羅　丹　申　艷　閭堅(jiān)強(qiáng)　范永前　黃東輝　林偉龍　沈海敏　徐滬濟(jì)　管劍龍

(1 復(fù)旦大學(xué)附屬華東醫(yī)院免疫風(fēng)濕科, 3骨科　上海　200040; 2 上海體育學(xué)院運(yùn)動(dòng)科學(xué)學(xué)院　上海　200433;4 第二軍醫(yī)大學(xué)長(zhǎng)征醫(yī)院風(fēng)濕免疫科　上海 200003)

機(jī)械應(yīng)力聯(lián)合維拉帕米對(duì)骨關(guān)節(jié)炎軟骨細(xì)胞-瓊脂糖三維模型蛋白聚糖代謝的作用

羅丹1▲申艷1▲閭堅(jiān)強(qiáng)2范永前3黃東輝3林偉龍3沈海敏3徐滬濟(jì)4管劍龍1△

(1復(fù)旦大學(xué)附屬華東醫(yī)院免疫風(fēng)濕科,3骨科上海200040;2上海體育學(xué)院運(yùn)動(dòng)科學(xué)學(xué)院上海200433;4第二軍醫(yī)大學(xué)長(zhǎng)征醫(yī)院風(fēng)濕免疫科上海 200003)

【摘要】目的通過(guò)對(duì)骨關(guān)節(jié)炎軟骨細(xì)胞-瓊脂糖施壓模型機(jī)械施壓及聯(lián)合維拉帕米干預(yù),研究軟骨細(xì)胞聚集蛋白聚糖(aggrecan,AGC)代謝的變化。方法選取膝關(guān)節(jié)炎患者關(guān)節(jié)置換術(shù)后脛骨平臺(tái)軟骨,分離軟骨細(xì)胞,制作軟骨細(xì)胞-瓊脂糖三維模型,設(shè)置空白對(duì)照組、施壓組和施壓聯(lián)合維拉帕米組。采用Flexcell FX-5000TM細(xì)胞加力儀施壓(24 kPa,0.33 Hz,2 h),維拉帕米干預(yù)濃度設(shè)定為40 μmol/L。采用RT-PCR法和ELISA法分別檢測(cè)AGC、ADAMTS-4、ADAMTS-5 mRNA和蛋白質(zhì)水平。結(jié)果與空白對(duì)照組比較,施壓組AGC mRNA水平顯著升高(P=0.000),ADAMTS-5 mRNA水平顯著降低(P=0.000),施壓聯(lián)合維拉帕米組AGC mRNA水平顯著升高(P=0.012),ADAMTS-4和ADAMTS-5 mRNA水平均顯著降低(P=0.000)。與空白對(duì)照組比較,施壓組AGC mRNA水平顯著增高(P=0.000)、ADAMTS-5水平顯著降低(P=0.000);與施壓組比較,施壓聯(lián)合維拉帕米組AGC水平顯著增高(P=0.008),ADAMTS-4 和ADAMTS-5 水平均顯著降低(P=0.000)。結(jié)論機(jī)械應(yīng)力可促進(jìn)骨關(guān)節(jié)炎軟骨細(xì)胞AGC合成,維拉帕米可以加強(qiáng)機(jī)械應(yīng)力的作用。

【關(guān)鍵詞】骨關(guān)節(jié)炎;軟骨細(xì)胞;蛋白聚糖;機(jī)械應(yīng)力;維拉帕米

骨關(guān)節(jié)炎(osteoarthritis,OA)是一種中老年人最常見(jiàn)的骨關(guān)節(jié)病,以關(guān)節(jié)軟骨進(jìn)行性破壞、骨贅形成為病理特征。隨著社會(huì)人口老齡化,OA已成為影響老年人健康的常見(jiàn)疾病之一[1-2]。OA在肥胖、運(yùn)動(dòng)員等人群中發(fā)病率高于普通人群,說(shuō)明機(jī)械應(yīng)力在OA發(fā)病過(guò)程中可能發(fā)揮重要作用。除磨損造成的軟骨直接損傷外,長(zhǎng)期應(yīng)力刺激造成軟骨細(xì)胞外基質(zhì)(extracellular matrix,ECM)合成與分解失衡是OA發(fā)病機(jī)制之一。軟骨ECM主要由Ⅱ型膠原和聚集蛋白聚糖(aggrecan,AGC)組成,Ⅱ型膠原纖維網(wǎng)和富有彈性的AGC共同維持軟骨細(xì)胞內(nèi)環(huán)境穩(wěn)定[3-4]。ADAMTS-4和ADAMTS-5為AGC裂解的主要酶類(lèi),在軟骨破壞中發(fā)揮重要作用[5]。既往研究表明,拉伸力影響軟骨ECM代謝[6],但有關(guān)機(jī)械應(yīng)力的作用研究較少。本實(shí)驗(yàn)應(yīng)用軟骨細(xì)胞-瓊脂糖三維模型模擬關(guān)節(jié)軟骨,研究機(jī)械應(yīng)力以及聯(lián)合鈣離子通道阻滯劑維拉帕米對(duì)軟骨細(xì)胞AGC代謝的影響。

材 料 和 方 法

關(guān)節(jié)軟骨來(lái)源復(fù)旦大學(xué)附屬華東醫(yī)院骨科提供行膝關(guān)節(jié)置換術(shù)的膝OA患者9例,男4例,女5例,平均年齡(66.6±8.4)歲,平均病程(9.3±9.4)年。入選病例均得到醫(yī)院倫理委員會(huì)和患者知情同意,診斷符合1986年ACR的OA分類(lèi)標(biāo)準(zhǔn)[7]。

試劑和儀器胰蛋白酶和甲苯胺藍(lán)染色液(北京雷根生物技術(shù)有限公司),Ⅱ型膠原酶、DMEM和胎牛血清(GlBICO),鹽酸維拉帕米注射液(2 mL,5 mg,上海禾豐制藥有限公司),ELISA檢測(cè)試劑盒[基爾頓生物科技(上海)有限公司],SYBR Green PCR試劑盒(Thermo Fisher Scientific),逆轉(zhuǎn)錄試劑盒(MBI Fermentas),酶標(biāo)儀(Labsystems Multiskan MS),洗板機(jī)(Thermo Labsystems),real-time PCR檢測(cè)儀(ABI),Flexcell FX-5000TM細(xì)胞加力儀和BioPress 6孔壓力板(Flexcell International Corporation)。

軟骨細(xì)胞分離與培養(yǎng)膝關(guān)節(jié)置換術(shù)后將關(guān)節(jié)放入無(wú)菌生理鹽水清洗后,在4 h內(nèi)于超凈臺(tái)內(nèi)選取脛骨平臺(tái)軟骨損傷較輕的軟骨組織,切成1 mm2大小軟骨粒,PBS清洗3次,加入0.25%胰蛋白酶,37 ℃消化30 min。棄去消化液,將軟骨粒置入0.25%Ⅱ型膠原酶,37 ℃振蕩消化14 h,70μm濾器過(guò)濾,收集軟骨細(xì)胞懸液離心,PBS清洗3次,10%FBS的DMEM懸浮接種到培養(yǎng)瓶中,于5% CO237 ℃培養(yǎng)箱中培養(yǎng),軟骨細(xì)胞生長(zhǎng)至單層時(shí)用甲苯胺藍(lán)染色鑒定。

軟骨細(xì)胞-瓊脂糖模型制作及施壓培養(yǎng)以5×細(xì)胞培養(yǎng)液按1:4比例與液態(tài)瓊脂糖混勻,以2×106的軟骨細(xì)胞濃度接種,取8 mL倒入60 mm培養(yǎng)皿中,室溫凝固,制作成高3 mm、直徑5 mm的軟骨細(xì)胞-瓊脂糖模型,轉(zhuǎn)移至6孔板,加入1.5 mL細(xì)胞培養(yǎng)液。9例患者隨機(jī)分為空白對(duì)照組、施壓組和施壓聯(lián)合維拉帕米組,每組3例。調(diào)整維拉帕米終濃度為40μmol/L[8-9]。采用Flexcell FX-5000TM細(xì)胞加力儀對(duì)施壓組、施壓聯(lián)合維拉帕米組三維模型施以動(dòng)態(tài)循環(huán)機(jī)械應(yīng)力(24 kPa,0.33 Hz,2 h)。施壓完成后,放入5% CO2培養(yǎng)箱繼續(xù)培養(yǎng)24 h,收集上清液及瓊脂糖三維模型。

RNA提取和RT-PCR檢測(cè)取出軟骨細(xì)胞-瓊脂糖模型于1 mL的Trizol勻漿管中勻漿20 s;溫育5 min,4 ℃下12 000×g離心10 min;吸取上清于1.5 mL離心管加入200μL氯仿,搖勻,室溫靜置2 min,4 ℃下12 000×g 離心10 min。再吸取上清于1.5 mL離心管加入600μL異丙醇混合均勻,室溫靜置15 min,4 ℃下12 000×g離心15 min,棄上清加75%無(wú)水乙醇(750μL無(wú)水乙醇+250μL DEPC水)漂洗沉淀,4 ℃下12 000×g離心5 min;加入1 mL無(wú)水乙醇,漂洗沉淀,4 ℃下12 000×g離心5 min;后棄上清,室溫干燥10 min;加入40μL DEPC水溶解RNA,置于-80 ℃冰箱保存?zhèn)溆谩T-PCR檢測(cè)AGC、ADAMTS-4和ADAMTS-5 mRNA,所用引物見(jiàn)表1,實(shí)驗(yàn)數(shù)據(jù)采用儀器自帶軟件ABI Prism 7300 SDS Software分析,通過(guò)2-ΔΔCt計(jì)算cDNA相對(duì)轉(zhuǎn)錄水平。

表1　AGC、ADAMTS-4和ADAMTS-5實(shí)時(shí)PCR引物序列

ELISA檢測(cè)瓊脂糖三維模型上清液-80 ℃保存,然后按照ELISA試劑盒要求和步驟檢測(cè)AGC、ADAMTS-4和ADAMTS-5蛋白水平。

統(tǒng)計(jì)分析采用SPSS17.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行方差分析,兩組之間比較采用LSD法,P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

軟骨細(xì)胞分離、培養(yǎng)與鑒定甲苯胺藍(lán)染色顯示軟骨細(xì)胞為三角形或多邊型,呈紫藍(lán)色(圖1)。軟骨細(xì)胞-瓊脂糖三維模型見(jiàn)圖2。

RT-PCR檢測(cè)與空白對(duì)照組相比,施壓組AGC mRNA水平(0.007 9±0.000 1vs.0.006 2±0.000 2,P=0.000)顯著升高,ADAMTS-5 mRNA水平(0.044 4±0.000 5vs.0.062 0±0.001 5,P=0.000)顯著降低,ADAMTS-4 mRNA水平差異(0.015 2±0.001 2vs.0.015 4±0.000 9,P=0.721)無(wú)統(tǒng)計(jì)學(xué)意義;與施壓組相比,施壓聯(lián)合維拉帕米組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)均顯著降低(圖3)。

ELISA檢測(cè)收集軟骨細(xì)胞-瓊脂糖模型6孔板內(nèi)上清液,ELISA 法檢測(cè)空白對(duì)照組、施壓組和施壓聯(lián)合維拉帕米組AGC、ADAMTS-4和ADAMTS-5表達(dá)(表2)。施壓組、施壓聯(lián)合維拉帕米組和空白對(duì)照組3組相比,AGC、ADAMTS-4和ADAMTS-5蛋白水平均存在顯著差異。

圖3空白對(duì)照組、施壓組和施壓聯(lián)合維拉帕米組

AGC,ADAMTS-4,ADAMTS-5mRNA水平比較

Fig3ExpressionsofAGC,ADAMTS-4and

ADAMTS-5mRNAinplacebo,stressaloneand

stresscombinedwithverapamilgroups

與空白對(duì)照組比較,施壓組AGC生成增多(P=0.000),ADAMTS-5水平降低(P=0.000),ADAMTS-4水平無(wú)明顯變化(P=0.510);與施壓組比較,施壓聯(lián)合維拉帕米組AGC生成增多(P=0.008),ADAMTS-4水平降低(P=0.000),ADAMTS-5水平進(jìn)一步降低(P=0.000)。

討論

OA是影響老年人健康的常見(jiàn)疾病之一,關(guān)節(jié)疼痛和功能受限嚴(yán)重影響老年OA患者的生活質(zhì)量。OA發(fā)病過(guò)程中,軟骨細(xì)胞產(chǎn)生金屬蛋白酶和ADAMTSs[10],使關(guān)節(jié)軟骨 ECM合成和降解失衡,軟骨細(xì)胞內(nèi)環(huán)境進(jìn)一步破壞,引起一系列生物學(xué)反應(yīng)。

表2　空白對(duì)照組、施壓組和施壓聯(lián)合維拉帕米組AGC,ADAMTS-4,ADAMTS-5水平

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

AGC是關(guān)節(jié)軟骨ECM的重要成分之一,賦予軟骨組織的抗壓性能。近年來(lái),ADAMTSs已經(jīng)成為OA關(guān)節(jié)軟骨破壞的研究熱點(diǎn),ADAMTS-4和ADAMTS-5為AGC裂解的酶類(lèi),在軟骨破壞的早期發(fā)揮重要作用[5];進(jìn)一步研究提示,ADAMTS-4和ADAMTS-5的表達(dá)和活性調(diào)節(jié)為OA治療提供了理論基礎(chǔ)[11-12]。Glasson等[13]發(fā)現(xiàn),ADAMTS-5基因敲除小鼠不能被誘導(dǎo)為OA模型。Gendron 等[14]發(fā)現(xiàn),生理?xiàng)l件下ADAMTS-5降解AGC的活性至少為ADAMTS-4的1 000倍,因此ADAMTS-5在OA關(guān)節(jié)軟骨破壞中發(fā)揮更重要的作用。瓊脂糖-軟骨細(xì)胞三維模型可模擬關(guān)節(jié)軟骨,已成為研究軟骨細(xì)胞生物力學(xué)的常用模型[15]。有學(xué)者研究表明,機(jī)械應(yīng)力負(fù)荷可抑制iNOS、COX-2,促進(jìn)關(guān)節(jié)ECM合成[16-17]。也有研究[6,18]發(fā)現(xiàn),低強(qiáng)度的機(jī)械應(yīng)力可以通過(guò)抑制IL-1β和TNF-α誘導(dǎo)的多種炎性介質(zhì),從而抑制軟骨基質(zhì)降解,上調(diào)AGC和Ⅱ型膠原表達(dá),發(fā)揮修復(fù)和保護(hù)關(guān)節(jié)軟骨的作用;高強(qiáng)度的機(jī)械應(yīng)力是致炎過(guò)程,抑制基質(zhì)合成,導(dǎo)致軟骨破壞,這一過(guò)程可能與NF-κB調(diào)節(jié)相關(guān)。Raveenthiran 等[19]研究發(fā)現(xiàn),動(dòng)態(tài)施壓抑制纖維蛋白片段對(duì)體外培養(yǎng)軟骨細(xì)胞-瓊脂糖模型ECM降解的刺激作用。張巍等[20]對(duì)大鼠脊柱終板軟骨細(xì)胞的研究發(fā)現(xiàn),隨著壓力刺激(10%,1 Hz)時(shí)間延長(zhǎng),Ⅱ型膠原和AGC合成相應(yīng)增加。本實(shí)驗(yàn)在前期工作基礎(chǔ)上,調(diào)整壓力參數(shù)為24 kPa時(shí),既可為軟骨細(xì)胞-瓊脂糖模型提供充分的壓力刺激,又能維護(hù)瓊脂糖模型的完整性。對(duì)軟骨細(xì)胞-瓊脂糖三維模型施加動(dòng)態(tài)循環(huán)機(jī)械應(yīng)力(24 kPa,0.33 Hz,2 h)發(fā)現(xiàn),ADAMTS-5水平顯著降低,AGC水平升高,結(jié)果提示,適當(dāng)?shù)臋C(jī)械應(yīng)力可抑制ADAMTS-5分泌,從而促進(jìn)軟骨細(xì)胞AGC合成。

離子通道與軟骨細(xì)胞的生物功能有關(guān),鈣離子參與機(jī)械調(diào)控離子通道開(kāi)放和結(jié)構(gòu)型NO合酶活化,與NO的產(chǎn)生和AGC基因表達(dá)密切相關(guān)[21]。鈣離子參與機(jī)械應(yīng)力調(diào)控的離子通道開(kāi)放,在OA軟骨ECM代謝中可能發(fā)揮作用。Prehm 等[22-23]發(fā)現(xiàn),鈣通道阻滯劑維拉帕米可抑制透明質(zhì)酸的轉(zhuǎn)運(yùn),抑制OA關(guān)節(jié)軟骨透明質(zhì)酸產(chǎn)生過(guò)多和AGC丟失。最近的研究顯示,Wnt/β-catenin信號(hào)通路激活通過(guò)上調(diào)金屬蛋白酶和ADAMTSs,與OA病情進(jìn)展密不可分[22-23]。Takamatsu等[26]發(fā)現(xiàn),維拉帕米(50μmol/L)可上調(diào)FRZB基因表達(dá),抑制Wnt/β-catenin信號(hào)傳導(dǎo),進(jìn)一步抑制ECM降解,改善大鼠OA模型軟骨損傷。然而,Chowdhury等[27]對(duì)瓊脂糖-軟骨細(xì)胞模型進(jìn)行動(dòng)態(tài)壓縮(15%,1 Hz,48 h)后發(fā)現(xiàn),機(jī)械負(fù)荷可以抑制NO釋放、促進(jìn)AGC合成,而釓(張力激活的鈣離子通道抑制劑)可以通過(guò)抑制嘌呤信號(hào)通路拮抗機(jī)械負(fù)荷的效應(yīng)。我們?cè)谇捌谘芯恐邪l(fā)現(xiàn),40μmol/L維拉帕米以減少機(jī)械應(yīng)力對(duì)軟骨細(xì)胞MCP-1的促進(jìn)作用[9]。本實(shí)驗(yàn)結(jié)果顯示,維拉帕米聯(lián)合機(jī)械應(yīng)力可以抑制ADAMTS-4和ADAMTS-5分泌,促進(jìn)AGC合成。然而,臨床上關(guān)節(jié)腔內(nèi)注射維拉帕米需要考慮對(duì)血壓等心血管系統(tǒng)的影響,具體結(jié)果有待進(jìn)一步實(shí)驗(yàn)。本研究提示,適當(dāng)?shù)臋C(jī)械應(yīng)力和維拉帕米對(duì)關(guān)節(jié)軟骨ECM中AGC合成有促進(jìn)作用,為OA的治療提供了新的理論基礎(chǔ)。

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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

【中圖分類(lèi)號(hào)】R681.3

【文獻(xiàn)標(biāo)識(shí)碼】A

doi:10.3969/j.issn.1672-8467.2016.03.002

(收稿日期:2015-09-14;編輯:段佳)

國(guó)家自然科學(xué)基金面上項(xiàng)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).