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      小鼠SDF-1/CXCR4介導(dǎo)的AKT與ERK通路抑制對(duì)延緩骨折愈合的影響

      2020-09-02 06:39:27唐昊陳順有盧曉坤陳福明
      中外醫(yī)學(xué)研究 2020年19期
      關(guān)鍵詞:成骨細(xì)胞股骨通路

      唐昊 陳順有 盧曉坤 陳福明

      【摘要】 目的:探討SDF-1/CXCR4介導(dǎo)通路對(duì)小鼠骨折愈合的影響。方法:40只C57BL雄性小鼠隨機(jī)分為抑制組與對(duì)照組,采用切開(kāi)暴露股骨中段制造股骨干橫行骨折,插入髓內(nèi)針制作小鼠骨折愈合模型,抑制組小鼠每次每只每12小時(shí)1.25mg/kg局部皮下注射CXCR4抑制劑AMD3100,對(duì)照組小鼠注射等量磷酸緩沖鹽溶液;在模型構(gòu)建后第1、3、5、8周處死小鼠,采用放射學(xué)X線片觀測(cè)其影像學(xué)改變;采用免疫組織化學(xué)觀測(cè)指標(biāo)SDF-1、ERK、AKT在組織中的表達(dá)水平;采用實(shí)時(shí)定量熒光PCR觀測(cè)指標(biāo)COL1、COL2、CXCR4、VEGF在組織中的表達(dá)水平。結(jié)果:小鼠單側(cè)開(kāi)放性股骨干橫形骨折+髓內(nèi)針固定模型構(gòu)建成功,適用于小鼠骨折愈合的研究。兩組COL1、CXCR4、VEGF在組織中表達(dá)均逐漸上升,至第3周時(shí)達(dá)到高峰,后逐漸下降,且同時(shí)間對(duì)照組均高于抑制組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05);兩組COL2均在第1周即達(dá)到高峰,后快速下降,且同時(shí)間對(duì)照組均高于抑制組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。兩組SDF-1、ERK、AKT在組織中表達(dá)均逐漸上升,至第3周時(shí)達(dá)到高峰,后逐漸下降,且同時(shí)間對(duì)照組均高于抑制組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論:在小鼠中抑制SDF-1/CXCR4通路將會(huì)延緩骨折愈合。

      【關(guān)鍵詞】 骨折愈合 AMD3100 SDF-1 CXCR4

      doi:10.14033/j.cnki.cfmr.2020.19.003 文獻(xiàn)標(biāo)識(shí)碼 A 文章編號(hào) 1674-6805(2020)19-000-04

      Effect of SDF-1/CXCR4 Mediated AKT and ERK Pathway Inhibition on Delayed Fracture Healing in Mice/TANG Hao, CHEN Shunyou, LU Xiaokun, CHEN Fuming. //Chinese and Foreign Medical Research, 2020, 18(19): -9

      [Abstract] Objective: To investigate the the effect of SDF-1/CXCR4 pathway on fracture healing in mice. Method: A total of 40 C57BL male mice were randomly divided into the inhibition group and the control group. All mice operated by exposing the middle part of femur and making transverse fracture of femoral shaft, inserting intramedullary needle to make model of fracture healing in mice. The inhibition group was injected CXCR4 inhibitor AMD3100 1.25 mg/kg every 12 hours at a time by partial subcutaneous. The control group was injected with the same amount of phosphate buffer solution. The mice were sacrificed at week 1, 3, 5 and 8 after model construction, and radiological X-ray was used to observe the imaging changes. The expression levels of SDF-1, ERK and AKT in tissues were used as immunohistochemical observation indexes. Real-time quantitative fluorescence PCR was used to observe the expression levels of COL1, COL2, CXCR4 and VEGF in tissues. Result: The model of fracture healing in mice was established successfully by unilateral open transverse fracture of femoral shaft+intramedullary needle fixation. The expressions of COL1, CXCR4 and VEGF in the tissues of the two groups increased gradually, reached the peak at week 3, and then decreased gradually. In the same period, the control group was higher than the inhibition group, the differences were statistically significant (P<0.05). The COL2 in both groups reached its peak in the first week, and then decreased rapidly. In the same period, the COL2 in the control group was higher than that in the inhibition group, the differences were statistically significant (P<0.05). The expressions of SDF-1, ERK and AKT in the tissues of the two groups increased gradually, reached the peak at week 3, and then decreased gradually. Moreover, the expression of SDF-1, ERK and AKT in the control group were higher than those in the inhibition group at that time, the differences were statistically significant (P<0.05). Conclusion: Inhibition of SDF-1/CXCR4 pathway in mice will delay fracture healing.

      [Key words] Fracture healing AMD3100 SDF-1 CXCR4

      First-authors address: Fuzhou Second Hospital Affiliated to Xiamen University, Fuzhou 350007, China

      隨著年齡的增長(zhǎng)、骨質(zhì)的疏松,骨折的發(fā)生率將逐漸增高。根據(jù)流行病學(xué)調(diào)查,在中國(guó),全年齡骨折發(fā)病率為3.21%,由此,可推測(cè)大約有4.39億人發(fā)生過(guò)骨折[1]。骨折的成功愈合依賴于炎癥、血管形成、軟骨形成和成骨細(xì)胞的募集、遷移和歸巢[2]。有文獻(xiàn)[3]指出,將小鼠CXCR4基因敲除,在骨折愈合早期骨與血管新生都會(huì)下降。因此,SDF-1/CXCR4通路可能參與骨折愈合和血管新生。本實(shí)驗(yàn)旨在探究SDF-1/CXCR4通路可能對(duì)骨折預(yù)后的影響,具體如下。

      1 材料與方法

      1.1 實(shí)驗(yàn)材料

      實(shí)驗(yàn)動(dòng)物:SPF級(jí)C57BL雄性小鼠40只(福建中醫(yī)藥大學(xué)實(shí)驗(yàn)中心提供),體重20~25 g。

      1.2 小鼠骨折模型

      使用5%水合氯醛(6 μl/g,本實(shí)驗(yàn)約0.15 ml/只)麻醉C57BL小鼠,剃去體毛,擺好體位。75%酒精消毒3次,上至腹股溝下至腳趾,無(wú)菌紗布中央剪1.5 cm左右孔洞,覆蓋操作區(qū)。于股骨外側(cè)中部做一橫行切口,長(zhǎng)度大約1.5 cm,暴露股四頭肌肌間隙,用無(wú)齒鑷鈍性分離,暴露股骨,于膝關(guān)節(jié)處使用24 G注射器針頭鉆入股骨形成髓內(nèi)針,使用線剪在股骨中段垂直股骨干做一骨折端,斷端可見(jiàn)注射器針頭,則表示模型構(gòu)建成功。術(shù)中使用無(wú)菌紗布?jí)浩戎寡褂蒙睇}水稀釋注射用青霉素鈉粉末沖洗傷口,生理鹽水清洗傷口3次,用絲線縫合傷口處皮膚。

      1.3 SDF-1/CXCR4通路抑制

      對(duì)40只C57BL小鼠進(jìn)行隨機(jī)分組,抑制組20只,對(duì)照組20只,使用苦味酸標(biāo)記小鼠。抑制組術(shù)后每12小時(shí)右側(cè)股骨骨折斷端局部注射CXCR4抑制劑AMD3100(1.25 mg/kg,本實(shí)驗(yàn)約0.15 ml/只),分別持續(xù)1、3、5、8周;對(duì)照組在同樣部位同樣時(shí)間注射等量PBS。

      1.4 放射學(xué)分析

      術(shù)后第1、3、5、8周使用相同方法麻醉小鼠,雙下肢外展外旋體位拍攝X線片,請(qǐng)兩位骨科醫(yī)生評(píng)估骨痂、骨折線等,以評(píng)估骨折愈合情況。

      1.5 標(biāo)本處理

      術(shù)后第1、3、5、8周,每組每周處死5只,共40只,麻醉后脫頸處死小鼠,取右下肢,除去周圍軟組織,剝離出股骨,拔出髓內(nèi)針。PCR組織迅速放入凍存管,投入液氮中。完成取材后迅速放入-80 ℃冰箱保存。常規(guī)脫鈣。

      1.6 組織形態(tài)學(xué)測(cè)量與分析

      免疫組化:切片60 ℃恒溫箱烤制過(guò)夜,常規(guī)脫蠟至水;微波抗原修復(fù);磷酸緩沖鹽溶液漂洗3次,5 min/次;內(nèi)源性過(guò)氧化酶阻斷劑浸泡10 min;PBS漂洗3次,5 min/次;血清封閉30 min,4 ℃冰箱孵育一抗過(guò)夜(抗體濃度:AKT 1∶200,ERK 1∶200,SDF-1 1∶140);磷酸緩沖鹽溶液漂洗3次,5 min/次;DAB顯影2 min,超純水終止顯影;蘇木精染30 s,3道超純水漂洗;晾干封片。

      1.7 實(shí)時(shí)定量PCR檢測(cè)

      使用Vazyme公司實(shí)時(shí)定量PCR檢測(cè)試劑盒,具體詳見(jiàn)試劑盒說(shuō)明書(shū),目的基因引物序列見(jiàn)表1。

      1.8 統(tǒng)計(jì)學(xué)處理

      采用SPSS 20.0統(tǒng)計(jì)軟件進(jìn)行處理,計(jì)量資料以(x±s)表示,均采用正態(tài)性檢驗(yàn)后兩獨(dú)立樣本t檢驗(yàn)。P<0.05為有統(tǒng)計(jì)學(xué)意義。

      2 結(jié)果

      2.1 放射學(xué)觀察

      術(shù)后第1周,兩組骨折線均明顯,有骨痂形成,抑制組稍少于對(duì)照組;術(shù)后第3周,對(duì)照組有大量骨痂生成;術(shù)后第5周,抑制組仍可見(jiàn)骨折線,對(duì)照組骨折線幾乎不可見(jiàn),骨折基本愈合;術(shù)后第8周,抑制組與對(duì)照組骨折線均不明顯,骨折基本愈合,見(jiàn)圖1。

      2.2 兩組各周實(shí)時(shí)定量熒光PCR檢測(cè)結(jié)果比較

      兩組COL1、CXCR4、VEGF在組織中表達(dá)均逐漸上升,至第3周時(shí)達(dá)到高峰,后逐漸下降,且同時(shí)間對(duì)照組均高于抑制組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05);兩組COL2均在第1周即達(dá)到高峰,后快速下降,且同時(shí)間對(duì)照組均高于抑制組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05),見(jiàn)表2。

      2.3 兩組各周免疫組織化學(xué)檢測(cè)結(jié)果比較

      兩組SDF-1、ERK、AKT在組織中表達(dá)均逐漸上升,至第3周時(shí)達(dá)到高峰,后逐漸下降,且同時(shí)間對(duì)照組均高于抑制組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05),見(jiàn)表3。

      3 討論

      骨折的修復(fù)是一個(gè)復(fù)雜的生物學(xué)過(guò)程,依賴于間充質(zhì)干細(xì)胞激活后在骨折間隙中積累,增殖分化形成成骨細(xì)胞系,最后形成骨痂,重塑為新生骨。許多因素都會(huì)對(duì)骨折修復(fù)產(chǎn)生影響,包括祖細(xì)胞的缺乏或受損,其激活,歸巢,增殖和分化為成骨細(xì)胞譜系的分子調(diào)控紊亂,或缺乏適當(dāng)?shù)拇x環(huán)境來(lái)進(jìn)行骨折修復(fù)等[4]。

      本研究的目的在于研討SDF-1/CXCR4通路對(duì)骨折愈合的影響,通過(guò)抑制此通路發(fā)現(xiàn),骨折愈合相關(guān)指標(biāo)均在前3周中明顯升高,信號(hào)介導(dǎo)的下游通路也隨之升高,證明SDF-1/CXCR4通路通過(guò)下游AKT與ERK等通路影響骨折愈合,而同時(shí)間段對(duì)照組均高于抑制組,證實(shí)抑制SDF-1/CXCR4通路將會(huì)抑制骨折愈合。SDF-1/CXCR4通路在許多生理與病理過(guò)程中起重要作用,Wei等[5]研究表明,缺氧激活SDF-1/CXCR4通路,動(dòng)員和募集祖細(xì)胞調(diào)節(jié)缺氧的肺動(dòng)脈高壓與血管重塑,而使用AMD3100會(huì)抑制抑制SDF-1/CXCR4通路,抑制細(xì)胞周期與肺動(dòng)脈平滑肌的細(xì)胞增殖。Kucia等[6]認(rèn)為SDF-1在調(diào)節(jié)正常造血干細(xì)胞(HSC)的運(yùn)輸及其在骨髓中的歸巢中起著關(guān)鍵作用。SDF-1/CXCR4通路在骨折修復(fù)中除了對(duì)細(xì)胞增殖、歸巢有促進(jìn)作用以外,對(duì)細(xì)胞募集同樣也有促進(jìn)作用,文獻(xiàn)[7-9]報(bào)道,SDF-1在缺氧缺血的環(huán)境下高表達(dá),在機(jī)體出現(xiàn)損傷時(shí),SDF-1會(huì)在損傷部位高表達(dá)。COL1、COL2與軟骨形成有關(guān)[10-11],本研究中COL2表達(dá)量在第1周即達(dá)到頂峰,之后快速下降,第5、8周幾乎無(wú)表達(dá),COL1表達(dá)量在前3周較多,說(shuō)明在前3周中有大量軟骨細(xì)胞形成,之后迅速分化形成成骨細(xì)胞。COL1與成骨仍有相關(guān)[12],故在第5、8周仍有表達(dá)。VEGF是調(diào)節(jié)血管發(fā)育和血管生成最重要的生長(zhǎng)因子之一,由于骨是高度血管化的器官,血管的發(fā)育和生成對(duì)骨骼發(fā)育早期至關(guān)重要,VEGF能夠增強(qiáng)成骨細(xì)胞活性、刺激增殖、分化從而加速骨形成[13]。Berendsen等[14]認(rèn)為,成骨細(xì)胞衍生的VEGF會(huì)刺激間充質(zhì)細(xì)胞向成骨細(xì)胞分化,抑制其向脂肪細(xì)胞分化,維持人體骨穩(wěn)態(tài)。在本實(shí)驗(yàn)VEGF表達(dá)量對(duì)照組高于抑制組,與骨生成趨勢(shì)一致,這與上述文獻(xiàn)所表達(dá)的VEGF可以促進(jìn)骨質(zhì)生成相一致。但是也有文獻(xiàn)顯示使用VEGF與AMD3100作用于小鼠股骨干骨折愈合模型可以加快骨折愈合[15-19],與本研究結(jié)果相悖的原因可能是由于使用VEGF與AMD3100同時(shí)作用,VEGF對(duì)骨折的正向作用超過(guò)AMD3100對(duì)骨折愈合的負(fù)面作用。

      總而言之,SDF-1/CXCR4通路通過(guò)介導(dǎo)ERK和AKT通路調(diào)節(jié)骨折處血管新生、骨質(zhì)生成等生理過(guò)程,AMD3100作為CXCR4的抑制劑抑制了SDF-1/CXCR4通路導(dǎo)致血管新生、骨質(zhì)生成等相關(guān)指標(biāo)同時(shí)降低,故SDF-1/CXCR4通路可作為研究骨折愈合的關(guān)鍵靶點(diǎn),為研究骨折愈合提供一個(gè)思路。本研究的優(yōu)勢(shì)在于通過(guò)建立骨折后4個(gè)時(shí)間點(diǎn)觀察骨折愈合情況,前后對(duì)比,更具有說(shuō)服力,小鼠骨折愈合模型建立簡(jiǎn)單便捷,快速有效,很好地模擬了人體骨折。但本研究的不足之處在于無(wú)客觀指標(biāo)觀察骨痂新生、骨質(zhì)生成,僅通過(guò)X線觀察骨折端,由經(jīng)驗(yàn)豐富的臨床骨科醫(yī)生進(jìn)行判斷,具有一定的偏倚性。本研究下一階段將從基因?qū)用孢M(jìn)一步觀測(cè)通路介導(dǎo)信號(hào),并通過(guò)客觀指標(biāo)觀察骨痂新生、骨質(zhì)生成的具體數(shù)值。

      參考文獻(xiàn)

      [1] Lin X,Xiong D,Peng Y Q,et al.Epidemiology and management of osteoporosis in the Peoples Republic of China: current perspectives[J].Clinical Interventions in Aging,2015,25(10):1017-1033.

      [2] Marsell R,Einhorn T A.The biology of fracture healing[J].Injury,2011,42(6):551-555.

      [3] Kawakami Y,Ii M,Matsumoto T,et al.SDF-1/CXCR4 Axis in Tie2-lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing[J].Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research,2015,30(1):95-105.

      [4] Grzegorz S.Fracture Healing and its Disturbances. A literature Review[J].Ortopedia Traumatologia Rehabilitacja,2015,17(5):437-454.

      [5] Wei L,Zhang B,Cao W,et al.Inhibition of CXCL12/CXCR4 suppresses pulmonary arterial smooth muscle cell proliferation and cell cycle progression via PI3K/AKT pathway under hypoxia[J]. Journal of Receptors and Signal Transduction,2015,35(4):329-339.

      [6] Kucia M,Reca R,Miekus K,et al.Trafficking of Normal Stem Cells and Metastasis of Cancer Stem Cells Involve Similar Mechanisms: Pivotal Role of the SDF-1-CXCR4 Axis[J].Stem Cells,2005,23(7):879-894.

      [7] Kucia M,Ratajczak J,Reca R,et al.Tissue-specific muscle,neural and liver stem/progenitor cells reside in the bone marrow, respond to an SDF-1 gradient and are mobilized into peripheral blood during stress and tissue injury[J].Blood Cells Mol Dis,2004,32(1):52-57.

      [8] Devine M J,Mierisch C M,Jang E,et al.Transplanted bone marrow cells localize to fracture callus in a mouse model[J].Journal of Orthopaedic Research,2002,20(6):1232-1239.

      [9] Granero-Moltó F,Weis J A,Miga M I,et al.Regenerative Effects of Transplanted Mesenchymal Stem Cells in Fracture Healing[J].Stem Cells,2009,27(8):1887-1898.

      [10] Haltmayer E,Ribitsch I,Gabner S,et al.Co-culture of osteochondral explants and synovial membrane as in vitro model for osteoarthritis[J].PloS One,2019,14(4):e0214709.

      [11] Dong R,Ying J,Xu T,et al.Bushenhuoxue Formula Facilitates Articular Cartilage Repair and Attenuates Matrix Degradation by Activation of TGF-β Signaling Pathway[J].Evid Based Complement Alternat Med,2018,2018:2734581.

      [12] Aghajanian P,Xing W,Cheng S,et al.Epiphyseal bone formation occurs via thyroid hormone regulation of chondrocyte to osteoblast transdifferentiation[J].Scientific Reports,2017,7(1):10432.

      [13] Liu Y,Olsen B R.Distinct VEGF Functions During Bone Development and Homeostasis[J].Archivum Immunologiae et Therapiae Experimentalis,2014,62(5):363-368.

      [14] Berendsen A D,Olsen B R.How Vascular Endothelial Growth Factor-A (VEGF) Regulates Differentiation of Mesenchymal Stem Cells[J].Journal of Histochemistry & Cytochemistry,2014,62(2):103-108.

      [15] Meeson R,Sanghani-Keri A,Coathup M,et al.VEGF with AMD3100 endogenously mobilizes mesenchymal stem cells and improves fracture healing[J].J Orthop Res,2019,37(6):1294-1302.

      [16]Xu J,Mora A L,Shim H,et al.298 role of the SDF-1/CXCR4 axis in the pathogenesis of lung injury and pulmonary fibrosis[J].Journal of Investigative Medicine,2006,54(1):S309.

      [17]Thomas M N,Kalnins A,Andrassy M,et al. SDF-1/CXCR4/CXCR7 is pivotal for vascular smooth muscle cell proliferation and chronic allograft vasculopathy[J].Transplant International,2015,28(12):1426-1435.

      [18]Liao A,Shi R,Jiang Y,et al.Retracted Article:SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Up-regulation of Survivin in Glioblastoma[J].Molecular Neurobiology,2016,53(1):210-215.

      [19]Zhang J,Ren X,Shi W,et al.Small molecule Me6TREN mobilizes hematopoietic stem/progenitor cells by activating MMP-9 expression and disrupting SDF-1/CXCR4 axis[J].Blood,2014,123(3):428.

      (收稿日期:2020-04-08) (本文編輯:馬竹君)

      *基金項(xiàng)目:福建省衛(wèi)生計(jì)生科研人才培養(yǎng)項(xiàng)目(2017-CX-46)

      ①?gòu)B門大學(xué)附屬福州第二醫(yī)院 福建 福州 350007

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