布洛芬与氨基葡萄糖对膝骨关节炎患者滑膜细胞增殖及软骨寡聚基质蛋白表达影响的比较

目的 探讨布洛芬与氨基葡萄糖对膝骨关节炎(OA)滑膜细胞增殖及软骨寡聚基质蛋白(COMP)表达的影响.方法 布洛芬与氨基葡萄糖含药血清培养早期和晚期滑膜细胞,四唑氮化合物/黄嘌呤氧化酶(MTS/PMS)法测定吸光度(A)值,hCOMP定量试剂盒测定COMP含量.采用等方差假设双侧t检验进行统计学处理.结果 通过MTS/PMS法观察确定布洛芬与氨基葡萄糖含药血清培养滑膜细胞观察点在第5～7天;氨基葡萄糖含药血清培养滑膜细胞A值[晚期组(0.054±0.021),早期组(0.777±0.034)]低于正常血清对照组(P＜0.05).布洛芬[晚期组(35.4±1.9),早期组(46.0±2.2)]与氨基葡萄糖含药血清[晚期组(36.6±1.3),早期组(48.8±1.3)]对软骨寡聚基质蛋白的表达也有明显降低作用(P＜0.05).结论 氨基葡萄糖可以抑制体外培养早期和晚期膝OA患者滑膜细胞增殖,而布洛芬与氨基葡萄糖皆可抑制体外培养滑膜细胞分泌COMP.     

柔肝中药对体外培养软骨细胞增殖能力及关节软骨低聚基质蛋白分泌的影响

目的 探讨柔肝中药对体外培养软骨细胞增殖能力及关节软骨低聚基质蛋白（COMP）分泌的影响。方法 采用分阶段酶消化法体外培养兔软骨细胞，以2×10^4／ml密度接种3代内细胞，以Ⅱ型胶原的免疫组织化学研究鉴定细胞。给予家兔临床等效剂量灌胃后，抽取含药血清培养细胞，分别用5％、10％的柔肝方含药血清（分给药后1、3、5h3个时间点）以及正常兔血清、小牛血清干预7d，采用四甲基偶氮唑蓝（MTF）法观察中药含药血清对体外培养的软骨细胞增殖的影响；体外培养人软骨细胞，采用柔肝复方组及单味柔肝药提取物直接添加体外培养体系3d，添加终浓度为10mg／ml。采用酶联免疫吸附试验（ELISA）检测药物对体外培养的人软骨细胞COMP分泌的影响。结果 培养细胞经Ⅱ型胶原的免疫组织化学染色后有阳性表现，在对软骨细胞增殖的影响方面，柔肝方含药血清各组均优于兔血清组和小牛血清组，其中含药血清组的3h时间点总体优于1h及5h时间点（P〈0．05）；在对软骨细胞上清COMP分泌方面，柔肝复方及单方提取物组均有促进细胞分泌COMP的作用（P〈0．05），但两组之间差异无统计学意义（P〉0．05）。结论 柔肝方中药含药血清可以促进体外培养软骨细胞增殖；在药物直接干预的条件下，柔肝复方及单方提取物可以促进软骨细胞分泌COMP。     

氟对体外培养乳鼠软骨细胞增殖的影响

目的 探讨氟对体外培养乳鼠软骨细胞增殖的影响.方法 采用细胞培养的方法,原代培养昆明乳鼠软骨细胞,传第3代后按染氟剂量不同分为O(对照)、5、10、20、40 ms/L组,10 d后光、电镜观察软骨细胞形态学变化;采用生长曲线、噻唑蓝(MTT)方法,在染氟24、48、72 h测定细胞数量变化及细胞增殖率.结果 染氟10 d后,镜下0、5、10mg/L组软骨细胞表现为增殖,细胞生长旺盛,均可见部分细胞核中核仁数增加:40mg/L组部分软骨细胞中有染色质固缩或凝结成块状,可见到凋亡细胞.在染氟24 h,细胞增殖活力各染氟组组间比较差异无统计学意义(F=2.313,P＞0.05).在染氟48、72 h,0 mg/L组[(23.5±4.6)%、(29.9±1.7)%]、5mg/L组[(34.6±4.7)%、(45.3±5.9)%]、10mg/L组[(39.9±4.8)%、(56.8±5.5)%]、20mg/L组[(31.8±4.1)%、(38.3±6.5)%]、40 mg/L组[(28.3±4.3)%、(33.4±4.8)%]组问比较差异有统计学意义(F值分别为11.401、25.671,P＜0.05);各染氟组细胞增殖活力与对照组比较差异均有统计学意义(P＜0.05),其中5、10ms/L组明显高于40mg/L组(P＜0.05).结论 低剂量氟在较短作用时间内可以促进体外培养小鼠软骨细胞的增殖,剂量升高时表现为抑制.     

不同的跑步运动模式对T-2毒素诱导的大鼠关节软骨损伤的影响

目的 了解在关节软骨损伤的情况下,不同的跑步运动模式对关节软骨的影响,以评价运动在骨关节病发生发展中的作用.方法 100只Wistar大鼠,按体质量随机分为5组:阴性对照组(笼内自由活动),阳性对照组(笼内自由活动),高规组(规律运动,跑台速度24 m/min),低规组(规律运动,跑台速度12 m/min)和随机组(不规律运动,跑台速度12、24 m/min).阴性对照组食用正常饲料,其他各组均食用经T-2毒素染毒的饲料,实验5 ～ 10周,进行关节软骨的组织病理观察和血清软骨寡聚基质蛋白(COMP)的检测.结果 各实验组大鼠关节软骨病变明显,表现为软骨细胞变性、坏死,细胞核固缩、深染,细胞排列紊乱,细胞增生,基质胶原显现.与阳性对照组相比,运动组大鼠软骨表层细胞变性、坏死和缺失比较明显.不同运动组间比较可见,高规组软骨损伤最为严重,表层及中间层病变明显,表现为大面积软骨细胞坏死、缺失和基质胶原显现;随机组病变以软骨细胞变性、极性消失、排列紊乱以及细胞增生为主.随着实验周期的延长,各组大鼠软骨损害有加重的趋势.5周时,大鼠血清COMP水平组间比较差异有统计学意义(F=15.733,P＜ 0.05).高规组、低规组、随机组COMP水平[(13.95±1.23)、(14.96±1.29)、(12.99±1.43)μg/L]均高于阴性对照组[(11.55±0.89)μg/L,P均＜0.05],高规组、低规组高于阳性对照组[(12.32±1.38)μg/L,P均＜0.05],低规组高于高规组(P＜0.05);10周时,各组大鼠血清COMP变化趋势与5周时相同,组间比较差异有统计学意义(F=6.144,P＜0.05).其中高规组、低规组、随机组[(13.72±2.67)、(14.94±1.06)、(13.21±1.58)μg/L]高于阴性对照组[(10.59±1.93 )ug/1,P均＜0.05],低规组高于阳性对照组[(11.45±0.12)μg/L,P均＜0.05],但不同强度跑步组间比较,差异无统计学意义(P均＞ 0.05).结论 高强度规律跑步与不规律跑步运动都可以加重关节软骨损伤,而低强度规律跑步运动对关节软骨损伤的影响作用不显著.     

Ⅱ型胶原在T-2毒素诱导大鼠关节软骨早期损伤中的干预作用

目的 观察Ⅱ型胶原在T-2毒素诱导大鼠关节软骨早期损伤中的干预作用,在分子水平上寻找软骨损伤及修复的分子学生物标志,为探讨关节软骨损伤疾病的防治措施提供理论依据.方法 Wistar大鼠80只,按体质量随机分为4组:阴性对照组、阳性对照组、高剂量干预组、低剂量干预组,每组20只.阴性对照组食用常规成品颗粒饲料,其他3组食用含100 ng/kg T-2毒素染毒饲料;阴性对照组和阳性对照组饮自来水;低、高剂量干预组饮用含Ⅱ型胶原0.5、5.0 g/L的自来水.在3、5个月时处死大鼠,光镜下观察大鼠透明软骨的组织病理学改变,用酶联免疫吸附试验(ELISA法)检测大鼠血清Ⅱ型胶原羧基末端肽(CTX-Ⅱ)、软骨寡聚基质蛋白(COMP)及尿中吡啶啉(DPD)含量.结果 光镜下阳性对照组大鼠关节软骨细胞排列紊乱,软骨细胞变形、变性,可见大面积的软骨细胞坏死,而高、低剂量干预组表现为软骨表面原纤维形成,表层软骨细胞肿胀变圆,扁平的软骨细胞减少,软骨细胞簇集等骨关节炎早期病理改变.在3、5个月时,阴性对照组、阳性对照组、高剂量干预组、低剂量干预组大鼠血清CTX-Ⅱ含量分别为(18.77±4.61)、(25.07±9.17),(24.43±5.23)、(39.17±10.49),(21.11±5.02),(33.20±9.74),(19.87±4.53)、(29.73±9.32)μg/L;血清COMP含量分别为(5.43±2.75)、(6.38±2.23),(21.37±4.72)、(24.52±4.26),(17.27±4.77)、(20.32±4.74),(20.13±5.07)、(19.44±4.92)μg/L.其中,3个月时,与阴性对照组比较,阳性对照组血清CTX-Ⅱ含量明显升高(P＜0.05),而低、高剂量干预组未见明显改变(P均＞ 0.05);5个月时,与阴性对照组比较,其他3组血清CTX-Ⅱ含量明显升高(P均＜ 0.05),而高、低剂量干预组明显低于阳性对照组(P均＜0.05).3个月时,与阴性对照组比较,其他3组血清COMP含量明显升高(P均＜0.05),而与阳性对照组比较,高剂量干预组血清COMP含量明显降低(P＜0.05);5个月时,与阴性对照组比较,其他3组血清COMP含量明显升高(P均＜0.05),而与阳性对照组比较,高、低剂量干预组血清COMP含量明显降低(P均＜0.05).3、5个月时,上述4组大鼠尿液DPD含量分别为( 3.47±2.20)、(4.14±1.06),(4.09±2.48)、(4.33±3.43),(3.86±2.31)、(5.72±3.89),(3.58±2.77)、(4.23±2.90)μg/L,组间比较,差异无统计学意义(F值分别为2.608、2.436,P均＞0.05).结论 Ⅱ型胶原能拮抗T-2毒素的软骨损伤作用,延缓关节软骨的破坏进程,降低大鼠血清中CTX-Ⅱ及COMP水平.     

氨基葡萄糖对IL-1β诱导人骨关节炎软骨细胞蛋白聚糖分解代谢的影响

目的 通过体外培养骨关节炎(OA)软骨细胞体系,了解氨基葡萄糖对白细胞介素(IL)-1β诱导OA软骨细胞蛋白聚糖分解代谢的影响.方法 软骨细胞采自OA患者,采用分阶段酶消化法体外原代培养人软骨细胞.在培养液中加入IL-1β诱导剂,设立兔血清对照组、IL-1β实验对照组和氨基葡萄糖加药实验组;给予实验兔以氨基葡萄糖等效剂量灌胃后,抽取含药血清培养细胞.采用3种方法了解蛋白聚糖的代谢水平:①用二甲基亚甲蓝分光光度法(DMMB法)检测细胞中及培养液中的糖胺多糖(GAG)含量;②酶联免疫吸附法检测培养液中蛋白聚糖的不同代谢片段(Mab-3 B3和5D4);③反转录聚合酶链反应(RT-PCR)半定量法检测软骨细胞的蛋白聚糖、可聚蛋白聚糖酶-1和可聚蛋白聚糖酶-2mRNA的表达水平.结果 各浓度氨基葡萄糖含药血清组体外培养的软骨细胞释放入培养液中GAG百分比均值明显低于对照组(P＜0.01),随氨基葡萄糖浓度的增加,释放入培养液中GAG的百分比逐渐降低;3B3含量的均值较对照组相比明显增高(P＜0.01),与氨基葡萄糖浓度呈正相关,释放入培养液中5D4含量的均值则明显降低(P＜0.05),与氨基葡萄糖浓度呈负相关;氨基葡萄糖可以增加OA患者软骨细胞和IL-1β诱导的OA患者软骨细胞蛋白聚糖mRNA表达,减低可聚蛋白聚糖酶-1、可聚蛋白聚糖酶-2mRNA的表达.结论 氨基葡萄糖可以抑制IL-1β对OA患者软骨细胞蛋白聚糖分解代谢的促进作用,达到保护软骨,防止OA的目的.     

壳聚糖膜对体外培养大鼠软骨细胞增殖活性及分泌细胞外基质的影响

目的 探讨壳聚糖膜对体外培养大鼠软骨细胞增殖活性及分泌细胞外基质的影响.方法 制备壳聚糖膜材料.体外分离培养大鼠膝关节软骨细胞,随机分为6孔板细胞组、种植于壳聚糖膜细胞组、PBS处理组、壳聚糖膜浸出液处理组.通过CCK-8法检测培养不同时间软骨细胞的增殖活性,并通过Real-time PCR法检测不同培养时间软骨细胞分泌Ⅱ型胶原及聚集蛋白聚糖的量.结果 通过Ⅱ型胶原免疫组化染色发现,本实验培养的细胞呈阳性,表明该细胞为软骨细胞;CCK-8检测结果表明,种植于壳聚糖膜细胞组、壳聚糖膜浸出液处理组的软骨细胞增殖活性分别高于6孔板细胞组、PBS处理组(P均＜0.05);Real-time PCR检测结果表明,种植于壳聚糖膜细胞组、壳聚糖膜浸出液处理组的软骨细胞分泌Ⅱ型胶原及聚集蛋白聚糖的量分别高于6孔板细胞组、PBS处理组(P均＜0.05).结论 壳聚糖膜对体外培养大鼠软骨细胞增殖及分泌细胞外基质具有促进作用.     

Luminex液相蛋白芯片检测一氧化氮合酶抑制剂对骨关节炎患者软骨基质金属蛋白酶表达的影响

目的 应用Luminex液相蛋白芯片检测一氧化氮合酶(NOS)抑制剂对骨关节炎(OA)患者软骨基质金属蛋白酶(MMPs)表达的影响,以及NOS抑制剂改善OA代谢的途径.方法 研究经本院医学伦理委员会批准并获取患者的知情同意.无菌条件下,取15例重度OA需行关节置换术患者的关节软骨,置入体外培养系统.应用随机数目表法分为:①对照组:不加药物干预;②L-N6-亚氨乙基-赖氨酸(L-NIL)组:加入NOS抑制剂L-NIL干预.培养72 h后,通过检测硝酸盐和亚硝酸盐的含量来观察软骨一氧化氮(NO)的释放量和NOS的活性;应用Luminex液相蛋白芯片检测OA患者软骨中MMPs(MMP-1,MMP-2,MMP-3,MMP-9,MMP-13)表达量的变化.数据采用配对t检验作均数的显著性检验.结果 对照组软骨培养72 h后,在其上清液中可检测到高浓度NO[(216±47)μmol/L]和高活性的NOS[(5.7±1.3)U/ml],L-NIL组NO释放量[(55±20)μmol/L]较对照组明显减少,NOS活性[(1.7±0.7)U/m1]显著降低(P均<0.01).Luminex液相蛋白芯片显示对照组OA软骨中MMPs[(MMP-1 (10.8±5.4)ng/ml,MMP-2 (9.2±3.3)ng/ml,MMP-3(11.6±4.2)ng/ml,MMP-9(1.27±1.07)ng/ml,MMP-13(3.6±1.3)ng/ml)的表达异常,而L-NIL组OA软骨中MMPs表达量明显被抑制[分别为(3.6±1.8)ng/ml,(2.3±1.2)ng/ml,(3.6±1.4)ng/ml,(0.65±0.21)ng/ml,(1.8±0.5)ng/ml,P均<0.05].结论 Luminex液相蛋白芯片检测系统表明NOS 抑制剂通过减少NO的过度释放和降低NOS活性,进而抑制MMPs的过度表达来改善OA软骨的代谢.     

骨关节炎兔血清中软骨寡聚基质蛋白和基质金属蛋白酶-3水平

目的 探讨血清软骨寡聚基质蛋白(COMP)与基质金属蛋白酶(MMP)-3检测应用于临床评估骨关节炎(OA)软骨病理改变的可能性.方法 伸直位石膏管型制动16只兔右后膝关节制作OA模型.以造模时间不同分为造模2周、造模6周,左后膝关节未造模故为对照组.X线影像学与病理观察模型关节的变化;评估关节软骨降解程度(OA积分);ELISA检测兔血清COMP、MMP-3水平;分析血清COMP、MMP-3水平与OA积分间的相关性.结果 (1)造模2周影像学变化较造模前不明显;造模6周兔胫骨平台边缘不光滑,关节间隙变窄,表面有毛刺样增生,胫骨平台及股骨内髁外侧可见唇样增生.(2)OA关节病变的形态学观察:造模2周兔关节软骨表面粗糙,表层裂隙;软骨细胞弥漫增多,排列紊乱;OA积分为(4.000±2.204)分.造模6周兔关节软骨表层可见较多裂隙延伸向下深达辐射层;裂隙周围可见脱水固缩坏死的软骨细胞且排列紊乱,部分成簇增生,各层结构不易分辨,有血管翳通过;OA积分为(10.620±1.408)分,与造模2周比,P=0.000.(3)造模2周兔血清COMP[(3.64±0.18)μg/L]、MMP-3[(1.99±0.81)μg/L]水平高于造模前[COMP(3.35±0.20)μg/L,MMP-3(1.61±0.71)μg/L];造模6周兔血清COMP[(3.96±0.44)μg/L]、MMP-3[(3.44±0.91)μg/L]水平高于造模前和造模2周,差异有统计学意义(P值均＜0.05).血清COMP、MMP-3水平与OA积分呈线性相关关系(r值均＞0.710,P值均小于0.05).结论 OA血清中COMP和MMP-3水平对评估OA软骨降解程度具有重要意义.

Abstract：

Objectiye To study the levels of cartilage oligomeric matrixprotein (COMP) and matrix metalloproteinase-3 (MMP-3) in the serum fluid of osteoarthritic rabbit models and their relationships with the severity of pathological changes, so as to investigate their correlation with osteoarthritis(OA). Methods The osteoarthritic animal models were get from immobilizing the right knees of 18 rabbits in full extension using plaster cast. Knee joint pathological changes of 2,6 weeks were examined for pathological severity of OA; ELISA sandwich method was used to measure the levels of COMP and MMP-3 in serum before and after modeling( at 2, 6 weeks respectively); X ray of model keens was also obtained in different period.Correlation analysis was performed to demonstrate the relationship between the levels of COMP, MMP-3 in the serum and the pathological severity of OA. Results ( 1 ) Morphological observations: immobilizing the right knees of rabbits in full extension using plaster cast was a reliable methed for osteoarthritic animal models and the typical histopathologic character was seen; the severity of osteoarthritisgradually increased with time extended. (2) The levels of COMP[(3.64 ±0. 18)μg/L], MMP-3 [(1.99 ±0. 81 ) μg/L]in the serum of 2 weeks osteoarthritic animal models were higher than those before immobilizing with plaster cast [COMP(3.35 ±0. 20) μg/L,MMP-3( 1.61 ±0. 71 ) μg/L]. The levels of COMP[(3.96 ±0. 44) μg/L],MMP-3[(3.44 ±0. 91) μg/L] of 6 weeks were much higher,with a significant difference(P ＜0.05). The levels of COMP, MMP-3 in serum had a linear correlation with the pathological severity of OA (r ＞0. 710,and P ＜ 0. 05 ). Conclusion The levels of COMP and MMP-3 in serum can help to predict and evaluate the progression of OA.     

软骨寡聚基质蛋白对骨关节炎软骨破坏早期诊断价值的研究

目的 探讨软骨寡聚基质蛋白(COMP)对骨关节炎软骨破坏早期诊断价值.方法 兔右后膝伸直位石膏管型固定法制作骨关节炎模型;形态学方法观察造模不同时期关节病理切片;免疫组织化学方法检测软骨内COMP水平;酶联免疫吸附试验(ELISA)方法检测血清COMP水平.应用t检验,Pearson相关性分析.结果 ①伸直位石膏管型制动2周,模型关节呈现早期骨关节炎改变,制动6周呈现典型的中晚期骨关节炎特征;②造模前、模型2周、模型6周血清COMP含量分别是[(3.35±0.20)、(3.64±0.18)、(3.96±0.44)μg/L,P均＜0.05];③未造模、模型2周、模型6周关节软骨内COMP表达强度分别是[(2.7±1.8)%,(5.7±0.7)%,(7.6±0.7)%,P均＜0.05];④模型2周血清COMP水平与模型2周组、模型6周组OA病理评分存在线性相关关系(r均＞0.770,P均＜0.05).结论 骨关节炎血清COMP检测对早期诊断骨关节炎软骨破坏具有重要的意义.

Abstract：

Objective To study the diagnostic value of cartilage oligomeric matrix protein for early cartilage destruction in osteoarthritis and assess its value in the prediction of the disease progression.Methods The osteoarthritis animal models were developed by immobilizing the right knees of 18 rabbits in full extension position using plaster East.Knee joint pathological changes at week 2 and 6 were examined for pathological severity evaluation of osteoarthritis.ELISA sandwich method was used to measure the levels of cartilage oligomeric matrix protein(COMP) in serum before and after modeling(at week 2 and 6 respectively) and immunohistolgy method was used to examine the levels of COMP in knee articular cartilage of osteoarthritis animal models.Correlation analysis was performed to demonstrate the relationship between the levels of COMP in the serum and the pathological severity of osteoarthritis.Pearson's test and t-test were used for correlation analysis.Results ①) Osteoarthritis animal models could be successfully developed by immobilizing the right knees of rabbits in full extension position using plaster east for 2 weeks.Early histopathological changes in the articular cartilage could be observed,At week 6,the typical histopathological characteristics could be seen.②With the extension of modeling time,serum COMP levels persistently increased.The serum COMP levels before modeling,at modeling week 2,week 6 were (3.35±0.20),(3.64±0.18),(3.96±0.44) μg/L respectively,the difference was significant (P＜0.05).③ The level of COMP in the articular cartilage of non-osteoarthritis animal models,models at week 2,week 6 were (2.7±1.8 )％ ,(5.7±0.7)％,(7.6±0.7)％ respectively (P＜0.05 for all).④ The level of COMP in the serum was linearily correlated with the pathological severity of osteoarthritis(r＞0.770 for all,and P＜0.05 for all).Conclusion Levels of COMP in the serum can help to make early diagnosis of osteoarthritis,and elevated COMP level can predict the progression of osteoarthritis.     

性激素对脂肪细胞瘦素和脂联素分泌的影响

目的 观察性激素对前脂肪细胞增殖与分化及脂肪细胞瘦素脂联素分泌的影响.方法 原代培养人大网膜前脂肪细胞,观察其增殖、分化过程.性激素作用前脂肪细胞增殖和分化过程,检测瘦素、脂联素分泌及mRNA水平.结果 成功地培养出人大网膜前脂肪细胞.雌二醇促进前脂肪细胞增殖(0.823±0.059对0.276±0.032,P＜0.05)、抑制分化(P＜0.05);睾酮对前脂肪细胞增殖无明显作用,但抑制分化(P＜0.05).前脂肪细胞增殖及分化期均分泌瘦素.雌二醇促进瘦素分泌,而睾酮抑制(均P＜0.05).脂联素仅在分化期分泌,且性激素抑制其分泌.雌二醇促进瘦素mRNA表达但抑制脂联素mRNA表达;睾酮抑制瘦素、脂联素mRNA表达(均P＜0.05).结论 雌二醇促进脂肪细胞瘦素分泌及mRNA表达,抑制脂联素分泌及mRNA表达;睾酮抑制二者分泌及mRNA表达.     

Fibroblast-like synoviocyte-chondrocyte interaction in cartilage degradation

OBJECTIVE: In vitro models for joint diseases often focus on a single cell type, such as chondrocytes in osteoarthritis (OA) or fibroblast-like synoviocytes (synoviocytes) in rheumatoid arthritis (RA). However, these joint diseases affect the whole joint and interaction between chondrocytes and synoviocytes may play an important role in disease pathology. The current study was designed to study the use of the alginate recovered chondrocyte method as a model for cartilage degradation and to study interaction between chondrocytes and synoviocytes. METHODS: Bovine chondrocytes were cultured in alginate beads for 1 week, subsequently chondrons were retrieved and seeded into transwells. Every two days cartilage-slices were analysed for proteoglycan content (colorimetric, Blyscan GAG kit), collagen content (HPLC) and collagen HP and LP crosslinking (HPLC). For degradation experiments, monocultures of cartilage-slices labelled with (35)S and cocultures with synoviocytes were stimulated with IL-1beta or TNF-alpha. After 7 days, (35)S release was measured taken as a measure of cartilage degradation. RESULTS: After biochemical analysis, three week old cartilage-like slices were chosen to perform cartilage-degradation experiments. Synoviocytes were able to induce cartilage degradation only in the presence of living chondrocytes. In addition, the cytokines interleukin 1 (IL-1beta) and tumor necrosis factor (TNF-alpha) were only able to induce cartilage degradation by chondrocytes, not by synoviocytes. CONCLUSION: These data indicate that the alginate recovered chondrocyte method provides a novel model for cartilage degradation in which the interaction between synoviocytes and chondrocytes can be studied.     

Cartilage oligomeric matrix protein increases in serum after the start of growth hormone treatment in prepubertal children

Both GH and IGF-I stimulate bone growth, but the molecular mechanisms mediating their effects on the growth plate are not fully understood. We measured gene expression by microarray analysis in primary cultured human chondrocytes treated with either GH or IGF-I. One of the genes found to be up-regulated by both GH and IGF-I was that encoding cartilage oligomeric matrix protein (COMP). This protein is predominantly found in the extracellular matrix of cartilage. Mutations in the COMP gene have been associated with syndromes of short stature. To verify that COMP is regulated by GH in vivo, we measured COMP levels in serum in short children treated with GH. The study included 113 short prepubertal children (14 girls and 99 boys) with a mean (+/- sd) age of 8.84 +/- 2.76 yr, height sd score of -2.74 +/- 0.67, and IGF-I sd score of -1.21 +/- 1.07 at the start of GH administration. Serum levels of COMP were 1.58 +/- 0.28, 1.83 +/- 0.28 (P < 0.0001), 1.91 +/- 0.28 (P < 0.0001), 1.78 +/- 0.28 (P < 0.001), and 1.70 +/- 0.24 (P < 0.05) microg/ml at baseline and after 1 wk and 1, 3, and 12 months, respectively.In conclusion, we have demonstrated that COMP expression is up-regulated by both GH and IGF-I in primary cultured human chondrocytes. Furthermore, serum levels of COMP increase after the start of GH treatment in short children.     

透明质酸对体外培养大骨节病软骨细胞增殖与凋亡的影响

目的 通过观察透明质酸(HA)对体外培养的大骨节病(Kashin-Beck disease,KBD)软骨细胞增殖、凋亡的影响,为临床上HA治疗KBD提供实验依据.方法 依据<大骨节病诊断标准>(GB 16003-1995)收集KBD患者和遭遇意外事故的病人(对照组)关节软骨,分离、体外培养关节软骨细胞.选用第2代细胞进行实验.两组软骨细胞分别给予不同剂量的HA,按HA剂量分为0、100、500 mg/L组.通过二苯甲唑溴盐(MTT)实验,测定第2、4、6天HA对KBD组、对照组软骨细胞增殖的影响.并通过流式细胞检测观察HA对软骨细胞凋亡的影响.结果 对照组在第4天时,500 mg/L组(0.140 ±0.049)促软骨细胞增殖作用大于0 mg/L组(0.116±0.021);KBD组在第6天时,500 mg/L组(0.179±0.081)与0 mg/L组(0.128 ±0.017)比较,显示了明显的促增殖作用(P<0.05).KBD组细胞凋亡率100、500 mg/L组(10.458±1.143、7.877±1.346)均较0 mg/L组(12.860±2.159)下降(P<0.05);对照组500 ms/L组(4.045±1.204)较0 mg/L组(7.128±1.244)细胞凋亡率下降(P<0.05).结论 HA对KBD软骨细胞具有促进增殖和抑制软骨细胞凋亡的作用,其中500 mg/L的HA改善KBD软骨细胞代谢的作用较100 mg/L明显.     

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.     

Differential regulation of lubricin/superficial zone protein by transforming growth factor beta/bone morphogenetic protein superfamily members in articular chondrocytes and synoviocytes

OBJECTIVE: To investigate the role of transforming growth factor beta (TGFbeta)/bone morphogenetic protein (BMP) superfamily members on accumulation of superficial zone protein (SZP) in articular chondrocytes and synoviocytes. METHODS: Chondrocytes and synoviocytes were isolated from articular cartilage and synovium from calf stifle joints and cultured as monolayers in serum-free chemically defined medium. Articular chondrocytes were isolated from 3 distinct zones of the cartilage: superficial, middle, and deep. Accumulation of SZP in the culture medium in response to various members of the TGFbeta/BMP superfamily was demonstrated by immunoblotting and quantified by enzyme-linked immunosorbent assay. RESULTS: TGFbeta stimulated SZP accumulation in both superficial zone chondrocytes and synoviocytes. The 3 isoforms of TGFbeta elicited a similar dose response. Inhibition of TGFbeta receptor type I kinase by the specific inhibitor SB431542 abolished the TGFbeta-stimulated accumulation of SZP. BMPs up-regulated SZP accumulation in the superficial zone; however, the magnitude of the effects was not as great as was observed with TGFbeta. There was an additive action between TGFbeta and BMP on SZP accumulation. The response of synoviocytes to BMP was stronger than that of superficial zone chondrocytes. Activin up-regulated SZP accumulation in synoviocytes, but not in chondrocytes. CONCLUSION: TGFbeta is a critical regulator of SZP accumulation in both superficial zone articular chondrocytes and synoviocytes. TGFbeta and BMP have an additive effect. Synoviocytes are more sensitive to BMP family members and activins than are superficial zone articular chondrocytes. Thus, regulation of SZP accumulation by TGFbeta /BMP superfamily members is regulated differently in articular chondrocytes and synoviocytes.     

High binding capacity of cyclophilin B to chondrocyte heparan sulfate proteoglycans and its release from the cell surface by matrix metalloproteinases: possible role as a proinflammatory mediator in arthritis

OBJECTIVE: To study cyclophilin B, a protein newly identified as a secretion product of cultured chondrocytes, in the context of chondrocyte pathobiology. METHODS: Cyclophilin B was purified by sequential chromatographic processing of the secretion medium of cultured guinea pig chondrocytes. Its presence both at the surface of chondrocyte monolayers and in cartilage was demonstrated by immunohistochemistry. Binding sites at the surface of chondrocytes were characterized by Scatchard plot analysis using (125)I-labeled cyclophilin B, and by glycosidase treatments. The release of cyclophilin B from chondrocytes by activated matrix metalloproteinases (MMPs) was studied by Western blot analysis. RESULTS: Cyclophilin B was present at the surface of cultured chondrocytes and within cartilage, both in cells and in the extracellular matrix, with a particularly intense staining in the superficial layer. It was secreted constitutively by chondrocytes and cartilage explants. Its secretion was enhanced after treatment with its pharmacologic binding partner, cyclosporin A (CSA). Experiments with (125)I-labeled cyclophilin B demonstrated the presence of high-capacity, low-affinity, NaCl-sensitive binding sites at the surface of chondrocytes. Cell-bound cyclophilin B could be released by heparinase treatment, demonstrating binding to pericellular heparan sulfate proteoglycans (HSPGs). Chondroitinase or keratanase treatments had no effect. MMPs 1, 2, 3, 9, and 13 released intact cyclophilin B from the cell surface, probably by cleavage of HSPGs. This effect was reversed by the broad-spectrum MMP inhibitor, marimastat. CONCLUSION: Cyclophilin B is a secreted CSA-binding protein involved in inflammatory events. It can induce chemotaxis in human neutrophils and T lymphocytes. The finding that cyclophilin B is an intrinsic component of cartilage and that it can be released by MMPs suggests that it has a role in the pathogenesis of arthritic diseases, even more so since its signaling receptor is present within the inflamed joint both on T cells and in the rheumatoid synovium.     

应用离心管技术以异体微粒软骨脱细胞基质为支架体外构建组织工程软骨

目的将软骨细胞与异体软骨微粒脱细胞基质相结合．构建组织工程软骨。方法分别利用氯化钾、胰蛋白酶和乙二胺四乙酸钠对绵羊关节软骨进行脱细胞，并制成直径0．100—0．154mm的微粒。先分离异体关节软骨细胞并进行体外扩增，再将异体软骨细胞与软骨微粒脱细胞基质混合培养，离心后应用离心管体外培养。结果本脱细胞方法可以使关节软骨细胞完全脱落，且软骨细胞紧紧围绕于异体软骨微粒脱细胞基质四周，生长和分泌功能良好。结论软骨细胞与异体软骨微粒脱细胞基质有良好的生物相容性．可于体外形成软骨样组织。