機械應力聯合維拉帕米對骨關節炎軟骨細胞-瓊脂糖三維模型蛋白聚糖代謝的作用

羅　丹　申　艷　閭堅強　范永前　黃東輝　林偉龍　沈海敏　徐滬濟　管劍龍

(1 復旦大學附屬華東醫院免疫風濕科, 3骨科　上?！?00040; 2 上海體育學院運動科學學院　上?！?00433;4 第二軍醫大學長征醫院風濕免疫科　上海 200003)

機械應力聯合維拉帕米對骨關節炎軟骨細胞-瓊脂糖三維模型蛋白聚糖代謝的作用

羅丹1▲申艷1▲閭堅強2范永前3黃東輝3林偉龍3沈海敏3徐滬濟4管劍龍1△

(1復旦大學附屬華東醫院免疫風濕科,3骨科上海200040;2上海體育學院運動科學學院上海200433;4第二軍醫大學長征醫院風濕免疫科上海 200003)

【摘要】目的通過對骨關節炎軟骨細胞-瓊脂糖施壓模型機械施壓及聯合維拉帕米干預,研究軟骨細胞聚集蛋白聚糖(aggrecan,AGC)代謝的變化。方法選取膝關節炎患者關節置換術后脛骨平臺軟骨,分離軟骨細胞,制作軟骨細胞-瓊脂糖三維模型,設置空白對照組、施壓組和施壓聯合維拉帕米組。采用Flexcell FX-5000TM細胞加力儀施壓(24 kPa,0.33 Hz,2 h),維拉帕米干預濃度設定為40 μmol/L。采用RT-PCR法和ELISA法分別檢測AGC、ADAMTS-4、ADAMTS-5 mRNA和蛋白質水平。結果與空白對照組比較,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)。結論機械應力可促進骨關節炎軟骨細胞AGC合成,維拉帕米可以加強機械應力的作用。

【關鍵詞】骨關節炎;軟骨細胞;蛋白聚糖;機械應力;維拉帕米

骨關節炎(osteoarthritis,OA)是一種中老年人最常見的骨關節病,以關節軟骨進行性破壞、骨贅形成為病理特征。隨著社會人口老齡化,OA已成為影響老年人健康的常見疾病之一[1-2]。OA在肥胖、運動員等人群中發病率高于普通人群,說明機械應力在OA發病過程中可能發揮重要作用。除磨損造成的軟骨直接損傷外,長期應力刺激造成軟骨細胞外基質(extracellular matrix,ECM)合成與分解失衡是OA發病機制之一。軟骨ECM主要由Ⅱ型膠原和聚集蛋白聚糖(aggrecan,AGC)組成,Ⅱ型膠原纖維網和富有彈性的AGC共同維持軟骨細胞內環境穩定[3-4]。ADAMTS-4和ADAMTS-5為AGC裂解的主要酶類,在軟骨破壞中發揮重要作用[5]。既往研究表明,拉伸力影響軟骨ECM代謝[6],但有關機械應力的作用研究較少。本實驗應用軟骨細胞-瓊脂糖三維模型模擬關節軟骨,研究機械應力以及聯合鈣離子通道阻滯劑維拉帕米對軟骨細胞AGC代謝的影響。

材 料 和 方 法

關節軟骨來源復旦大學附屬華東醫院骨科提供行膝關節置換術的膝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。入選病例均得到醫院倫理委員會和患者知情同意,診斷符合1986年ACR的OA分類標準[7]。

試劑和儀器胰蛋白酶和甲苯胺藍染色液(北京雷根生物技術有限公司),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),逆轉錄試劑盒(MBI Fermentas),酶標儀(Labsystems Multiskan MS),洗板機(Thermo Labsystems),real-time PCR檢測儀(ABI),Flexcell FX-5000TM細胞加力儀和BioPress 6孔壓力板(Flexcell International Corporation)。

軟骨細胞分離與培養膝關節置換術后將關節放入無菌生理鹽水清洗后,在4 h內于超凈臺內選取脛骨平臺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濾器過濾,收集軟骨細胞懸液離心,PBS清洗3次,10%FBS的DMEM懸浮接種到培養瓶中,于5% CO237 ℃培養箱中培養,軟骨細胞生長至單層時用甲苯胺藍染色鑒定。

軟骨細胞-瓊脂糖模型制作及施壓培養以5×細胞培養液按1:4比例與液態瓊脂糖混勻,以2×106的軟骨細胞濃度接種,取8 mL倒入60 mm培養皿中,室溫凝固,制作成高3 mm、直徑5 mm的軟骨細胞-瓊脂糖模型,轉移至6孔板,加入1.5 mL細胞培養液。9例患者隨機分為空白對照組、施壓組和施壓聯合維拉帕米組,每組3例。調整維拉帕米終濃度為40μmol/L[8-9]。采用Flexcell FX-5000TM細胞加力儀對施壓組、施壓聯合維拉帕米組三維模型施以動態循環機械應力(24 kPa,0.33 Hz,2 h)。施壓完成后,放入5% CO2培養箱繼續培養24 h,收集上清液及瓊脂糖三維模型。

RNA提取和RT-PCR檢測取出軟骨細胞-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%無水乙醇(750μL無水乙醇+250μL DEPC水)漂洗沉淀,4 ℃下12 000×g離心5 min;加入1 mL無水乙醇,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 ℃冰箱保存備用。RT-PCR檢測AGC、ADAMTS-4和ADAMTS-5 mRNA,所用引物見表1,實驗數據采用儀器自帶軟件ABI Prism 7300 SDS Software分析,通過2-ΔΔCt計算cDNA相對轉錄水平。

表1　AGC、ADAMTS-4和ADAMTS-5實時PCR引物序列

ELISA檢測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AGC、ADAMTS-4和ADAMTS-5蛋白水平。

統計分析采用SPSS17.0統計軟件對數據進行方差分析,兩組之間比較采用LSD法,P<0.05為差異有統計學意義。

結果

軟骨細胞分離、培養與鑒定甲苯胺藍染色顯示軟骨細胞為三角形或多邊型,呈紫藍色(圖1)。軟骨細胞-瓊脂糖三維模型見圖2。

RT-PCR檢測與空白對照組相比,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)無統計學意義;與施壓組相比,施壓聯合維拉帕米組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檢測收集軟骨細胞-瓊脂糖模型6孔板內上清液,ELISA 法檢測空白對照組、施壓組和施壓聯合維拉帕米組AGC、ADAMTS-4和ADAMTS-5表達(表2)。施壓組、施壓聯合維拉帕米組和空白對照組3組相比,AGC、ADAMTS-4和ADAMTS-5蛋白水平均存在顯著差異。

圖3空白對照組、施壓組和施壓聯合維拉帕米組

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水平無明顯變化(P=0.510);與施壓組比較,施壓聯合維拉帕米組AGC生成增多(P=0.008),ADAMTS-4水平降低(P=0.000),ADAMTS-5水平進一步降低(P=0.000)。

討論

OA是影響老年人健康的常見疾病之一,關節疼痛和功能受限嚴重影響老年OA患者的生活質量。OA發病過程中,軟骨細胞產生金屬蛋白酶和ADAMTSs[10],使關節軟骨 ECM合成和降解失衡,軟骨細胞內環境進一步破壞,引起一系列生物學反應。

表2　空白對照組、施壓組和施壓聯合維拉帕米組AGC,ADAMTS-4,ADAMTS-5水平

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

AGC是關節軟骨ECM的重要成分之一,賦予軟骨組織的抗壓性能。近年來,ADAMTSs已經成為OA關節軟骨破壞的研究熱點,ADAMTS-4和ADAMTS-5為AGC裂解的酶類,在軟骨破壞的早期發揮重要作用[5];進一步研究提示,ADAMTS-4和ADAMTS-5的表達和活性調節為OA治療提供了理論基礎[11-12]。Glasson等[13]發現,ADAMTS-5基因敲除小鼠不能被誘導為OA模型。Gendron 等[14]發現,生理條件下ADAMTS-5降解AGC的活性至少為ADAMTS-4的1 000倍,因此ADAMTS-5在OA關節軟骨破壞中發揮更重要的作用。瓊脂糖-軟骨細胞三維模型可模擬關節軟骨,已成為研究軟骨細胞生物力學的常用模型[15]。有學者研究表明,機械應力負荷可抑制iNOS、COX-2,促進關節ECM合成[16-17]。也有研究[6,18]發現,低強度的機械應力可以通過抑制IL-1β和TNF-α誘導的多種炎性介質,從而抑制軟骨基質降解,上調AGC和Ⅱ型膠原表達,發揮修復和保護關節軟骨的作用;高強度的機械應力是致炎過程,抑制基質合成,導致軟骨破壞,這一過程可能與NF-κB調節相關。Raveenthiran 等[19]研究發現,動態施壓抑制纖維蛋白片段對體外培養軟骨細胞-瓊脂糖模型ECM降解的刺激作用。張巍等[20]對大鼠脊柱終板軟骨細胞的研究發現,隨著壓力刺激(10%,1 Hz)時間延長,Ⅱ型膠原和AGC合成相應增加。本實驗在前期工作基礎上,調整壓力參數為24 kPa時,既可為軟骨細胞-瓊脂糖模型提供充分的壓力刺激,又能維護瓊脂糖模型的完整性。對軟骨細胞-瓊脂糖三維模型施加動態循環機械應力(24 kPa,0.33 Hz,2 h)發現,ADAMTS-5水平顯著降低,AGC水平升高,結果提示,適當的機械應力可抑制ADAMTS-5分泌,從而促進軟骨細胞AGC合成。

離子通道與軟骨細胞的生物功能有關,鈣離子參與機械調控離子通道開放和結構型NO合酶活化,與NO的產生和AGC基因表達密切相關[21]。鈣離子參與機械應力調控的離子通道開放,在OA軟骨ECM代謝中可能發揮作用。Prehm 等[22-23]發現,鈣通道阻滯劑維拉帕米可抑制透明質酸的轉運,抑制OA關節軟骨透明質酸產生過多和AGC丟失。最近的研究顯示,Wnt/β-catenin信號通路激活通過上調金屬蛋白酶和ADAMTSs,與OA病情進展密不可分[22-23]。Takamatsu等[26]發現,維拉帕米(50μmol/L)可上調FRZB基因表達,抑制Wnt/β-catenin信號傳導,進一步抑制ECM降解,改善大鼠OA模型軟骨損傷。然而,Chowdhury等[27]對瓊脂糖-軟骨細胞模型進行動態壓縮(15%,1 Hz,48 h)后發現,機械負荷可以抑制NO釋放、促進AGC合成,而釓(張力激活的鈣離子通道抑制劑)可以通過抑制嘌呤信號通路拮抗機械負荷的效應。我們在前期研究中發現,40μmol/L維拉帕米以減少機械應力對軟骨細胞MCP-1的促進作用[9]。本實驗結果顯示,維拉帕米聯合機械應力可以抑制ADAMTS-4和ADAMTS-5分泌,促進AGC合成。然而,臨床上關節腔內注射維拉帕米需要考慮對血壓等心血管系統的影響,具體結果有待進一步實驗。本研究提示,適當的機械應力和維拉帕米對關節軟骨ECM中AGC合成有促進作用,為OA的治療提供了新的理論基礎。

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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

【中圖分類號】R681.3

【文獻標識碼】A

doi:10.3969/j.issn.1672-8467.2016.03.002

(收稿日期:2015-09-14;編輯:段佳)

國家自然科學基金面上項目(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).