，Ⅱ型胶原支架立体培养＋rhTGF-β1（10ng／m1）组。利用倒置显微镜、扫描电镜、RT-PCR、^3H-proline掺入法观察髓核细胞形态学、基因表达水平和总胶原合成的变化。结果：B、C组兔髓核细胞由A组的多角形转为类圆形；与A组相比，B组Ⅱ型胶原、集聚蛋白多糖基因表达水平升高（P〈0．05），总胶原合成升高（P〈0．01）。与B组相比，C组Ⅱ型胶原、集聚蛋白多糖、核心蛋白多糖基因表达水平增高（P〈0．01、P〈0．01、P〈0．05），总胶原合成升高（P〈0．01）。结论：兔髓核细胞由单层培养转到Ⅱ型胶原支架上培养时其基因表达和总胶原合成增强。rhTGF-β1（10ng／ml）增强立体培养的兔髓核细胞基因表达和总胶原合成。     

冻椎间盘复合表达hBMP7的髓核细胞体外构建生物椎间盘的可行性研究

目的 探讨利用液氮储存的椎间盘与表达人BMP7的髓核细胞体外构建生物活性椎间盘器官的可行性.方法 取液氮储存2个月的犬椎间盘48个,分为EGFP对照组、1×10^4、1×10^5和1×106共四组,每组12个椎间盘.对照组用22 G注射器自椎间盘后正中注入20 μL 含有1×10^5表达EGFP的髓核细胞,1×10^4组注入20 μL含有1×10^4 PKH-26标记的表达hBMP7的髓核细胞,1×10^5组注入20 μL含1×106KH-26标记的表达hBMP7的髓核细胞,1×106组注入20 μL含1×10^5KH-26标记的表达hBMP7的髓核细胞.注射后立即置入50 mL离心管中,加入30 mL完全培养基,分别于培养第4、7、14 d,从外源细胞存活、存活细胞量、hBMP7的表达、蛋白多糖及总胶原含量变化对体外构建的生物活性椎间盘进行评价.结果 在椎间盘培养的各时间点均可见PKH-26红色荧光和表达绿色荧光蛋白的髓核细胞存在.对不同组、不同时间点的椎间盘中荧光强度定量后发现：培养第7天、14天时1×10^5组荧光强度明显高于1×106组和1×10^4组（P〈0.05）.1×10^5组在第7、14天时hBMP7 mRNA表达量明显高于1×106组和1×10^4组（P〈0.05）,EGFP组未见表达;且1×10^5组在培养的第7天、14天时蛋白多糖及总胶原含量均较另外三组明显增高（P〈0.05）.结论 应用液氮冻存的椎间盘与1×10^5个表达hBMP7的髓核细胞复合,外源性髓核细胞可较长时间存活,并能增加蛋白多糖及胶原含量,是一种体外构建生物活性椎间盘的可行性方法.     

人转化生长因子β1重组腺病毒转染传代髓核细胞对细胞外基质合成的影响

目的探讨人转化生长因子融（transforming growthfactorβ，TGF-β1）对传代羊髓核细胞的细胞外基质（extracellular matrix，ECM）和DNA合成调节因子的作用。方法取1岁龄成年山羊腰椎间盘，体外分离培养羊髓核细胞，传至第3代后以携人TGF-β1（humanTGF-β1，hTGF-β1）或lacZ基因的复制缺陷型腺病毒（Ad／hTGF—β1及Ad／lacZ）感染，分别为实验组和阴性对照组；未加病毒液的细胞为空白对照组；原代髓核细胞为原代组。然后继续单层或藻酸钙凝胶三维（3-D）培养10d。对两种系统培养的细胞分别行DNA荧光定量、Westernblot分析和蛋白多糖（glycosaminoglycan，GAG）定量检测。结果DNA荧光定量显示，单层培养时实验组细胞的DNA合成显著高于两对照组（P〈0．05），藻酸钙凝胶3-D培养各组间比较差异无统计学意义（P〉0．05）。Western blot检测hTGF—β1、Ⅱ型胶原、Ⅰ型胶原和Aggrecan的表达显示，两种培养系统中，实验组hTGF—β1、Ⅱ型胶原和Aggrecan的表达均显著高于两对照组（P〈0．05），Ⅰ型胶原的表达显著低于两对照组（P〈0．05），实验组Ⅱ型胶原／Ⅰ型胶原比值较两对照组显著增高（P〈0．05）。GAG定量结果显示，两种培养系统中实验组细胞的GAG合成均显著高于两对照组（P〈0．05）。结论hTGF-β1在很大程度上可起到维持髓核细胞表型，并在细胞传代后仍发挥表型的调节作用。通过基因工程方法使髓核细胞表达hTGF—β1，有望遏制、甚至逆转椎间盘退变；而以Ad／hTGF—β1感染过的髓核细胞，在藻酸钙凝胶3-D培养系统中培养则表现出原始表型。     

人髓核细胞诱导人尿源性干细胞向髓核样细胞分化作用的研究

【摘要】 目的：探讨人髓核细胞（NPCs）诱导对人尿源性干细胞（USCs）向髓核样细胞分化的作用。方法：手术时获取腰椎间盘突出症患者L4/5的髓核组织,并采用贴壁法体外分离培养NPCs;从健康成年人尿液中获取USCs并进行体外培养,通过倒置显微镜观察细胞形态,并采用成骨、成脂、成软骨三系诱导分化及蛋白免疫印迹技术（Western blot,WB）对所获取的USCs进行形态、分化潜能及细胞表面标志蛋白鉴定。使用Transwell小室将P3代NPCs与USCs培养以建立共培养体系,设实验组、对照组及NPCs组,实验组为NPCs与USCs共培养组,对照组为单独USCs培养,NPCs组为单独NPCs培养;培养14d后应用倒置显微镜观察细胞形态;应用定量实时聚合酶链反应（qRT-PCR）及WB分别检测实验组USCs、对照组USCs与NPCs组NPCs中蛋白多糖（ACAN）、SOX-9（SRY-related high mobility groupbox gene 9）、Ⅱ型胶原（COL2）及缺氧诱导因子1α（HIF-1α）的mRNA及蛋白的表达情况;免疫荧光染色观察3组 COL2A1及ACAN荧光表达。结果：培养的USCs成骨、成脂、成软骨三系分化实验结果均为阳性;USCs中干细胞阳性标志物CD29、CD44、CD73和CD90呈高表达,未检出干细胞阴性标志物CD34和CD45。培养14d后倒置显微镜下对照组及NPCs组细胞形态无变化,实验组USCs向髓核样细胞分化,形态变化明显。经共培养诱导14d后实验组及NPCs组中的ACAN、SOX-9、COL2A1及HIF-1α基因mRNA及蛋白表达均显著高于对照组（P<0.05）,ACAN及COL2A1荧光强度明显高于对照组;实验组上述各种mRNA及蛋白表达与NPCs组比较均无统计学差异（P＞0.05）,实验组荧光强度与NPCs组比较无明显差异。结论：在体外实验中,人NPCs可通过共培养的方式诱导人USCs分化为髓核样细胞,可为椎间盘组织工程研究提供NPCs来源。     

新型丝素蛋白多孔支架复合兔髓核细胞体外构建组织工程化髓核的实验研究

[目的]观察以新型丝素蛋白多孔支架复合兔髓核细胞体外构建组织工程化髓核的可行性。[方法]分离培养兔髓核细胞,与丝素蛋白多孔支架在体外复合培养,建立组织工程化髓核模型,通过扫描电镜、HE染色、甲苯胺蓝染色、Ⅱ型胶原免疫组化以及酶联免疫吸附测定观察细胞在支架上1周和3周的生长及增殖情况。[结果]培养1周后,扫描电镜显示细胞呈球状均匀地贴附在支架内部。细胞-支架复合体HE染色可见支架内部有大量髓核细胞,甲苯胺兰染色阳性,Ⅱ型胶原免疫组化染色阳性。培养3周后,扫描电镜显示细胞成层黏附于支架表面,细胞重叠生长,分泌大量细胞外基质,HE染色可见支架内部有大量髓核细胞填满支架孔隙并分泌大量细胞外基质,甲苯胺兰染色阳性,Ⅱ型胶原免疫组化染色阳性。酶联免疫吸附测定:3周组蛋白多糖含量(52.4±4.5)ng/ml明显高于1周组(29.3±3.6)ng/ml,P<0.05,3周组的II型胶原含量(24.3±1.8)ng/ml明显高于1周组(15.16±1.5)ng/ml,P<0.05。[结论]新型丝素蛋白多孔支架复合兔髓核细胞体外培养生长良好,分泌大量类似髓核样细胞外基质,可以用于体外构建组织工程化髓核。     

四环素联合桂枝加葛根汤对髓核细胞增殖和相关因子表达的影响

目的:探讨四环素(TET)联合桂枝加葛根汤(CK)对髓核细胞增殖和蛋白聚糖(aggrecan)、Ⅱ型胶原蛋白(typeⅡcollagen,Col2a)、基质金属蛋白酶13(matrix metalloproteinase-13,MMP-13)和诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)表达的影响。方法:体外分离培养8周龄健康雄性SD大鼠髓核细胞并进行鉴定。然后用药物处理分离成功的髓核细胞:TET组分别应用不同浓度(5,10,15,20,25μg/ml)的TET处理,CK组分别应用低、中、高剂量TET处理,TET+CK组应用20μg/ml TET和中剂量CK处理,对照组不添加药物。采用CCK8方法检测不同药物处理组细胞增殖活力;实时荧光定量PCR(q RT-PCR)检测TET+CK组髓核细胞的aggrecan、Col2a、i NOS和MMP-13的mRNA表达量,免疫印迹(Western blot)检测Aggrecan、Col2a、i NOS和MMP-13的蛋白表达量。利用pc DNA3.1-CMV(+)构建重组质粒pc DNA3.1-i NOS,转染组用pc DNA3.1-i NOS转染髓核细胞,应用20μg/ml TET和中剂量CK处理,空载体组用空载体(pc DNA3.1)转染细胞,应用20μg/ml TET和中剂量CK处理,对照组不添加药物且不转染,应用q RT-PCR检测各组i NOS和MMP-13的mRNA表达量,Western blot检测各组i NOS和MMP-13的蛋白表达量。结果:分离培养的髓核细胞Col2a和Aggrecan免疫组织化学染色阳性细胞比例分别为96%和98%。不同浓度TET或CK处理髓核细胞后细胞活力与对照组比较无显著性差异(P0.05)。20μg/ml TET和中剂量CK联合作用后髓核细胞的活力、Aggrecan和Col2a的mRNA和蛋白表达量均较对照组显著性升高(P0.05),i NOS、MMP-13的mRNA和蛋白表达量较对照组显著性下降(P0.05)。转染pc DNA3.1-i NOS重组质粒后髓核细胞i NOS和MMP-13的m NRA和蛋白表达与对照组和空载体组均显著性升高(P0.05)。结论:TET联合CK可促进髓核细胞增殖,增加髓核细胞Aggrecan和Col2a mRNA和蛋白的表达,同时可以通过抑制i NOS减少MMP-13 mRNA和蛋白的表达量,为药物预防和治疗椎间盘退变提供了理论依据。     

地塞米松对兔髓核细胞增殖及聚集蛋白聚糖表达的影响

目的 观察地塞米松对兔椎间盘髓核细胞增殖的影响,以及对髓核细胞分泌聚集蛋白聚糖(Aggrecan)的影响.方法 无菌条件下取兔椎间盘,常规分离消化髓核细胞进行培养;传代培养第2代髓核细胞7 d,随机分为两组,实验组给于1～10000 nmol/L地塞米松进行培养,对照组不给于地塞米松,分别培养不同的时间段.采用噻唑蓝(MTT)比色法检测地塞米松对兔髓核细胞增殖的影响;采用逆转录-聚合酶链反应(RT-PCR)法检测地塞米松对兔髓核细胞内Aggrecan mRNA表达的影响.结果 MTT检测结果表明地塞米松对兔髓核细胞增殖有促进作用,最佳作用浓度为100nmol/L,最佳作用时间为48 h;RT-PCR检测结果表明经地塞米松处理的髓核细胞其Aggrecan表达明显较对照组高,是对照组的2.04倍(0.92/0.45),差异有统计学意义(P<0.05).结论 地塞米松可促进兔椎间盘髓核细胞的增殖,并可使兔髓核细胞内Aggrecan表达增高.     

富含血小板血浆凝胶复合脂肪间充质干细胞构建可注射组织工程髓核

目的:探讨以自体富含血小板血浆(PRP)凝胶复合脂肪间充质干细胞(ADSCs)构建可注射组织工程髓核的可行性。方法:将体外扩增的第3代兔ADSCs经流式细胞仪鉴定后接种至自体PRP凝胶中,体外立体培养2周、4周、8周时分别行大体观察、组织学检查、5-溴-2-脱氧尿嘧啶(BrdU)免疫荧光法观察细胞在PRP凝胶中的分布及存活情况;分光光度法检测PRP凝胶-细胞复合体中糖胺聚糖(GAG)含量,实时荧光定量PCR法检测低氧诱导因子(HIF-1α)、蛋白多糖(Aggrecan)、Ⅱ型胶原(CollagenⅡ)基因表达情况。结果:流式细胞仪检测显示体外扩增的第3代细胞CD90阳性率为95.2%,CD45阳性率为0.9%。培养2、4、8周时PRP凝胶细胞复合体均为表面光滑的凝胶状,弹性较好;番红O染色2周时细胞外基质几乎不着色,4周时可见细胞周围呈粉色的弱阳性染色,8周时多数细胞周围呈红色阳性染色。HE染色和扫描电镜各时间点均可见细胞均匀分布于网络状支架内;BrdU免疫荧光法显示细胞在支架中生存状态良好,培养4周时阳性细胞数较培养2周时明显增多(P<0.05),8周时较4周时明显增多(P<0.05)。培养4周时GAG含量较培养2周时明显增高(P<0.05),8周时较4周时明显增高(P<0.05);培养4周时与培养2周时比较3个目的基因mRNA表达量均增高,差异有显著性(P<0.05),8周时与4周时比较亦明显增高(P<0.05)。结论:以兔自体PRP凝胶与ADSCs构建的复合体在体外培养时细胞可以向类髓核样细胞分化,用此方法构建可注射组织工程髓核具有可行性。     

Physical limitations to tissue engineering of intervertebral disc cells: effect of extracellular osmotic change on glycosaminoglycan production and cell metabolism. Laboratory investigation

OBJECT: In this study, the authors examined how physiological levels of extracellular osmolality influence proteoglycan accumulation in nucleus pulposus cells in a 3D culture system. METHODS: Cells were isolated from the nucleus pulposus of caudal discs obtained from 18- to 24-month-old bovines. They were cultured for 6 days in alginate beads at 4 million cells/ml in Dulbecco modified Eagle medium containing 6% fetal bovine serum under 21% O2. Medium osmolality was altered by NaCl addition between 270 and 570 mOsm and monitored using a freezing point osmometer. The cell viability profile was determined by manual counting after trypan blue staining. Profiles across intact beads were determined by manual counting by using fluorescent probes and a transmission electron microscope. Lactate production was measured enzymatically, and glycosaminoglycan (GAG) accumulation was measured using a dimethylmethylene blue assay. Rate of sulfate GAG synthesis was measured using a standard [35S]sulfate radioactive method. RESULTS: The cell viability was similar for the high- and low-osmolality cultures. However, confocal microscopy showed that the cells were the largest at 270 mOsm and became smaller with increasing osmotic pressure. The GAG production was largest at 370 mOsm, the capacity for GAG production and cell metabolism (lactate production) was low under hypoosmolality and hyperosmolality, and cell death was observed on electron microscopy. CONCLUSIONS: In the authors' model, the prevailing osmolality was a powerful regulator of GAG accumulation by cultured nucleus cells. Thus, these results indicate that GAG synthesis rates are regulated by GAG concentration, with implications both for the cause of degeneration and for tissue engineering.     

地塞米松对兔髓核细胞增殖及增殖细胞核抗原表达的影响

目的 观察地塞米松对兔椎间盘髓核细胞增殖的影响,以及对髓核细胞表达增殖细胞核抗原(PCNA)的影响.方法 无菌条件下取兔椎间盘,常规分离消化髓核细胞进行培养;传代培养第2代髓核细胞7 d,随机分为4组,实验组给予l～100 nmol/L地塞米松进行培养,对照组给与等量磷酸盐缓冲液(PBS),培养5 d后收集细胞.采用逆转录-聚合酶链反应(RT-PCR)法检测地塞米松对兔髓核细胞内PCNA mRNA表达的影响;并提取蛋白用Western blot方法检测PCNA蛋白表达.结果 RT-PCR及Western blot检测结果表明,1～100 nmol/L地塞米松可增加兔髓核细胞内PCNA的表达,当地塞米松浓度为100 nmol/L时效果最显著,分别增加了0.42和1.37倍,差异有统计学意义(P<0.05).结论 地塞米松可以促进兔椎间盘髓核细胞的增殖,并可使兔髓核细胞内PCNA的表达增高.     

Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells

STUDY DESIGN: With the heterogeneity of the intervertebral disc as the focus, intervertebral discs from normal young rabbits were separated into nucleus pulposus (NP), inner anulus fibrosus (IAF), and outer anulus fibrosus (OAF) zones. Disc cells from each zone were isolated and propagated under monolayer and within agarose gel culture. The metabolism of these cultured disc cells was examined in terms of glycosaminoglycan (GAG) accumulation. OBJECTIVES: The object was to study the metabolism of disc cells derived from each zone and characterize them on the basis of GAG accumulation and composition. SUMMARY OF BACKGROUND DATA: It has been shown that three-dimensional culture systems, such as within-agarose gels or in alginate beads, permit long-term maintenance of the articular chondrocyte phenotype in vitro. However, little has been reported on how the metabolism of intervertebral disc cells, especially GAG accumulation, is affected by different culture conditions. METHODS: Cells from each zone were subjected to monolayer or three-dimensional culture for up to 12 days. GAG accumulation in the different culture systems was analyzed using chemical, histologic, and immunohistologic methods. Differences of GAG and DNA content among NP, IAF, and OAF cells were statistically evaluated by analysis of variance. The data of keratin sulfate content in three-dimensional culture were compared with that in monolayer culture using nonparametric Mann-Whitney U test. RESULTS: Monolayer culture revealed that increases in GAG content were significantly higher in IAF cells than in OAF cells. However, in three-dimensional culture GAG content was similar in the two groups. AF cells in three-dimensional cultures showed immunohistochemical localization of chondroitin sulfate and keratan sulfate, suggesting the existence of pericellular matrix. High performance liquid chromatography confirmed the expression of keratan sulfate in cultured cells. CONCLUSIONS: GAG accumulation in cultures of cells from different zones of the intervertebral disc varied according to the culture conditions used. The importance of choosing the appropriate culture systems to meet the objectives of a study should be emphasized.     

羧甲基壳聚糖对体外培养髓核细胞增殖及硝普钠诱导凋亡的影响

[目的]研究不同浓度羧甲基壳聚糖对体外培养椎间盘髓核细胞增殖及硝普钠诱导细胞凋亡的保护作用.[方法]体外培养大鼠椎间盘髓核细胞,Ⅱ型胶原免疫组化染色鉴定;分别加入不同浓度羧甲基壳聚糖培养24h,通过CCK-8细胞计数法检测髓核细胞的增殖情况;采用不同浓度硝普钠诱导髓核细胞凋亡,通过流式细胞仪检测早期凋亡细胞比例,并通过Hoechst 33342荧光染色检测髓核细胞凋亡核的形态学变化.[结果]CCK-8检测结果表明10～500μg/ml羧甲基壳聚糖作用髓核细胞24 h对髓核细胞增殖无明显作用(P＞0.05);流式细胞检测结果表明1～3mmol/L的硝普钠可诱导髓核细胞发生早期凋亡,加入50～200μg/ml羧甲基壳聚糖后硝普钠诱导的髓核细胞凋亡有不同程度的降低(P＜0.05).Hoechst 33342染色结果表明羧甲基壳聚糖可降低硝普钠诱导髓核细胞凋亡.[结论]一定浓度的羧甲基壳聚糖对髓核细胞增殖无明显影响,羧甲基壳聚糖对硝普钠诱导下髓核细胞凋亡有保护作用.     

Regulation of apoptosis in nucleus pulposus cells by optimized exogenous Bcl‐2 overexpression

Although the etiology of intervertebral disc degeneration is poorly understood, one possible approach to regulate the process of intervertebral disc degeneration may include the inhibition of apoptosis. We investigated the anti‐apoptotic effects of bcl‐2 in nucleus pulposus cells to enhance disc cell survival. Rat nucleus pulposus cells were transfected in vitro with a codon optimized rat bcl‐2 gene. Forty‐eight hours after transfection, cells were cultured in serum‐deprived medium. After serum withdrawal, the cells were evaluated for bcl‐2 protein levels and cell apoptosis. To investigate the effects of bcl‐2 overexpression on the final apoptotic pathways and on basic genes important for nucleus pulposus homeostasis, mRNA levels of caspase‐3, type II collagen, and aggrecan were also quantified. Nucleus pulposus cells were successfully transfected with codon optimized bcl‐2 gene, which effectively reduced serum starvation‐induced cell apoptosis. Overexpression of bcl‐2 also reduced the mRNA expression level of caspase‐3. mRNA levels of type II collagen and aggrecan were significantly higher in bcl‐2 transfected groups compared to control plasmid vector groups after serum withdrawal. We firstly showed that bcl‐2 overexpression in intervertebral disc cells was effective in preventing in vitro apoptotic cell death, indicating the potential advantages of this therapeutic approach in regulating disc degeneration. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1608–1613, 2010     

兔腰椎间盘髓核细胞的培养及形态观察

目的：通过对兔腰椎间盘髓核细胞的培养,观察细胞内的演变,探讨髓核细胞的生物学行为及影响因素。方法：取兔腰椎间盘髓核细胞,在加10％灭活胎牛血清的F12－DMEM液中培养,建立体外髓核细胞培养模型。通过光镜、电镜观察,同时进行细胞活力测定。结果：（1）原代细胞生物学性状最接近体内细胞,传代后细胞呈现衰老现象。（2） 活力测定提示随着培养时间的延长及传代,活力逐渐降低。（3）髓核细胞内细胞器的变化与其生物学活性变化相一致。结论：兔腰椎髓核细胞的体外培养成功为人腰椎髓核细胞的培养奠定了基础。     

An in-vitro study on regeneration of human nucleus pulposus by using gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold

Tissue engineering approaches for treating degenerative intervertebral discs aim to promote tissue regeneration then retard or even reverse the degenerative process. A gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer was developed to serve as a bioactive scaffold that could help human nucleus pulposus (NP) cells to preserve their cell viability/proliferation and promote matrix synthesis. Each scaffold was seeded with 1 x 10(6) monolayer-expanded human NP cells and then cultured in vitro. Over a 4-week cultivation period, cell-scaffold hybrids demonstrated active cell viability/proliferation and a progressive increase in net production of glycosaminoglycans. In comparison to monolayer cells, scaffold-cultured cells showed significantly higher mRNA expression in collagen II, aggrecan, Sox9, TGFbeta1, and TIMP1. Expression of mRNA was significantly suppressed in collagen I, collagen X, IL1, and Fas-associating death domain protein. Histological studies showed newly synthesized glycosaminoglycans deposits and collagen II in scaffolds. These results indicate that the tri-copolymer scaffold could be considered as a promising bioactive scaffold for regenerating human NP.