賈玲玲 萬乾炳

口腔疾病研究國家重點(diǎn)實(shí)驗(yàn)室，國家口腔疾病臨床研究中心，四川大學(xué)華西口腔醫(yī)院口腔修復(fù)科，成都 610041

·綜述·

基質(zhì)金屬蛋白酶抑制劑的研究進(jìn)展

賈玲玲 萬乾炳

口腔疾病研究國家重點(diǎn)實(shí)驗(yàn)室，國家口腔疾病臨床研究中心，四川大學(xué)華西口腔醫(yī)院口腔修復(fù)科，成都 610041

牙本質(zhì)粘接混合層的長期穩(wěn)定性不佳，基質(zhì)金屬蛋白酶等內(nèi)源性酶降解膠原是導(dǎo)致混合層破壞的重要因素。使用酶抑制劑抑制膠原降解，維持膠原結(jié)構(gòu)的完整性，是提高混合層穩(wěn)定性的關(guān)鍵。本文重點(diǎn)總結(jié)了基質(zhì)金屬蛋白酶抑制劑（包括氯己定、乙二胺四乙酸、季銨鹽類、鋅離子和氧化鋅、四環(huán)素類及其衍生物、異羥肟酸類抑制劑、二磷酸鹽衍生物、交聯(lián)劑等）的研究進(jìn)展，并對未來的發(fā)展進(jìn)行展望。

基質(zhì)金屬蛋白酶； 半胱氨酸組織蛋白酶； 牙本質(zhì)； 酶抑制劑； 生物降解； 耐久性

在牙本質(zhì)粘接過程中，酸蝕劑或者酸性樹脂單體使牙本質(zhì)脫礦，粘接劑單體滲入脫礦的膠原纖維網(wǎng)中，包裹膠原纖維形成混合層，這是牙本質(zhì)粘接的基礎(chǔ)[1]。由于膠原纖維網(wǎng)結(jié)構(gòu)的復(fù)雜性，現(xiàn)有的粘接劑難以對膠原纖維形成完全的滲入和包裹[2-3]，且樹脂的水解也會導(dǎo)致原本被包裹的膠原暴露[4]。

基質(zhì)金屬蛋白酶（matrix metalloproteinases，MMPs）是一類鈣、鋅離子依賴的蛋白水解酶。研究表明，牙本質(zhì)中含有明膠酶（MMP-2和MMP-9）[5-6]、間質(zhì)溶解素1（MMP-3）[7]、釉質(zhì)素（MMP-20）[8]和膠原酶-2（MMP-8）[9]等。全酸蝕和自酸蝕粘接系統(tǒng)都能夠激活MMPs[10-11]，活化的MMPs能降解暴露的膠原纖維[12-14]，導(dǎo)致混合層完整性被破壞。Tersariol等[15]發(fā)現(xiàn)，牙本質(zhì)中存在另一個重要的酶家族—半胱氨酸組織蛋白酶。研究[16-17]發(fā)現(xiàn)，齲壞牙本質(zhì)中的組織蛋白酶含量明顯高于正常牙本質(zhì)，提示組織蛋白酶可能也參與了牙本質(zhì)膠原的降解。使用酶抑制劑抑制膠原降解，維持膠原結(jié)構(gòu)的完整性，是提高混合層穩(wěn)定性的關(guān)鍵。目前，使用酶抑制劑來保護(hù)混合層的完整性是研究的熱點(diǎn)，本文對基質(zhì)金屬蛋白酶抑制劑的研究進(jìn)展進(jìn)行綜述。

1 氯己定（clorhexidine，CHX）

CHX是目前研究最多的非特異性MMPs抑制劑，能有效抑制MMP-2、-9和-8[18]。其機(jī)制可能是CHX通過螯合作用結(jié)合鈣、鋅離子，從而抑制MMPs催化區(qū)域的活性[19]。Scaffa等[20]研究發(fā)現(xiàn)，CHX對半胱氨酸組織蛋白酶也有很強(qiáng)的抑制作用。CHX的有效濃度為0.002%～4%[21]，最常用的濃度是0.2%和2%。CHX可以單獨(dú)作為一種預(yù)處理劑[22-24]，但其研究結(jié)果尚存在爭議。Stanislawczuk等[22]研究發(fā)現(xiàn)，2.0%CHX預(yù)處理能夠有效減緩牙本質(zhì)粘接混合層的降解。但Mobarak[23]和Lührs等[24]的研究顯示，2.0%CHX預(yù)處理雖然不影響即刻粘接強(qiáng)度，但并不能減緩粘接強(qiáng)度的降低。也有研究者[25-26]嘗試將CHX加入到粘接劑或處理劑中，但研究結(jié)果尚無統(tǒng)一的定論。Sabatini[25]將0.2%CHX加入到Peak Universal Bond的粘接劑中，發(fā)現(xiàn)0.2%CHX的加入不影響即刻粘接強(qiáng)度，且能抑制MMPs活性，但是經(jīng)6個月水老化后，0.2% CHX組與對照組之間的粘接強(qiáng)度并無統(tǒng)計(jì)學(xué)差異，提示0.2%CHX不能提高粘接耐久性。Zhou等[26]將CHX加入Clearfil SE Bond的處理劑中，形成含有不同濃度CHX的處理劑，結(jié)果發(fā)現(xiàn)雖然0.05%CHX不能減少粘接強(qiáng)度的降低，但0.1%、0.5%和1.0%CHX能夠有效減少粘接強(qiáng)度的降低。

Hebling等[27]最早發(fā)現(xiàn)，使用2.0%葡萄糖酸氯己定（chlorhexidine digluconate）作為一種治療性的預(yù)處理劑能夠減少粘接界面的降解。隨后大量的臨床研究也得出相同的結(jié)論[28-30]。但是Sartori等[31]經(jīng)過3年的臨床研究發(fā)現(xiàn)，2.0%葡萄糖酸氯己定預(yù)處理牙本質(zhì)并不能提高粘接耐久性，其成功率為88%，對照組成功率為76%，二者間無統(tǒng)計(jì)學(xué)差異；且二者在術(shù)后敏感性、邊緣變色、邊緣完整性、繼發(fā)齲發(fā)生率等方面也都無統(tǒng)計(jì)學(xué)差異。產(chǎn)生這種差異的原因可能與CHX和膠原的結(jié)合機(jī)制有關(guān)。CHX通過靜電作用結(jié)合到膠原上[32]，這種結(jié)合并不穩(wěn)定，CHX逐漸被來自牙本質(zhì)小管液或者唾液中的競爭性離子取代，從粘接界面溢出而失去對酶的抑制作用?；谶@些特性，短期臨床研究取得較好的結(jié)果，而長期研究則顯示葡萄糖酸氯己定并不能提高牙本質(zhì)粘接的耐久性。目前，對于CHX的臨床研究絕大多數(shù)是針對全酸蝕系統(tǒng)，對于自酸蝕系統(tǒng)的研究還比較缺乏。

2 乙二胺四乙酸（ethylene diamine tetraphosphonic acid，EDTA）

EDTA是一種鈣離子螯合劑，含有4個羧基基團(tuán)，可與鈣離子結(jié)合形成可溶性鈣鹽。Thompson等[33]研究發(fā)現(xiàn)，17%EDTA能抑制rhMMP-9和牙本質(zhì)MMPs，并且呈時(shí)間依賴性。EDTA酸蝕后的牙本質(zhì)，其粘接混合層抗降解能力增強(qiáng)[34-36]，并且與乙醇濕粘接技術(shù)聯(lián)合運(yùn)用可獲得更大的即刻粘接強(qiáng)度[37]。但是EDTA酸蝕作用緩慢，且與牙本質(zhì)結(jié)合疏松，容易被水洗脫，故其臨床適用性還有待進(jìn)一步的研究。

3 季銨鹽類

季銨鹽類化合物是一類抗菌試劑，帶正電荷。MMPs活性區(qū)域含有半胱氨酸重復(fù)序列，重復(fù)序列中的谷氨酸殘基對酶的活性至關(guān)重要。谷氨酸為二元羧酸，在中性pH條件下含有一個自由羧基，并且在肽鍵形成后保留了一個負(fù)電荷。帶正電的季銨鹽通過靜電作用與這些負(fù)電荷結(jié)合，阻止酶的活性區(qū)域與膠原的結(jié)合，從而抑制酶的活性[38]。甲基丙酰氧十二烷基溴吡啶（12-methacryloyloxydodecylpyrid inium bromide，MDPB）是一類可聚合的甲基丙烯酸酯季銨鹽，具有抗菌性和可與樹脂單體共聚的特性，已被應(yīng)用到自酸蝕粘接劑Clearfil Protect Bond中[39]。體內(nèi)和體外研究[40-41]都表明，使用含MDPB的粘接劑能夠獲得耐久性更長的粘接界面。Tezvergil-Mutluay等[38]研究發(fā)現(xiàn)，5.0%MDPB能夠有效抑制rhMMP-9和與牙本質(zhì)膠原結(jié)合的MMPs。最近研究[42]表明，MDPB還能夠抑制半胱氨酸組織蛋白酶的活性。相比于其他的抑制劑，MDPB的最大優(yōu)勢是能夠與粘接劑單體共聚，這使得MDPB不易從粘接界面中溢出，從而保持更長久的MMP抑制性[41]，因而提高牙本質(zhì)粘接的耐久性。苯扎氯銨（benzalkonium chloride，BAC）是具有不同長度烷基鏈的烷基二甲胺鹽酸鹽混合物，含有季銨基團(tuán)，擁有廣譜抗菌活性。研究[43]表明，BAC在37%磷酸中能保持穩(wěn)定并且能夠抑制MMPs。Sabatini等[44-45]將BAC加入粘接劑中，使其濃度分別為0.25%、0.5%、1.0%和2.0%，結(jié)果表明所有濃度都能夠抑制膠原降解。最近一項(xiàng)研究[46]表明，在粘接劑Adper Single Bond Plus中加入0.5%和1%BAC，結(jié)果發(fā)現(xiàn)含0.5%BAC組粘接強(qiáng)度在一年內(nèi)僅降低9%，而不含BAC的對照組粘接強(qiáng)度降低44%。甲基丙烯酸二甲氨基十二烷基酯（dimethylaminododecyl methacrylate，DMADDM）是一種新合成的季銨單體，具有抗菌活性[47]。Li等[48]研究發(fā)現(xiàn)，0.1%、1%、2.5%、5%、7.5%和10%DMADDM能抑制rhMMP-8和rhMMP-9，并且呈濃度依賴性，其中5%DMADDM能抑制90%的rhMMP-8和rhMMP-9，同時(shí)還能抑制牙本質(zhì)彈性模量的降低（實(shí)驗(yàn)組降低34%，對照組降低73%）。

4 鋅離子和氧化鋅

MMPs是一類鈣、鋅離子依賴性的蛋白水解酶，微量的鋅是保持MMPs活性所必需的[19]。過量濃度的鋅離子能夠抑制MMPs[49-50]。Souza等[50]通過酶譜法研究發(fā)現(xiàn)，ZnCl2能夠有效抑制MMP-2和MMP-9。研究[51-53]表明，ZnCl2能夠有效抑制MMPs介導(dǎo)的膠原降解。其作用機(jī)制可能是過量的鋅與MMPs的多肽結(jié)合，改變了酶的三維空間構(gòu)象，從而抑制了MMPs的活性[53]。Toledano等[54]分別將10%ZnO和2%ZnCl2加入Single Bond Plus的粘接劑中，發(fā)現(xiàn)二者都能夠保存混合層的完整性。鋅離子除了能抑制MMPs活性外，還具有誘導(dǎo)牙本質(zhì)再礦化的能力。Osorio等[55]研究發(fā)現(xiàn)，鋅離子能夠誘導(dǎo)牙本質(zhì)再礦化中羥磷灰石和磷鈣鋅礦的形成。Toledano等[56]研究表明，鋅能夠提高齲壞牙本質(zhì)的功能性再礦化能力。Osorio等[57]研究發(fā)現(xiàn)，在使用Single Bond之前，用負(fù)載鋅的納米粒子的乙醇懸浮液預(yù)處理粘接界面，不會影響Single Bond的即刻粘接強(qiáng)度，且有利于保存粘接界面的完整性；二甲苯酚染色反應(yīng)表明，負(fù)載鋅的納米粒子還有助于粘接混合層底部的功能性再礦化。利用鋅對MMPs的抑制作用和誘導(dǎo)再礦化能力，將Zn2+或者ZnO結(jié)合到粘接劑中，可能是提高混合層穩(wěn)定性的一種新方法。

5 四環(huán)素類及其衍生物

四環(huán)素及其半合成類似物強(qiáng)力霉素和米諾環(huán)素，以及化學(xué)修飾類四環(huán)素，是有效的MMPs抑制劑[58]。四環(huán)素類除了鋅螯合作用，還能夠下調(diào)MMP mRNA的表達(dá)[59]，干擾蛋白的活化過程，使MMPs更容易水解[60]。Osorio等[51]和Toledano等[52]研究發(fā)現(xiàn)，在體外實(shí)驗(yàn)中，5 mg·mL-1強(qiáng)力霉素能抑制牙本質(zhì)基質(zhì)中的MMPs和rhMMP-2。Stanislawczuk等[61]研究發(fā)現(xiàn)，用2%米諾環(huán)素預(yù)處理37%磷酸酸蝕后的牙本質(zhì)，不會影響即刻粘接強(qiáng)度，并且能夠減少納米滲漏?；瘜W(xué)修飾類四環(huán)素（chemically modified tetracyclines，CMTs）是一種廣譜的MMPs抑制劑。CMT-3（aka Metastat，COL-3）是抑制膠原酶作用最強(qiáng)的CTMs，同時(shí)也能夠抑制明膠酶，能夠有效減少齲壞牙本質(zhì)中的基質(zhì)降解[58]。關(guān)于CMT的作用機(jī)制目前尚不十分清楚，可能與其螯合作用有關(guān)。CMTs通過與酶活性位點(diǎn)的鋅結(jié)合，能夠改變酶前體分子的構(gòu)象，抑制其對細(xì)胞外基質(zhì)的催化活性[62]。研究者[63]用埃洛石納米管（halloysite nanotube）負(fù)載強(qiáng)力霉素，并將這種復(fù)合物加入粘接劑中，成功實(shí)現(xiàn)了強(qiáng)力霉素的緩慢釋放，并且能夠抑制13.4%的MMP-1；但是其只研究了14 d內(nèi)強(qiáng)力霉素的釋放，更長時(shí)間的釋放量以及其濃度能否起到抑制作用還有待進(jìn)一步的研究。

6 異羥肟酸類抑制劑

異羥肟酸類抑制劑是一類廣譜的合成類MMP抑制劑，已經(jīng)被用于抗癌治療中[64]。加拉定是一種代表性的異羥肟酸類MMP抑制劑，酶譜法已證實(shí)了加拉定對MMP-2和MMP-9的抑制性[65-66]。0.2 mmol·L-1加拉定預(yù)處理酸蝕后的牙本質(zhì)，能減緩牙本質(zhì)粘接強(qiáng)度的降低[65]。Almahdy等[66]將加拉定加入Optibond FL、Prime & Bond NT和G-Bond的處理劑中，發(fā)現(xiàn)含5 μmol·L-1加拉定的預(yù)處理劑能提高初始粘接強(qiáng)度和減少納米滲漏。但Lührs等[24]研究發(fā)現(xiàn)，0.2 mmol·L-1加拉定預(yù)處理并不能抑制粘接界面的降解。巴馬司他（Batimastat，aka BB94） 是另一類異羥肟酸類MMP抑制劑，其能夠抑制粘接界面中的MMPs活性[66-67]。目前關(guān)于異羥肟酸類MMP抑制劑在口腔中的研究還很少，其能否應(yīng)用于口腔臨床中尚需進(jìn)一步研究。

7 二膦酸鹽衍生物

二膦酸鹽是一類抗骨質(zhì)疏松的藥物，包括氯膦酸鹽、阿侖膦酸鈉、氨羥二磷酸二鈉以及唑來膦酸等[68]。Heikkil?等[69]發(fā)現(xiàn)，二膦酸鹽能抑制MMPs，其機(jī)制可能是通過螯合鋅離子和鈣離子。聚乙烯基磷酸（polyvinylphosphonic acid，PVPA）中含有–C–P膦酸酯鍵，與二膦酸鹽中的–C–O–P 酯鍵具有結(jié)構(gòu)相似性[70]。Tezvergil-Mutluay等[70]研究發(fā)現(xiàn)， PVPA能抑制rhMMP-9。與CHX類似，PVPA能通過靜電作用與膠原結(jié)合。但不同的是，PVPA能通過乙基二甲胺丙基碳化二亞胺[1-ethyl-3- （3-dimethylaminopropyl）carbodiimide，EDC]交聯(lián)到膠原基質(zhì)上。PVPA不僅能夠抑制MMPs，還能誘導(dǎo)牙本質(zhì)再礦化，作為牙本質(zhì)基質(zhì)蛋白（dentin matrix protein，DMP）的模板類似物，誘導(dǎo)無定形鈣磷納米顆粒進(jìn)入膠原基質(zhì)中，促進(jìn)牙本質(zhì)再礦化[71-72]。這些研究結(jié)果提示，PVPA或許能夠在抑制MMPs的同時(shí)，誘導(dǎo)牙本質(zhì)再礦化，從而提高牙本質(zhì)粘接的耐久性。在牙本質(zhì)粘接領(lǐng)域，目前關(guān)于PVPA的研究仍然較少，其能否抑制牙本質(zhì)粘接界面的降解還有待更多的研究。

8 交聯(lián)劑

常見的交聯(lián)劑分為化學(xué)交聯(lián)劑、天然交聯(lián)劑和物理交聯(lián)劑三類[73]。

8.1 化學(xué)交聯(lián)劑

戊二醛能減緩膠原降解的速率，提高牙本質(zhì)膠原的性能。研究[74]表明，5.0%戊二醛處理酸蝕后的牙本質(zhì)1 min，能增加牙本質(zhì)的彈性模量。近幾年，EDC因其性質(zhì)穩(wěn)定、毒性低而受到了越來越多的關(guān)注。研究[75]發(fā)現(xiàn)，0.1、0.3和0.5 mol·L-1EDC預(yù)處理酸蝕后的牙本質(zhì)，不會對成牙本質(zhì)樣細(xì)胞產(chǎn)生毒性。Tezvergil-Mutluay等[76]研究表明，0.3 mol·L-1EDC作用1 min即可抑制rhMMP-9的活性。Mazzoni等[77]用原位酶譜法證實(shí)，0.3 mol·L-1EDC能夠抑制MMP-2和MMP-9。Scheffel等[74,78]研究也表明，EDC能夠提高膠原強(qiáng)度，增強(qiáng)粘接界面穩(wěn)定性，延長粘接耐久性。丙酮作為溶劑能夠提高EDC的交聯(lián)能力，增強(qiáng)EDC抑制膠原降解的能力[79]。

8.2 天然交聯(lián)劑

天然交聯(lián)劑具有低毒性和可持續(xù)的特點(diǎn)，是近幾年研究的熱點(diǎn)。常見的天然交聯(lián)劑有京尼平、多酚類和原花青素（proanthocyanidins，PACs）等[73]。PACs是一種凝縮單寧類物質(zhì)，無毒性，是有效的膠原酶抑制劑[80]。Epasinghe等[81]研究發(fā)現(xiàn)，1.0%PACs能抑制超過90%的rhMMP-2、-8、-9以及75%～90%的半胱氨酸組織蛋白酶，其抑制作用明顯高于CHX。PACs作用非常迅速，3.75%PACs預(yù)處理37%磷酸酸蝕后的牙本質(zhì)10 s，即可提高膠原抗水解能力，且其交聯(lián)牙本質(zhì)膠原的能力強(qiáng)于戊二醛[82-83]。近幾年，研究者[84-86]嘗試將PACs加入粘接劑中。Epasinghe等[84]研究表明，將濃度低于2%的PACs加入粘接劑中，不會影響即刻粘接強(qiáng)度，但樹脂聚合度降低。其原因可能是PACs是游離基清除劑[87]，會干擾粘接劑單體的聚合。基于上述研究結(jié)果，用PACs作為處理劑可能是保護(hù)混合層完整性的一種新方法，且由于PACs交聯(lián)作用迅速，該方法具有一定的臨床可行性。

8.3 物理交聯(lián)劑

核黃素（riboflavin）是一種物理交聯(lián)劑，在紫外光照射下能夠產(chǎn)生氧自由基，氧自由基能夠使甘氨酸的氨基與相鄰鏈羥脯氨酸和脯氨酸的羰基形成共價(jià)鍵，產(chǎn)生交聯(lián)作用。目前，核黃素和紫外線A（ultra violet A，UVA）聯(lián)合應(yīng)用已成為治療眼科疾病（如角膜?。┑囊恍路椒?。在牙本質(zhì)粘接方面，核黃素作為交聯(lián)劑的研究取得了較好的結(jié)果[88-90]。Cova等[88]研究表明，核黃素/UVA能夠抑制牙本質(zhì)MMPs（特別是MMP-9），且能夠提高即刻粘接強(qiáng)度和粘接界面穩(wěn)定性。Fawzy等[89-90]研究發(fā)現(xiàn)，核黃素/UVA能夠增強(qiáng)牙本質(zhì)膠原的機(jī)械性能和抵抗水解的能力。核黃素具有良好的生物相容性和操作簡便性。雖然在實(shí)驗(yàn)室中證明核黃素/UVA有效[90]，但關(guān)于使用UVA的安全性和在牙科中的實(shí)用性還有待進(jìn)一步的研究。

9 其他外源性抑制劑

其他外源性抑制劑如二甲基亞砜[91]、特異性抑制劑SB-3CT[92]和金納米粒子[93]等也有報(bào)道，但是這些抑制劑能否應(yīng)用于口腔臨床還有待更加系統(tǒng)的研究。

綜上所述，利用酶外源性抑制劑抑制膠原降解，是提高混合層穩(wěn)定性的一種具有較好研究前景的方法。雖然CHX、MDPB等已應(yīng)用于臨床，但是其遠(yuǎn)期效果仍不佳。利用鋅離子和PVPA等抑制MMPs，同時(shí)誘導(dǎo)牙本質(zhì)再礦化，可能是一種保護(hù)混合層完整性的新思路。交聯(lián)劑除了能夠抑制MMPs外，還能夠交聯(lián)膠原纖維，提高膠原的機(jī)械性能和抵抗水解的性能，可能是未來外源性抑制劑研究的重點(diǎn)領(lǐng)域。由于單一種類抑制劑的作用有限，多種抑制劑聯(lián)合應(yīng)用可能是未來研究的趨勢。

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（本文編輯 李彩）

Progress on matrix metalloproteinase inhibitors

Jia Lingling, Wan Qianbing. (State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Dept. of Prosthetics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China)

Continuing advances in dentin bonding technology and adhesives revolutionized bonding of resin-based composite restorations. However, hybrid layers created by contemporary dentin adhesives present imperfect durability, and degradation of collagen matrix by endogenous enzymes is a significant factor causing destruction of hybrid layers. Bond durability can be improved by using enzyme inhibitors to prevent collagen degradation and to preserve integrity of collagen matrix. This review summarizes progress on matrix metalloproteinase inhibitors (including chlorhexidine, ethylenediaminetetraacetic acid, quaternary ammonium salt, tetracycline and its derivatives, hydroxamic acid inhibitors, bisphosphonate derivative, and cross-linking agents) and suggests prospects for these compounds.

matrix metalloproteinase; cysteine cathepsins; dentin; enzyme inhibitors; biodegradation; durability

R 783.2

A

10.7518/hxkq.2017.02.019

2016-08-11；

2016-12-09

賈玲玲，碩士，E-mail：jlingling0612@126.com

萬乾炳，教授，博士，E-mail：champion@scu.edu.cn

Correspondence: Wan Qianbing, E-mail: champion@scu.edu.cn.