大骨节病区FA对软骨细胞ALPase活性的影响

为探明大骨节病病区黄腐酸（Ｆｕｌｖｉｃａｃｉｄ，ＦＡ）对软骨内成骨过程中矿化的影响，采用４－甲基伞形酮磷酸酯（４－ＭｅｔｈｙｌｕｍｂｅｌｌｉｆｅｒｙｌＰｈｏｓｐｈａｔｅ，４－ＭＵＰ）为碱性磷酸酶（ＡｌｋａｌｉｎｅＰｈｏｓｐｈａｔａｓｅ，ＡＬａｓｅ）作底物，并结合Ｇｏｍｏｒｉ染色法，研究了ＦＡ对体外培养的肥大软骨细胞及基质在矿化过程中ＡＬＰａｓｅ活性影响。发现与视黄酸（Ｒｅｔｉｎｏｉｃａｃｉｄ，ＲＡ     

软骨细胞和骨髓间质干细胞混合培养构建组织工程软骨的实验研究

目的 探讨软骨细胞和骨髓间质干细胞(bone mesenchymal stem cells,BMSCs)混合培养对构建组织工程软骨的影响,并确定两者的最佳比例.方法 体外分离培养兔(1月龄)关节软骨细胞和BMSCs,按不同比例(软骨细胞和BMSCs的浓度比为:4:1、2:1、1:1、1:2、1:4及单纯软骨细胞)混合培养一代.以4×107/ml的细胞终浓度接种40μl于聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA:4mm×4mm×2mm),静态培养2 d,然后移至周期性压力场(压力0～200 kPa,频率:0.1 Hz,时间:8 h/d)培养3周.收获组织工程软骨行大体观察,组织切片,定量检测糖胺聚糖(glyeosaminoglycans,GAGs)含量、DNA含量及Ⅱ型胶原染色面积.结果 软骨细胞和BMSCs混合培养组与单纯软骨细胞组相比,体积较大,表面光滑,有弹性,有光泽.组织学检查显示混合培养组结构致密,细胞外基质分布更均匀,其中软骨细胞和BMSCs的浓度比为2:1组可见软骨陷窝.混合培养组的Ⅱ型胶原染色面积、GAGs含量、DNA含量高于单纯软骨细胞组,其中软骨细胞和BMSCs的浓度比为2:1组含量最高,与其他比例混合组比较,差异有统计学意义.结论 软骨细胞和BMSCs混合培养能提高组织工程软骨的质量,其中以软骨细胞和BMSCs的浓度比为2:1最佳.     

兔羊膜上皮细胞向兔软骨细胞诱导的研究

目的 观察兔羊膜上皮细胞(AECs)向兔软骨细胞诱导分化的特性.方法 获得兔新鲜羊膜组织,去除外膜组织,并剪碎,胶原酶消化法进行原代培养.经传代培养、细胞形态观察后,用软骨细胞诱导培养液使AECs向软骨细胞分化,并以组织化学和免疫组织化学染色进行鉴定,电镜观察细胞形态检测软骨细胞特征性表现、细胞活性噻唑蓝(MTT)比色法检测.结果 兔羊膜上皮细胞具有前期胚胎干细胞的多向分化潜能,采用酶消化法可快速分离培养原代细胞.MTT比色法显示第3代传代细胞活性、增殖能力较高,未进行软骨细胞诱导的细胞未显示出软骨细胞的特征性表现,经软骨细胞诱导分化后,组织化学和免疫组织化学检测表明其能较好的表现软骨细胞特征.结论 兔AECs无伦理学限制,且具有前期胚胎干细胞的多向分化潜能,有望成为软骨细胞组织工程学新型的种子细胞.     

硒对体外培养大骨节病软骨细胞生长及凋亡的影响

目的 观察硒对体外培养大骨节病(KBD)患者和正常人关节软骨细胞增殖和凋亡的影响,探索补硒防治KBD的作用,并为硒对正常软骨细胞生长的影响提供依据.方法 依据<大骨节病临床诊断标准>(GB 16003-1995),选择Ⅱ度和Ⅲ度KBD患者5例和非病区正常人意外事故者5例的关节软骨进行体外分离、培养.KBD组和对照组分别给予不同剂量的硒(0、0.0125、0.0250、0.0500、0.1000、0.2500、0.5000、1.0000 mg/L)进行干预,采用四氮唑蓝(MTT)法、流式细胞仪和免疫组化法观察细胞生长和凋亡情况.结果 对照组第6天时各剂量组的细胞增殖率(0.086±0.025、0.077±0.012、0.073±0.027、0.071±0.017、0.058±0.028、0.052±0.028和0.046±0.037)比0 mg/L组(0.138±0.026)明显降低(P均＜0.05);0.1000～1.0000 mg/L剂量组的平均细胞增殖率为负值(-0.001±0.001、-0.003±0.000、-0.003±0.001和-0.004±0.001),显著低于0 mg/L组(0.025±0.003,P均＜0.05);KBD组,与0 mg/L组(0.115±0.011)比较,0.2500 mg/L剂量组促进细胞增殖(0.128±0.037,P＜0.05),1.0000 mg/L剂量组细胞生长受到抑制(0.071±0.019,P＜0.05).对照组0.0500～1.0000mg/L剂量组的细胞凋亡率[(18.88±0.02)%、(17.58±0.01)%、(17.09±0.04)%、(56.00±0.02)%、(57.85±0.03)%]比0 mg/L组[(13.51±0.01)%]增高(P均＜0.05);KBD组,与0 mg/L组[(25.84±0.02)%]比较,0.0250～0.2500 mg/L剂量组的细胞凋亡率[(13.69±0.02)%、(15.96±0.03)%、(16.68±0.03)%、(16.67±0.02)%]降低,0.5000、1.0000 mg/L剂量组的细胞凋亡率[(59.58±0.03)%、(73.48±0.04)%]明显增高(P均＜0.05).KBD组0.0500～0.2500 mg/L剂量组的Fas表达[(41.2±1.5)%、(40.3±2.0)%、(50.2±2.5)%]低于同剂量硒干预的对照组[(52.4±1.0)%、(67.2±4.0)%、(75.1±5.0)%,P均＜0.05],0.0500、0.1000 mg/L剂量组的Caspase-3表达[(40.8±1.1)%、(45.1±2.1)%]低于同剂量硒干预的对照组[(68.0±3.0)%、(70.6±3.5)%,P均＜0.05].结论 适宜的补硒剂量(0.1000～0.2500 mg/L)具有促进KBD软骨细胞生长的作用,降低细胞凋亡率,但补硒剂量＞0.5000 mg/L时具有损伤作用;促进KBD软骨细胞生长的硒剂量并非也能促进正常人活体软骨细胞的生长.     

脱氢表雄酮对白细胞介素-1β诱导的人骨关节炎软骨细胞退变的影响

目的:研究脱氢表雄酮(DHEA)对白细胞介素(IL-1β)诱导的人骨关节炎软骨细胞退变的影响。方法:分离人骨关节炎软骨细胞传代培养并加入IL-1β,分别将50,25和12.5μmol/L的DHEA作用于该软骨细胞。其后检测细胞增殖、上清液糖胺聚糖(GAG)和NO含量、细胞凋亡以及质金属蛋白酶-1及其组织抑制剂-1(MMP-1和TIMP-1)含量。结果:50,25和12.5μmol/L的DHEA均能促进软骨细胞增殖和GAG合成(均P<0.01),抑制IL-1β诱导的软骨细胞凋亡(均P<0.01)以及MMP-1合成(均P<0.01);50和25μmol/L的DHEA可抑制IL-1β诱导的软骨细胞NO形成(均P<0.01),还可抑制软骨细胞自身的凋亡(均P<0.01)和MMP-1合成(均P<0.05),其效应呈浓度依赖性;50μmol/L的DHEA还可改善IL-1β对骨关节炎软骨细胞TIMP-1合成的抑制作用(P<0.05)。结论:脱氢表雄酮能在一定程度上对抗IL-1β诱导的人骨关节炎软骨细胞退变。     

治疗型关节炎护膝对实验性骨关节炎软骨细胞凋亡基因Bcl-2及p53的影响

目的：通过检测OA护膝对日本大耳白兔膝骨性关节炎软骨细胞凋亡基因Bcl-2、p53mRNA表达的影响,探讨OA护膝防治兔膝骨性关节炎软骨细胞凋亡的分子生物学机制。方法：健康6月龄日本大耳白兔54只,雌雄各半,空腹体重2～2．2kg,采用改良Huhh法复制膝骨性关节炎模型,随机分为6组,即正常组、模型组、对照组（微波组）、实验1组（电组）、实验2组（热组）、实验3组（护膝组）．正常组10只,常规饲养;模型组9只,造模后常规饲养;对照组9只,微波仪治疗30min,每日1次;实验1组9只,电（疏密波）治疗30min,每日1次;实验2组8只,热（热软膜）治疗30min,每日1次;实验3组9只,电热（OA护膝）治疗30min,每日1次,连续治疗16周时处死。采用荧光定量RT—PCR法检测各组膝关节软骨细胞Bcl-2、p53mRNA的表达水平。结果：16周时,各组所有抽提的兔关节软骨组织总RNA的OD260／OD280值均在1．80～2．00范围内,表明RNA纯度高：模型组、对照组、实验1组、实验2纽、实验3组关节软骨细胞p53的mRNA相对呈高表达,而关节软骨细胞Bcl-2的mRNA相对低表达,与正常组差异有统计学意义（P〈0．01）：关节软骨细胞Bcl-2、p53的mRNA相对表达水平,对照组、实验1组、实验2组、实验3组与模型组差异有统计学意义（P〈0．01）;对照组、实验1组、实验2组与实验3组差异有统计学意义（P〈0．01）。结论：OA护膝能提高关节软骨细胞Bcl-2 mRNA表达,减弱软骨细胞p53mRNA表达,从而抑制软骨细胞凋亡,延缓膝关节软骨的退变：     

Telopeptides of type II collagen upregulate proteinases and damage cartilage but are less effective than highly active fibronectin fragments

Objective and Design:  We hypothesize that N-telopeptide (NT) and C-telopeptides (CT) of type II collagen can enhance proteinases and cause cartilage damage and have compared damaging activities to an extensively characterized potent fibronectin fragment (Fn-f). Materials:  NT and CT peptides were synthesized. Methods:  Interaction of labeled peptides with chondrocytes was studied by fluorescence microscopy. Effects on the metalloproteinases (MMPs) MMP-3 and MMP-13 and on ADAMTS-5 were analyzed by western blotting. Cartilage damage was assayed by loss of proteoglycan (PG) from cultured explants. Results:  NT and CT peptides penetrated cartilage, bound to chondrocytes and enhanced proteinase release and cartilage PG depletion. Peptides had detectable activity at 0.3 μM (1 μg/ml) and were comparable at 30 μM (100 μg/ml) to 1 μM Fn-f (29 μg/ml). However, while the Fn-f enhanced IL-1β and TNF-α, the NT and CT peptides did not. Conclusions:  Collagen peptides containing NT and CT regions were less active on a molar basis than Fn-fs but were still potent damaging agents. Since collagen fragments are found in OA cartilage at μg/ml, they have the potential to play a role in physiologic cartilage damage. Received 7 May 2008; returned for revision 9 June 2008; received from final revision 8 July 2008; accepted by J. A. Battista 31 July 2008     

Prostaglandin E2 receptors EP1 and EP4 are up-regulated in rabbit chondrocytes by IL-1β, but not by TNFα

Prostaglandin E2 (PGE2) exerts its actions through the binding of the high affinity EP receptors. We wanted to evaluate the regulation of EP1 and EP4, and the expression of cyclooxigenase (COX)-2, main enzyme responsible for PGE2 synthesis in inflammatory situations, in healthy rabbit chondrocytes stimulated with inflammatory mediators locally increased during osteoarthritis. Articular chondrocytes obtained from healthy rabbits were stimulated with interleukin (IL)-1β (0.1–100 u/ml) or tumour necrosis factor (TNF)α (100 ng/ml). Where indicated, cells were preincubated with non-steroidal antiinflammatory drugs (NSAIDs) (10⁻⁶ M) to inhibit PGE2 synthesis. IL-1β induced a dose and time-dependent increase in EP1, EP4 and COX-2 expression. However, TNFα presence did not induce a significant modification in EP1, EP4 or COX-2 gene expression at any time of study. NSAID presence significantly inhibited PGE2 release but did not modify the EP receptors or COX-2 expression induced by IL-1β. Our results indicate that EP1 and EP4 receptors, and COX-2 are up-regulated in IL-1β-stimulated chondrocytes, while no significant modifications are observed in TNFα-stimulated cells. NSAIDs were unable to modify the expression of these mediators induced by IL-1β. Therefore, the increase in PGE2 synthesis, induced by IL-1β, does not seem to mediate the increase in EP receptor expression, in rabbit chondrocytes.     

膝关节滑膜间质干细胞分离、培养及其多向分化潜能特性的研究

目的 探讨晚期骨关节炎患者膝关节滑膜间质干细胞(synovium-derived mesenchymalstem cells,SMSCs)体外分离、培养的可行性及其在体外向脂肪细胞、成骨细胞和软骨细胞定向分化的特性.方法 取膝关节滑膜组织,胶原酶消化获得有核细胞.挑选单细胞克隆,筛选获得SMSCs.流式细胞技术检测细胞表面特异性抗原标志.培养至第三代,分别向脂肪细胞、成骨细胞和软骨细胞诱导分化.油红O染色鉴定向脂肪细胞分化;碱性磷酸酶染色、茜素红染色鉴定向成骨细胞分化;甲苯胺蓝染色鉴定向软骨细胞分化.RT-PCR检测脂肪细胞、成骨细胞标志基因.Ⅱ型胶原免疫组化染色检测软骨细胞Ⅱ型胶原的表达.结果 原代SMSCs体外培养呈葵花样细胞集落,传代后可见圆形巨噬样细胞和纺锤形成纤维样细胞,融合后呈成纤维细胞样生长.CD44、CD90呈阳性,CD34、CD71和CD45呈阴性.向脂肪细胞诱导21d,油红O染色阳性;RT-PCR检测有脂蛋白酶、乙二腈及PPARγ2表达;向成骨细胞诱导7、28 d,ALP,茜素红染色阳性,有ALP、Osteopontin及Osteocalcin表达;向软骨细胞诱导21d,甲苯胺蓝染色阳性,Ⅱ型胶原免疫组化染色阳性.结论 晚期骨关节炎患者膝关节滑膜组织可以分离、培养获得SMSCs. SMSCs具有向脂肪细胞、成骨细胞和软骨细胞发生定向分化的潜能.     

Fas Ligand gene transfer enhances the survival of tissue-engineered chondrocyte allografts in mini-pigs

BACKGROUND AND PURPOSE: Since the Fas/Fas Ligand (FasL) interaction has been recognized as an apoptotic pathway, it eliminates the activated T cells and promotes the survival of grafts. In this study, the effect of FasL transfection of pig chondrocytes on allogeneic transplantation was examined in vitro and in vivo. METHODS: Chondrocytes were isolated from articular and aural cartilages of anesthetized Guizhou Xiang (Gz) pig. The cells were transfected with G418 selected virus, packed from PA317 cells with a constructed plasmid using pig FasL (pGCEN-FasL). The apoptotic effect of FasL transfection was examined on Jurkat cells and activated recipient Gz T cells. The FasL expression was assessed by Western blot and flow cytometry. FasL+chondrocytes-Pluronic F-127 complex was injected into the right abdomen of recipient Gz pig. Histology and morphology of the engineered tissue were examined after 2 and 5 weeks of transplantation. RESULTS: The FasL expression was confirmed in pGCEN-FasL transfected chondrocytes. The expression of FasL of chondrocytes from Gz pig was analyzed by FACS. The apoptosis of Jurkat cells and activated recipient Gz T cells was increased by co-culture with FasL(+) chondrocytes (53.41% and 30.38% (E/T=10:1), in contrast of 32.27% and 13.16% with the control chondrocytes, respectively, P<0.01). FasL(+) chondrocytes-Pluronic F-127 implant expressed FasL and Type II collagen at the 5th week and survived until the 8th week. INTERPRETATION: The result indicates that the expression of FasL by chondrocytes is capable of inducing apoptosis of activated T cells. This suggests a potential role for allogeneic transplantation with chondrocytes.