机械应力联合维拉帕米对骨关节炎软骨细胞-琼脂糖三维模型蛋白聚糖代谢的作用

罗　丹　申　艳　闾坚强　范永前　黄东辉　林伟龙　沈海敏　徐沪济　管剑龙

(1 复旦大学附属华东医院免疫风湿科, 3骨科　上海　200040; 2 上海体育学院运动科学学院　上海　200433;4 第二军医大学长征医院风湿免疫科　上海 200003)

机械应力联合维拉帕米对骨关节炎软骨细胞-琼脂糖三维模型蛋白聚糖代谢的作用

罗丹1▲申艳1▲闾坚强2范永前3黄东辉3林伟龙3沈海敏3徐沪济4管剑龙1△

(1复旦大学附属华东医院免疫风湿科,3骨科上海200040;2上海体育学院运动科学学院上海200433;4第二军医大学长征医院风湿免疫科上海 200003)

【摘要】目的通过对骨关节炎软骨细胞-琼脂糖施压模型机械施压及联合维拉帕米干预,研究软骨细胞聚集蛋白聚糖(aggrecan,AGC)代谢的变化。方法选取膝关节炎患者关节置换术后胫骨平台软骨,分离软骨细胞,制作软骨细胞-琼脂糖三维模型,设置空白对照组、施压组和施压联合维拉帕米组。采用Flexcell FX-5000TM细胞加力仪施压(24 kPa,0.33 Hz,2 h),维拉帕米干预浓度设定为40 μmol/L。采用RT-PCR法和ELISA法分别检测AGC、ADAMTS-4、ADAMTS-5 mRNA和蛋白质水平。结果与空白对照组比较,施压组AGC mRNA水平显著升高(P=0.000),ADAMTS-5 mRNA水平显著降低(P=0.000),施压联合维拉帕米组AGC mRNA水平显著升高(P=0.012),ADAMTS-4和ADAMTS-5 mRNA水平均显著降低(P=0.000)。与空白对照组比较,施压组AGC mRNA水平显著增高(P=0.000)、ADAMTS-5水平显著降低(P=0.000);与施压组比较,施压联合维拉帕米组AGC水平显著增高(P=0.008),ADAMTS-4 和ADAMTS-5 水平均显著降低(P=0.000)。结论机械应力可促进骨关节炎软骨细胞AGC合成,维拉帕米可以加强机械应力的作用。

【关键词】骨关节炎;软骨细胞;蛋白聚糖;机械应力;维拉帕米

骨关节炎(osteoarthritis,OA)是一种中老年人最常见的骨关节病,以关节软骨进行性破坏、骨赘形成为病理特征。随着社会人口老龄化,OA已成为影响老年人健康的常见疾病之一[1-2]。OA在肥胖、运动员等人群中发病率高于普通人群,说明机械应力在OA发病过程中可能发挥重要作用。除磨损造成的软骨直接损伤外,长期应力刺激造成软骨细胞外基质(extracellular matrix,ECM)合成与分解失衡是OA发病机制之一。软骨ECM主要由Ⅱ型胶原和聚集蛋白聚糖(aggrecan,AGC)组成,Ⅱ型胶原纤维网和富有弹性的AGC共同维持软骨细胞内环境稳定[3-4]。ADAMTS-4和ADAMTS-5为AGC裂解的主要酶类,在软骨破坏中发挥重要作用[5]。既往研究表明,拉伸力影响软骨ECM代谢[6],但有关机械应力的作用研究较少。本实验应用软骨细胞-琼脂糖三维模型模拟关节软骨,研究机械应力以及联合钙离子通道阻滞剂维拉帕米对软骨细胞AGC代谢的影响。

材 料 和 方 法

关节软骨来源复旦大学附属华东医院骨科提供行膝关节置换术的膝OA患者9例,男4例,女5例,平均年龄(66.6±8.4)岁,平均病程(9.3±9.4)年。入选病例均得到医院伦理委员会和患者知情同意,诊断符合1986年ACR的OA分类标准[7]。

试剂和仪器胰蛋白酶和甲苯胺蓝染色液(北京雷根生物技术有限公司),Ⅱ型胶原酶、DMEM和胎牛血清(GlBICO),盐酸维拉帕米注射液(2 mL,5 mg,上海禾丰制药有限公司),ELISA检测试剂盒[基尔顿生物科技(上海)有限公司],SYBR Green PCR试剂盒(Thermo Fisher Scientific),逆转录试剂盒(MBI Fermentas),酶标仪(Labsystems Multiskan MS),洗板机(Thermo Labsystems),real-time PCR检测仪(ABI),Flexcell FX-5000TM细胞加力仪和BioPress 6孔压力板(Flexcell International Corporation)。

软骨细胞分离与培养膝关节置换术后将关节放入无菌生理盐水清洗后,在4 h内于超净台内选取胫骨平台软骨损伤较轻的软骨组织,切成1 mm2大小软骨粒,PBS清洗3次,加入0.25%胰蛋白酶,37 ℃消化30 min。弃去消化液,将软骨粒置入0.25%Ⅱ型胶原酶,37 ℃振荡消化14 h,70μm滤器过滤,收集软骨细胞悬液离心,PBS清洗3次,10%FBS的DMEM悬浮接种到培养瓶中,于5% CO237 ℃培养箱中培养,软骨细胞生长至单层时用甲苯胺蓝染色鉴定。

软骨细胞-琼脂糖模型制作及施压培养以5×细胞培养液按1:4比例与液态琼脂糖混匀,以2×106的软骨细胞浓度接种,取8 mL倒入60 mm培养皿中,室温凝固,制作成高3 mm、直径5 mm的软骨细胞-琼脂糖模型,转移至6孔板,加入1.5 mL细胞培养液。9例患者随机分为空白对照组、施压组和施压联合维拉帕米组,每组3例。调整维拉帕米终浓度为40μmol/L[8-9]。采用Flexcell FX-5000TM细胞加力仪对施压组、施压联合维拉帕米组三维模型施以动态循环机械应力(24 kPa,0.33 Hz,2 h)。施压完成后,放入5% CO2培养箱继续培养24 h,收集上清液及琼脂糖三维模型。

RNA提取和RT-PCR检测取出软骨细胞-琼脂糖模型于1 mL的Trizol匀浆管中匀浆20 s;温育5 min,4 ℃下12 000×g离心10 min;吸取上清于1.5 mL离心管加入200μL氯仿,摇匀,室温静置2 min,4 ℃下12 000×g 离心10 min。再吸取上清于1.5 mL离心管加入600μL异丙醇混合均匀,室温静置15 min,4 ℃下12 000×g离心15 min,弃上清加75%无水乙醇(750μL无水乙醇+250μL DEPC水)漂洗沉淀,4 ℃下12 000×g离心5 min;加入1 mL无水乙醇,漂洗沉淀,4 ℃下12 000×g离心5 min;后弃上清,室温干燥10 min;加入40μL DEPC水溶解RNA,置于-80 ℃冰箱保存备用。RT-PCR检测AGC、ADAMTS-4和ADAMTS-5 mRNA,所用引物见表1,实验数据采用仪器自带软件ABI Prism 7300 SDS Software分析,通过2-ΔΔCt计算cDNA相对转录水平。

表1　AGC、ADAMTS-4和ADAMTS-5实时PCR引物序列

ELISA检测琼脂糖三维模型上清液-80 ℃保存,然后按照ELISA试剂盒要求和步骤检测AGC、ADAMTS-4和ADAMTS-5蛋白水平。

统计分析采用SPSS17.0统计软件对数据进行方差分析,两组之间比较采用LSD法,P<0.05为差异有统计学意义。

结果

软骨细胞分离、培养与鉴定甲苯胺蓝染色显示软骨细胞为三角形或多边型,呈紫蓝色(图1)。软骨细胞-琼脂糖三维模型见图2。

RT-PCR检测与空白对照组相比,施压组AGC mRNA水平(0.007 9±0.000 1vs.0.006 2±0.000 2,P=0.000)显著升高,ADAMTS-5 mRNA水平(0.044 4±0.000 5vs.0.062 0±0.001 5,P=0.000)显著降低,ADAMTS-4 mRNA水平差异(0.015 2±0.001 2vs.0.015 4±0.000 9,P=0.721)无统计学意义;与施压组相比,施压联合维拉帕米组AGC mRNA水平(0.008 5±0.000 3vs.0.007 9±0.000 1,P=0.012)增加,ADAMTS-4(0.004 4±0.000 4vs.0.015 2±0.001 2,P=0.000)和ADAMTS-5 mRNA水平(0.014 2±0.000 9vs.0.044 4±0.000 5,P=0.000)均显著降低(图3)。

ELISA检测收集软骨细胞-琼脂糖模型6孔板内上清液,ELISA 法检测空白对照组、施压组和施压联合维拉帕米组AGC、ADAMTS-4和ADAMTS-5表达(表2)。施压组、施压联合维拉帕米组和空白对照组3组相比,AGC、ADAMTS-4和ADAMTS-5蛋白水平均存在显著差异。

图3空白对照组、施压组和施压联合维拉帕米组

AGC,ADAMTS-4,ADAMTS-5mRNA水平比较

Fig3ExpressionsofAGC,ADAMTS-4and

ADAMTS-5mRNAinplacebo,stressaloneand

stresscombinedwithverapamilgroups

与空白对照组比较,施压组AGC生成增多(P=0.000),ADAMTS-5水平降低(P=0.000),ADAMTS-4水平无明显变化(P=0.510);与施压组比较,施压联合维拉帕米组AGC生成增多(P=0.008),ADAMTS-4水平降低(P=0.000),ADAMTS-5水平进一步降低(P=0.000)。

讨论

OA是影响老年人健康的常见疾病之一,关节疼痛和功能受限严重影响老年OA患者的生活质量。OA发病过程中,软骨细胞产生金属蛋白酶和ADAMTSs[10],使关节软骨 ECM合成和降解失衡,软骨细胞内环境进一步破坏,引起一系列生物学反应。

表2　空白对照组、施压组和施压联合维拉帕米组AGC,ADAMTS-4,ADAMTS-5水平

(1)vs.placebo group,P<0.05;(2)vs.stress alone group,P<0.05.

AGC是关节软骨ECM的重要成分之一,赋予软骨组织的抗压性能。近年来,ADAMTSs已经成为OA关节软骨破坏的研究热点,ADAMTS-4和ADAMTS-5为AGC裂解的酶类,在软骨破坏的早期发挥重要作用[5];进一步研究提示,ADAMTS-4和ADAMTS-5的表达和活性调节为OA治疗提供了理论基础[11-12]。Glasson等[13]发现,ADAMTS-5基因敲除小鼠不能被诱导为OA模型。Gendron 等[14]发现,生理条件下ADAMTS-5降解AGC的活性至少为ADAMTS-4的1 000倍,因此ADAMTS-5在OA关节软骨破坏中发挥更重要的作用。琼脂糖-软骨细胞三维模型可模拟关节软骨,已成为研究软骨细胞生物力学的常用模型[15]。有学者研究表明,机械应力负荷可抑制iNOS、COX-2,促进关节ECM合成[16-17]。也有研究[6,18]发现,低强度的机械应力可以通过抑制IL-1β和TNF-α诱导的多种炎性介质,从而抑制软骨基质降解,上调AGC和Ⅱ型胶原表达,发挥修复和保护关节软骨的作用;高强度的机械应力是致炎过程,抑制基质合成,导致软骨破坏,这一过程可能与NF-κB调节相关。Raveenthiran 等[19]研究发现,动态施压抑制纤维蛋白片段对体外培养软骨细胞-琼脂糖模型ECM降解的刺激作用。张巍等[20]对大鼠脊柱终板软骨细胞的研究发现,随着压力刺激(10%,1 Hz)时间延长,Ⅱ型胶原和AGC合成相应增加。本实验在前期工作基础上,调整压力参数为24 kPa时,既可为软骨细胞-琼脂糖模型提供充分的压力刺激,又能维护琼脂糖模型的完整性。对软骨细胞-琼脂糖三维模型施加动态循环机械应力(24 kPa,0.33 Hz,2 h)发现,ADAMTS-5水平显著降低,AGC水平升高,结果提示,适当的机械应力可抑制ADAMTS-5分泌,从而促进软骨细胞AGC合成。

离子通道与软骨细胞的生物功能有关,钙离子参与机械调控离子通道开放和结构型NO合酶活化,与NO的产生和AGC基因表达密切相关[21]。钙离子参与机械应力调控的离子通道开放,在OA软骨ECM代谢中可能发挥作用。Prehm 等[22-23]发现,钙通道阻滞剂维拉帕米可抑制透明质酸的转运,抑制OA关节软骨透明质酸产生过多和AGC丢失。最近的研究显示,Wnt/β-catenin信号通路激活通过上调金属蛋白酶和ADAMTSs,与OA病情进展密不可分[22-23]。Takamatsu等[26]发现,维拉帕米(50μmol/L)可上调FRZB基因表达,抑制Wnt/β-catenin信号传导,进一步抑制ECM降解,改善大鼠OA模型软骨损伤。然而,Chowdhury等[27]对琼脂糖-软骨细胞模型进行动态压缩(15%,1 Hz,48 h)后发现,机械负荷可以抑制NO释放、促进AGC合成,而钆(张力激活的钙离子通道抑制剂)可以通过抑制嘌呤信号通路拮抗机械负荷的效应。我们在前期研究中发现,40μmol/L维拉帕米以减少机械应力对软骨细胞MCP-1的促进作用[9]。本实验结果显示,维拉帕米联合机械应力可以抑制ADAMTS-4和ADAMTS-5分泌,促进AGC合成。然而,临床上关节腔内注射维拉帕米需要考虑对血压等心血管系统的影响,具体结果有待进一步实验。本研究提示,适当的机械应力和维拉帕米对关节软骨ECM中AGC合成有促进作用,为OA的治疗提供了新的理论基础。

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The role of mechanical stress combined with verapamil on aggrecan metabolism in osteoarthritic chondrocytes-agarose constructs

LUO Dan1▲, SHEN Yan1▲,LYU Jian-qiang2, FAN Yong-qian3, HUANG Dong-hui3, LIN Wei-long3, SHEN Hai-min3, XU Hu-ji4, GUAN Jian-long1△

(1DepartmentofImmunologyandRheumatology,3DepartmentofOrthopedicSurgery,HuadongHospital,

FudanUniversity,Shanghai200040,China;2SchoolofKinesiology,ShanghaiUniversityofSport,Shanghai200433,China;4DepartmentofRheumatology,ChangzhengHospital,SecondMilitaryMedicalUniversity,Shanghai200003,China)

【Abstract】ObjectiveTo investigate the effect of mechanical stress and verapamil on aggrecan (AGC) metabolism by osteoarthritic chondrocytes-agarose constructs.MethodsPatients with osteoarthritis were treated with knee joint replacement.Chondrocytes were isolated to construct chondrocytes-agarose model,and then were divided into placebo group,stress group and stress combined with verapamil group.Flexcell FX-5000TMwas applied to impose cyclic mechanical stress (24 kPa,0.33 Hz,2 h),and verapamil (40 μmol/L) was added into stress combined with verapamil group.

RT-PCR and ELISA were performed to measure the expression of AGC,ADAMTS-4 and ADAMTS-5 at both mRNA and proteins levels,respectively.ResultsCompared with the control group,AGC mRNA level was increased (P=0.000),and ADAMTS-5 mRNA level was significantly decreased (P=0.000) in stress alone group.Compared with the stress group,the expression of AGC mRNA was significantly elevated (P=0.012),the expressions of ADAMTS-4 mRNA (P=0.000) and ADAMTS-5 mRNA (P=0.000) were decreased in stress combined with verapamil group.Compared with the placebo group,AGC mRNA level was increased (P=0.000),and ADAMTS-5 level was significantly decreased (P=0.000) in stress group.Compared with the stress group,AGC level was increased further (P=0.008),both ADAMTS-4 and ADAMTS-5 levels were decreased (P=0.000) in stress combined with the verapamil group.ConclusionsMechanical stress can promote chondrocyte AGC synthesis,and verapamil can strengthen the role of mechanical stress.

【Key words】osteoarthritis;chondrocytes;aggrecan;mechanical stress;verapamil

【中图分类号】R681.3

【文献标识码】A

doi:10.3969/j.issn.1672-8467.2016.03.002

(收稿日期:2015-09-14;编辑:段佳)

国家自然科学基金面上项目(81072478,81273298)

▲Co-first authors

△Corresponding authorE-mail:guanjianlong@medmail.com.cn

*This work was supported by the General Program of National Natural Science Foundation of China (81072478,81273298).