慢性牙周炎中腫瘤壞死因子ɑ對(duì)骨髓間充質(zhì)干細(xì)胞成骨分化的調(diào)控作用

李曉光 王一珠 郭斌

中國(guó)人民解放軍總醫(yī)院口腔醫(yī)學(xué)中心，北京 100853

慢性牙周炎中腫瘤壞死因子ɑ對(duì)骨髓間充質(zhì)干細(xì)胞成骨分化的調(diào)控作用

李曉光 王一珠 郭斌

中國(guó)人民解放軍總醫(yī)院口腔醫(yī)學(xué)中心，北京 100853

骨髓間充質(zhì)干細(xì)胞（BMSCs）在重構(gòu)牙周組織結(jié)構(gòu)和功能、促進(jìn)牙周炎好轉(zhuǎn)乃至愈合方面發(fā)揮重要作用，因此BMSCs的特性尤其是其成骨分化的調(diào)控機(jī)制是目前的研究熱點(diǎn)。腫瘤壞死因子α（TNF-α）是牙周組織炎癥微環(huán)境中的主要促炎因子，與BMSCs的成骨分化密切相關(guān)。探究TNF-α調(diào)控BMSCs成骨分化的機(jī)制有助于明確牙周炎的發(fā)病機(jī)制，尋找牙周疾病新的治療靶點(diǎn)，改善牙周炎的治療效果。本文將針對(duì)TNF-α在牙周炎發(fā)生發(fā)展過程中發(fā)揮的重要作用尤其是調(diào)控BMSCs成骨分化的可能機(jī)制作一綜述。

慢性牙周炎； 腫瘤壞死因子α； 骨髓間充質(zhì)干細(xì)胞； 成骨分化

慢性牙周炎是一種常見的以口腔致病微生物為始動(dòng)因子、以牙周袋形成和牙槽骨吸收為主要臨床特征的慢性炎性疾病。在牙周致病菌刺激下牙周組織中集聚大量的炎癥因子激活炎性反應(yīng)過程，并打破骨改建的內(nèi)穩(wěn)態(tài)，從而造成機(jī)體成骨能力下降和牙周骨組織損傷[1]。主要來源于骨髓間充質(zhì)干細(xì)胞（bone marrow mesenchymal stem cells，BMSCs）成骨向分化的成骨細(xì)胞是骨形成的重要細(xì)胞學(xué)基礎(chǔ)，炎癥等因素可以抑制BMSCs分化和增殖能力，使成骨細(xì)胞生成數(shù)量下降，最終導(dǎo)致骨量減少，這是牙周炎發(fā)生發(fā)展的重要機(jī)制之一[2]。腫瘤壞死因子α（tumor necrosis factor-α，TNF-α）是牙周病患者牙周組織中存在的一種主要促炎因子，其表達(dá)水平與牙周炎的活動(dòng)性密切相關(guān)。TNF-α還會(huì)影響B(tài)MSCs的成骨分化[3]，但是具體機(jī)制尚不明確。因此，探究TNF-α調(diào)控BMSCs成骨分化的機(jī)制，并在此基礎(chǔ)上逆向調(diào)控，促進(jìn)BMSCs成骨分化，改善機(jī)體骨代謝平衡，促進(jìn)慢性牙周炎的好轉(zhuǎn)乃至愈合成為研究的熱點(diǎn)。本文對(duì)TNF-α在BMSCs成骨分化過程中發(fā)揮的調(diào)控作用及可能機(jī)制作一綜述。

1 BMSCs是促進(jìn)牙周組織再生及慢性牙周炎愈合的基礎(chǔ)

重建正常的牙周組織是牙周治療的主要目標(biāo)，但是現(xiàn)有的牙周治療手段，如牙周序列治療、生長(zhǎng)因子或異種材料移植術(shù)以及引導(dǎo)組織再生術(shù)等只能部分修復(fù)牙周組織，不能實(shí)現(xiàn)牙周組織的生理和功能性再生且療效不穩(wěn)定[4]。組織工程技術(shù)的出現(xiàn)為牙周組織再生和牙周疾病的治療提供了新的思路，其核心要素是具有多向分化潛能的各類干細(xì)胞。相較于牙周膜干細(xì)胞等牙源性干細(xì)胞，BMSCs在牙周組織再生治療中具有獨(dú)特的優(yōu)勢(shì)。研究[5]發(fā)現(xiàn)，BMSCs具有免疫調(diào)節(jié)功能，減少炎性因子的產(chǎn)生和定向遷移，還可直接或間接發(fā)揮抗菌和保護(hù)組織的功能；還有研究[6]證實(shí)炎性狀態(tài)下BMSCs較牙周膜干細(xì)胞具有更強(qiáng)的成骨分化能力。因此，BMSCs是牙周組織再生治療中主要的干細(xì)胞來源，探索炎性條件下BMSCs成骨分化的調(diào)控機(jī)制，提高其成骨效率，促進(jìn)牙周骨組織結(jié)構(gòu)和功能重建已成為研究的熱點(diǎn)。

2 TNF-α在牙周炎發(fā)生發(fā)展過程中發(fā)揮重要作用

2.1 TNF-α是牙周組織炎癥微環(huán)境中的重要因子

慢性牙周炎是以菌斑為始動(dòng)因子，牙齦、牙槽骨、牙周膜等牙周組織破壞為臨床特征的慢性炎癥性疾病。細(xì)菌侵入對(duì)牙周組織的直接破壞作用是有限的，而由細(xì)菌激發(fā)的宿主免疫反應(yīng)使牙周組織的微環(huán)境發(fā)生炎性改變是造成牙周組織破壞的主要原因[7]。TNF-α是牙周組織炎性微環(huán)境中最重要的內(nèi)源性誘生型促炎因子，研究[8]發(fā)現(xiàn)牙周炎患者齦溝液中TNF-α含量明顯升高，并且其表達(dá)水平與牙周炎的嚴(yán)重程度密切相關(guān)。TNF-α與細(xì)胞膜上的腫瘤壞死因子受體結(jié)合后產(chǎn)生多種生物學(xué)效應(yīng)，與Ⅱ型腫瘤壞死因子受體結(jié)合后可調(diào)節(jié)炎癥反應(yīng)[9]。研究[10]發(fā)現(xiàn)TNF-α促進(jìn)炎癥細(xì)胞侵入牙周組織，釋放金屬蛋白酶，降解細(xì)胞外基質(zhì)，不僅導(dǎo)致牙槽骨吸收和膠原纖維的破壞，還會(huì)抑制牙齦和牙周膜成纖維細(xì)胞的增殖。因此，TNF-α在牙周組織微環(huán)境的炎性改變中發(fā)揮重要作用。

2.2 TNF-α通過調(diào)控BMSCs分化影響牙周骨組織改建

在生理?xiàng)l件下維持骨的正常強(qiáng)度和完整性需要BMSCs以合適的比例分化，繼而在成骨細(xì)胞導(dǎo)致的新骨形成和破骨細(xì)胞介導(dǎo)的舊骨吸收之間保持穩(wěn)定的平衡，這種平衡被稱為骨改建。但在炎癥微環(huán)境中，TNF-α破壞骨改建的內(nèi)穩(wěn)態(tài)，使骨代謝發(fā)生紊亂，加速骨組織的喪失。研究[11]證實(shí)TNF-α可增強(qiáng)破骨并抑制成骨，這種雙重作用會(huì)破壞骨微構(gòu)架，造成嚴(yán)重的骨吸收破壞。目前，針對(duì)TNF-α加速骨吸收的研究較多且形成了一定共識(shí)。首先，TNF-α可通過核因子κB受體活化因子配體（receptor activator for nuclear factor-κB ligand，RANKL）/骨保護(hù)素（osteoprotegerin，OPG）信號(hào)通路影響破骨細(xì)胞的激活分化。研究[12]發(fā)現(xiàn)TNF-α誘導(dǎo)成骨細(xì)胞分泌RANKL和巨噬細(xì)胞集落刺激因子（macrophage colony-stimulating factor，M-CSF），繼而激活核因子κB（nuclear factor κB，NF-κB）促進(jìn)破骨細(xì)胞的分化。有學(xué)者[13]指出TNF-α可能是通過促進(jìn)細(xì)胞內(nèi)活性氧（reactive oxygen species，ROS）積累和鈣離子振蕩影響RANKL對(duì)破骨細(xì)胞分化的調(diào)控。其次，TNF-α可誘導(dǎo)成熟的破骨細(xì)胞增加骨的再吸收循環(huán)[14]。

TNF-α不僅促進(jìn)骨吸收，還可抑制骨形成，從而加劇骨改建的失衡。TNF-α可能通過以下方式影響成骨細(xì)胞的分化、成熟和凋亡：1）抑制BMSCs的成骨分化。研究[15]發(fā)現(xiàn)在干細(xì)胞成骨分化過程中TNF-α可以抑制骨鈣蛋白、Ⅰ型膠原和成骨分化因子Runt相關(guān)轉(zhuǎn)錄因子2（Runt-related transcription factor 2，Runx2）的表達(dá)，但是具體機(jī)制尚未明確，這是因?yàn)門NF-α與Ⅰ型腫瘤壞死因子受體結(jié)合后參與多種信號(hào)通路對(duì)BMSCs成骨分化的調(diào)控[16]，厘清具體機(jī)制存在一定困難。2）TNF-α還可激活NF-κB信號(hào)通路加速成骨細(xì)胞凋亡[17]。但是，也有學(xué)者[18]認(rèn)為在炎性條件下TNF-α?xí)碳す堑男纬?，并推斷TNF-α具有不同的功能，可能與TNF-α的干預(yù)時(shí)間或骨形成的不同階段有關(guān)[19]。因此，TNF-α對(duì)骨改建發(fā)揮著復(fù)雜卻重要的調(diào)控作用，但TNF-α調(diào)控骨改建的具體機(jī)制尚需深入研究。

3 TNF-α調(diào)控BMSCs成骨分化的可能機(jī)制

3.1 通過Wnt信號(hào)通路影響B(tài)MSCs成骨分化

Wnt通路是目前已知的和BMSCs成骨分化關(guān)系最密切的信號(hào)通路之一。Wnt信號(hào)通路以是否有β連環(huán)蛋白（β-catenin）參與分為經(jīng)典Wnt信號(hào)通路和非經(jīng)典信號(hào)通路。研究發(fā)現(xiàn)經(jīng)典/非經(jīng)典Wnt信號(hào)通路均在BMSCs成骨分化過程中發(fā)揮重要作用，并且兩者之間還相互影響，例如Wnt3α促進(jìn)BMSCs的增殖，但Wnt5α可通過非經(jīng)典途徑拮抗Wnt3a的作用[20]。研究[21]發(fā)現(xiàn)經(jīng)典Wnt通路可通過增強(qiáng)成骨相關(guān)轉(zhuǎn)錄因子Runx2、Osterix的表達(dá)促使BMSCs向成骨細(xì)胞分化。目前針對(duì)非經(jīng)典Wnt通路的研究還較少，有學(xué)者[22]認(rèn)為非經(jīng)典Wnt通路配體Wnt5通過抑制成脂標(biāo)志物過氧化物酶體增殖受體的表達(dá)調(diào)節(jié)BMSCs的成骨分化。

研究[23]證實(shí)在BMSCs成骨分化過程中，TNF-α可在一定程度上抑制經(jīng)典Wnt通路的激活，但調(diào)控的具體機(jī)制仍無定論。有學(xué)者研究發(fā)現(xiàn)在某些慢性炎癥條件下，TNF-α通過抑制Wnt/β-catenin的表達(dá)使BMSCs由成骨分化向成脂分化轉(zhuǎn)變[24]，并推測(cè)TNF-α通過改變細(xì)胞的氧化應(yīng)激狀態(tài)抑制Wnt/β-catenin的表達(dá)[25]。還有學(xué)者[26]發(fā)現(xiàn)TNF-α可以增強(qiáng)Wnt通路抑制劑Dickkopf-1的表達(dá)，從而抑制Wnt/β-catenin的活性及間充質(zhì)干細(xì)胞的成骨分化，并且在降低TNF-α水平后可在一定程度上恢復(fù)細(xì)胞的成骨分化能力。研究[27]顯示糖原合成激酶-3β（glycogen synthase kinase 3β，GSK-3β）是TNF-α抑制Wnt/β-catenin活性調(diào)控干細(xì)胞成骨分化的關(guān)鍵因子，TNF-α可以增強(qiáng)GSK-3β活性，繼而使β-catenin磷酸化并降解，阻止其進(jìn)入細(xì)胞核與淋巴樣增強(qiáng)因子、T細(xì)胞因子形成聚合物后調(diào)節(jié)細(xì)胞生理功能[28]。但也有學(xué)者認(rèn)為TNF-α抑制GSK-3β、增強(qiáng)Wnt/β-catenin活性是其抑制細(xì)胞成骨分化的重要途徑[29]，這可能與干細(xì)胞的種類不同有關(guān)。而針對(duì)TNF-α與非經(jīng)典Wnt通路關(guān)系的研究還較少，研究[30]發(fā)現(xiàn)炎性狀態(tài)下高表達(dá)的β-catenin抑制非經(jīng)典Wnt/Ca2+通路進(jìn)而阻止牙周膜干細(xì)胞的成骨分化，也有學(xué)者[31]認(rèn)為TNF-α通過激活非經(jīng)典Wnt/Ca2+通路抑制細(xì)胞的成骨分化。但是TNF-α通過非經(jīng)典Wnt通路調(diào)控BMSCs成骨分化還鮮有報(bào)道。

3.2 通過骨形態(tài)發(fā)生蛋白（bone morphogentic protein，BMP）信號(hào)通路影響B(tài)MSCs成骨分化

BMP屬于轉(zhuǎn)化生長(zhǎng)因子β超家族，能誘導(dǎo)BMSCs骨向、軟骨向等分化，其中BMP2在BMSCs的生長(zhǎng)分化過程中起重要作用。而關(guān)于TNF-α對(duì)BMP2-Smad信號(hào)通路的影響，目前尚存在一定爭(zhēng)議。大量研究[32]顯示TNF-α可以通過絲裂原活化蛋白激酶（m itogenactivated protein kinase，MAPK）/ c-Jun氨基末端激酶（c-Jun N-term inal kinase，JNK）、NF-κB、P38/ MAPK等信號(hào)通路間接抑制BMP2的表達(dá)以及其介導(dǎo)的BMSCs的成骨分化，但有學(xué)者[33]提出TNF-α還可通過上述途徑促進(jìn)BMP2表達(dá)和成骨分化。TNF-α對(duì)BMP2不同的調(diào)控作用主要取決于TNF-α干預(yù)的濃度和時(shí)間[32]。研究[34]發(fā)現(xiàn)低濃度TNF-α干預(yù)較短時(shí)間，TNF-α?xí)龠M(jìn)BMP2、Smad1的表達(dá)及BMSCs的成骨分化，干預(yù)較長(zhǎng)時(shí)間后TNF-α的功能卻會(huì)逆轉(zhuǎn)為明顯抑制；較高濃度TNF-α干預(yù)時(shí)，BMP2表達(dá)增強(qiáng)，但Smad1的表達(dá)以及BMSCs的成骨分化受到明顯抑制。出現(xiàn)這一矛盾的原因可能是較高濃度TNF-α及其激活的NF-κB信號(hào)通路提升Smad7（BMP2抑制因子）的表達(dá)水平，從而使BMP2下游Smad1/5/8信號(hào)的磷酸化水平顯著減弱，并使細(xì)胞對(duì)BMP2刺激的反應(yīng)性降低[35]，也可能是因?yàn)門NF-α抑制Smad蛋白與下游靶基因的結(jié)合[36]。

3.3 通過MAPKs信號(hào)通路影響B(tài)MSCs成骨分化

MAPKs是細(xì)胞內(nèi)的一類絲氨酸/蘇氨酸蛋白激酶，與BMSCs成骨分化相關(guān)的主要是細(xì)胞外信號(hào)調(diào)節(jié)激酶（extracellular signal-regulated kinase，ERK）、JNK和p38MAPK通路。目前學(xué)者[37]認(rèn)為TNF-α主要通過ERK通路影響B(tài)MSCs的成骨分化。誘導(dǎo)BMSCs成骨分化時(shí)存在ERK通路的激活，而且抑制ERKs活性能阻斷BMSCs成骨分化。研究[38]發(fā)現(xiàn)TNF-α抑制MAPK激酶1的活性加速ERK的磷酸化，還有學(xué)者[39]證實(shí)TNF-α可上調(diào)神經(jīng)細(xì)胞黏附因子的表達(dá)激活ERK通路促進(jìn)BMSCs的遷移活化。近年來部分學(xué)者[40]認(rèn)為TNF-α還可以通過激活JNK和p38MAPK通路對(duì)成骨分化發(fā)揮正向調(diào)控作用。上述的研究表明TNF-α在體外可以激活MAPKs信號(hào)通路對(duì)干細(xì)胞成骨分化起到一定促進(jìn)作用而不是抑制作用，這可能與BMSCs來源部位、供體所患疾病、TNF-α的干預(yù)時(shí)間不同有關(guān)，因此在體內(nèi)BMSCs成骨分化過程中TNF-α與MAPK通路的關(guān)系還需進(jìn)一步驗(yàn)證。

3.4 通過m iRNA影響B(tài)MSCs成骨分化

m iRNA是一類參與基因轉(zhuǎn)錄后水平調(diào)控的小分子、非編碼的單鏈RNA，主要在轉(zhuǎn)錄后水平負(fù)向調(diào)控靶mRNA的表達(dá)，參與細(xì)胞增殖和分化的調(diào)控[41]，因其具有高度的保守性和特異性，且調(diào)控作用具有級(jí)聯(lián)放大效應(yīng)，因此日益得到學(xué)者關(guān)注。m iRNA不僅在牙周組織炎性微環(huán)境改變中發(fā)揮重要作用[42]，還參與調(diào)控BMSCs成骨分化，更與TNF-α關(guān)系密切。研究[43]發(fā)現(xiàn)m iR-155參與TNF-α介導(dǎo)的成骨分化，二者通過靶基因細(xì)胞因子信號(hào)抑制因子1（suppressor of cytokine signaling 1，SOCS1）和應(yīng)激活化蛋白激酶（stress activated protein kinase，SAPK）通路抑制成骨分化，m iR-30a也被證實(shí)有類似功能；而過表達(dá)m iR-21可抑制Spry1從而逆轉(zhuǎn)TNF-α對(duì)成骨分化的抑制作用[44]。但是也有學(xué)者[45]認(rèn)為m iRNA在TNF-α作用下隨環(huán)境變化對(duì)成骨分化發(fā)揮不同的調(diào)控作用。

綜上所述，TNF-α可以通過多種途徑調(diào)控BMSCs成骨分化，相關(guān)研究較多但難以形成共識(shí)，這可能是因?yàn)椋?）TNF-α參與多種信號(hào)通路形成錯(cuò)綜復(fù)雜的直接或間接影響B(tài)MSCs成骨分化的調(diào)控網(wǎng)絡(luò)，通路間相互作用，因此厘清TNF-α調(diào)控BMSCs成骨分化的機(jī)制尚有難度；2）影響因素較多，如BMSCs來源、TNF-α干預(yù)劑量和時(shí)間等都會(huì)對(duì)實(shí)驗(yàn)結(jié)果產(chǎn)生影響；3）倫理及炎癥模型難以建立等原因，目前大部分研究均為體外實(shí)驗(yàn)，難以精確模擬體內(nèi)炎性環(huán)境，也在一定程度上阻礙對(duì)TNF-α功能的認(rèn)識(shí)。

慢性牙周炎的現(xiàn)有治療手段難以重構(gòu)牙周組織正常組織結(jié)構(gòu)和生理功能，以BMSCs為主要細(xì)胞來源的牙周組織再生技術(shù)在促進(jìn)牙槽骨再生、加速牙周疾病的好轉(zhuǎn)乃至愈合等方面發(fā)揮著獨(dú)特的作用。TNF-α作為主要促炎因子，在牙周組織微環(huán)境的炎性改變及BMSCs成骨分化中發(fā)揮關(guān)鍵作用，通過對(duì)TNF-α調(diào)控BMSCs成骨分化機(jī)制的研究，將為恢復(fù)牙周組織形態(tài)和功能探尋新的途徑。

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（本文編輯 杜冰）

Tumor necrosis factor-α regulates the osteogenic differentiation of bone marrow mesenchym al stem cells in chronic periodontitis

Li Xiaoguang, Wang Yizhu, Guo Bin. (Institution of Stomatology, The PLA General Hospital, Beijing 100853,China)

Bone marrow mesenchymal stem cells (BMSCs) and ideal adult stem cells for alveolar bone regeneration considerably help restore the structure and function of the periodontium and promote the healing of periodontal disease. Thus, BMSC features, especially the mechanism of osteogenic differentiation, has recently become a research hotspot. Tumor necrosis factor-α (TNF-α), which is the main factor in the periodontal inflammatory m icroenvironment, is directly related to the osteogenic differentiation of BMSCs. Exploring the TNF-α-regulated differentiation mechanism of BMSCs aids in the search for new treatment targets. Such investigation also promotes the development of stem cell therapy for periodontal diseases. This article aims to describe the potential of TNF-α in regulating the osteogenic differentiation of stem cells.

chronic periodontitis; tumor necrosis factor-α; bone marrow mesenchymal stem cells; osteogenic differentiation

Q 257

A

10.7518/hxkq.2017.03.019

Supported by: The National Natural Science Foundation of China (81470754). Correspondence: Guo Bin, E-mail: guobin0408 @126.com.

2016-05-13；

2017-03-20

國(guó)家自然科學(xué)基金面上項(xiàng)目（81470754）

李曉光，住院醫(yī)師，博士，E-mail：xiaopa3084@126.com

郭斌，教授，博士，E-mail：guobin0408@126.com