鋰介導(dǎo)的Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路對骨髓間充質(zhì)干細(xì)胞的影響

翁詩陽　朱振中　湯春　張長青

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鋰介導(dǎo)的Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路對骨髓間充質(zhì)干細(xì)胞的影響

翁詩陽朱振中湯春張長青

人類骨髓間充質(zhì)干細(xì)胞來源于胚胎時期的中胚層，存在于骨髓、脂肪、皮膚等組織中，具有自我更新能力及多向分化潛能。研究表明，鋰劑可激活Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路而對骨髓間充質(zhì)干細(xì)胞增殖、分化等產(chǎn)生影響。該文就鋰介導(dǎo)的Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路對骨髓間充質(zhì)干細(xì)胞的影響作一綜述。

骨髓間充質(zhì)干細(xì)胞；鋰劑；Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路

鋰劑是治療抑郁癥和造血系統(tǒng)疾病最有效的藥物之一[1]，它還對神經(jīng)退行性疾病如阿爾茲海默病、帕金森病導(dǎo)致的腦損傷有明顯保護(hù)作用。大量研究表明，鋰劑可激活Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路[2]，Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路對骨髓間充質(zhì)干細(xì)胞增殖與分化起著舉足輕重的作用[3]，人類骨髓間充質(zhì)干細(xì)胞來源于胚胎時期的中胚層，存在于骨髓、脂肪、皮膚等組織中，具有自我更新能力及多向分化潛能，它在體外易分離獲得，并可通過特定的方法誘導(dǎo)成為各種組織細(xì)胞[4]。鋰劑是否可以通過Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路調(diào)控骨髓間充質(zhì)干細(xì)胞增殖及分化，從而促進(jìn)骨壞死的自我修復(fù)，近年來學(xué)者們對此進(jìn)行了許多研究。

1　鋰劑與Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路

鋰劑可以通過調(diào)控糖原合成酶激酶-3β(GSK-3β)影響Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路。GSK-3β是胞內(nèi)活躍的絲/蘇氨酸激酶，擔(dān)負(fù)著某些重要蛋白如Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路轉(zhuǎn)錄因子β-catenin磷酸化功能。正常情況下，Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路主要依靠β-catenin穩(wěn)定表達(dá)，β-catenin由GSK-3β磷酸化，隨后由泛素-蛋白酶體復(fù)合物降解而清除。研究表明，鋰劑可以通過抑制GSK-3β激活Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路[5]，其機(jī)制為鋰劑與鎂離子競爭性結(jié)合GSK-3β而直接發(fā)揮抑制作用，或通過激活磷脂酰肌醇-3-激酶(P13K)、蛋白激酶B1(Akt1)增強(qiáng)GSK-3β磷酸化作用[6]。理論上鋰劑抑制GSK-3β活性可造成胞質(zhì)內(nèi)β-catenin的蓄積，從而引起Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路的激活。有學(xué)者[7]實(shí)驗(yàn)研究發(fā)現(xiàn)，鋰劑可上調(diào)β-catenin蛋白表達(dá)，造成Wnt下游基因如Axin2、Ahr及Nkd2表達(dá)上調(diào)，從而激活Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路。

2　Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路與骨髓間充質(zhì)干細(xì)胞

Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路對于骨骼系統(tǒng)發(fā)育、骨形成、骨愈合、骨代謝平衡調(diào)節(jié)起著重要作用[8]，對骨髓間充質(zhì)干細(xì)胞也有重要影響，骨髓間充質(zhì)干細(xì)胞分化由信號通路調(diào)控，其中Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路是調(diào)控骨髓間充質(zhì)干細(xì)胞的主要信號通路之一，可在胚胎早期誘導(dǎo)骨髓間充質(zhì)干細(xì)胞成骨分化。研究[9]表明，腫瘤壞死因子通過激活Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路促進(jìn)骨髓間充質(zhì)干細(xì)胞成骨分化并抑制其成脂分化，從而緩解骨質(zhì)疏松。Weivoda等[10]研究表明，Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路可通過激活經(jīng)典及非經(jīng)典環(huán)磷酸腺苷(cAMP)/蛋白激酶A(PKA)信號轉(zhuǎn)導(dǎo)通路抑制破骨細(xì)胞分化，使體內(nèi)骨吸收減少，骨量上升。Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路是調(diào)控骨髓間充質(zhì)干細(xì)胞增殖及分化的樞紐，而其上游的GSK-3β則是該作用的開關(guān)。鋰劑憑借其組織分布特點(diǎn)在骨組織特異性聚集，抑制GSK-3β表達(dá)，從而維持骨髓間充質(zhì)干細(xì)胞增殖與分化，達(dá)到修復(fù)骨缺損的作用。

3　鋰劑對骨髓間充質(zhì)干細(xì)胞增殖及凋亡的影響

經(jīng)典的Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路可促進(jìn)骨髓間充質(zhì)干細(xì)胞增殖并抑制其凋亡，而鋰劑也有相同作用。體外研究表明，鋰劑可調(diào)節(jié)骨髓間充質(zhì)干細(xì)胞增殖。Zhu等[11]在實(shí)驗(yàn)中將骨髓間充質(zhì)干細(xì)胞分為兩組，實(shí)驗(yàn)組加入5 μm 氯化鋰，空白組中不加氯化鋰，3 d后使用CCK-8檢測細(xì)胞增殖情況，發(fā)現(xiàn)與對照組相比，實(shí)驗(yàn)組細(xì)胞數(shù)量明顯上升，以上作用在使用siRNA或β-catenin抑制劑Quercetin阻斷Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路后消失，表明鋰劑可促進(jìn)人類骨髓間充質(zhì)干細(xì)胞增殖。高麗娜等[12]的實(shí)驗(yàn)表明，在人類骨髓間充質(zhì)干細(xì)胞培養(yǎng)基中加入不同濃度的氯化鋰(5、10、20、40 mmol/L)后，通過MTT方法檢測細(xì)胞增殖情況，發(fā)現(xiàn)小劑量鋰劑(5 mmol/L)可促進(jìn)骨髓間充質(zhì)干細(xì)胞增殖,而大劑量鋰劑(20、40 mmol/L)則會抑制骨髓間充質(zhì)干細(xì)胞增殖。Dong等[13]應(yīng)用免疫熒光法發(fā)現(xiàn)，低濃度氯化鋰(0.1 mmol/L)可促進(jìn)骨髓間充質(zhì)干細(xì)胞增殖并分化為神經(jīng)細(xì)胞。

此外，鋰劑還可影響骨髓間充質(zhì)干細(xì)胞凋亡。研究[11]報道，采用5 mmol/L鋰劑干擾骨髓間充質(zhì)干細(xì)胞，4 d后使用流式細(xì)胞儀膜聯(lián)蛋白V與碘化丙啶標(biāo)記法檢測發(fā)現(xiàn)，鋰劑組骨髓間充質(zhì)干細(xì)胞凋亡比例(6.9%)小于空白組(7.2%)。然而，當(dāng)鋰劑劑量達(dá)到10 mmol/L時，骨髓間充質(zhì)干細(xì)胞凋亡率明顯上升，這可能是由于高濃度鋰劑會產(chǎn)生細(xì)胞毒性的緣故。

4　鋰劑對骨髓間充質(zhì)干細(xì)胞成骨分化的影響

Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路可調(diào)控骨髓間充質(zhì)干細(xì)胞分化方向，鋰劑同樣可以對體外骨髓間充質(zhì)干細(xì)胞分化產(chǎn)生影響。Yu等[14]使用不同濃度氯化鋰(1、5、10、20 mmol/L)干預(yù)小鼠骨髓間充質(zhì)干細(xì)胞成骨分化，21 d后通過聚合酶鏈?zhǔn)椒磻?yīng)(PCR)、免疫印跡技術(shù)法檢測骨鈣素(OCN)、Runt相關(guān)轉(zhuǎn)錄因子2(RUNX2)等成骨指標(biāo)，發(fā)現(xiàn)5 mmol/L氯化鋰可使細(xì)胞成骨特異性指標(biāo)表達(dá)上升，茜素紅染色顯示鈣結(jié)節(jié)數(shù)目上升，表明鋰劑可增加骨髓間充質(zhì)干細(xì)胞成骨能力；在使用Quercetin阻斷Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路后骨髓間充質(zhì)干細(xì)胞促成骨作用消失。有研究[6]使用不同濃度的鋰劑干預(yù)骨髓間充質(zhì)干細(xì)胞成骨分化，7、14 d后使用逆轉(zhuǎn)錄PCR 檢測成骨特異性蛋白OCN、RUNX2及堿性磷酸酶表達(dá)，發(fā)現(xiàn)實(shí)驗(yàn)組骨相關(guān)基因堿性磷酸酶、RUNX2、OCN表達(dá)水平均較空白組高，且呈濃度和時間依賴性。

5　鋰劑對骨髓間充質(zhì)干細(xì)胞成脂分化的影響

Tang等[15]研究表明，使用鋰劑干擾人類骨髓間充質(zhì)干細(xì)胞成脂分化，21 d后經(jīng)油紅染色觀察細(xì)胞內(nèi)脂滴數(shù)量，發(fā)現(xiàn)與對照組相比，鋰劑組脂滴數(shù)量明顯下降，此外鋰劑組成脂肪相關(guān)基因過氧化物酶體增殖物激活受體γ(PPARγ)表達(dá)下降，認(rèn)為鋰劑可抑制骨髓間充質(zhì)干細(xì)胞成脂分化。Visweswaran 等[16]的實(shí)驗(yàn)表明，使用鋰劑刺激骨髓間充質(zhì)干細(xì)胞成脂分化后，油紅染色顯示與空白組相比，實(shí)驗(yàn)組脂滴數(shù)量明顯減少，同時成脂特異性蛋白、PPARγ、CAAT區(qū)/增強(qiáng)子結(jié)合蛋白α(C/EBPα)和乙酰輔酶A羧化酶表達(dá)明顯下降，骨髓間充質(zhì)干細(xì)胞成脂分化受抑制。Zhang等[17]使用組織蛋白酶B阻斷Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路，結(jié)果鋰劑對骨髓間充質(zhì)干細(xì)胞成脂分化的抑制作用消失，表明鋰劑抑制骨髓間充質(zhì)干細(xì)胞成脂分化由Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路介導(dǎo)，且是可逆的。

6　鋰劑對骨髓間充質(zhì)干細(xì)胞成軟骨分化的影響

鋰劑可影響骨髓間充質(zhì)干細(xì)胞成軟骨分化。Eslaminejad等[18]研究表明，使用鋰劑5 mmol/L激活Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路后，骨髓間充質(zhì)干細(xì)胞成軟骨分化增加， 21 d后軟骨相關(guān)基因Sox9及聚蛋白多糖、Ⅱ型膠原蛋白表達(dá)上調(diào)，表明低濃度鋰劑可促進(jìn)骨髓間充質(zhì)干細(xì)胞成軟骨分化。然而，Kapadia等[19]的研究則發(fā)現(xiàn)15 mmol/L鋰劑可對大鼠胚胎細(xì)胞軟骨內(nèi)骨化產(chǎn)生抑制作用。Kawata等[20]的實(shí)驗(yàn)結(jié)果也得出相似結(jié)論，即小劑量鋰劑可促進(jìn)骨髓間充質(zhì)干細(xì)胞成軟骨分化，而大劑量鋰劑則抑制骨髓間充質(zhì)干細(xì)胞成軟骨分化。

7　鋰劑治療骨壞死

骨髓間充質(zhì)干細(xì)胞的存在對骨的生長發(fā)育及自我修復(fù)有很大作用[21-22]，是骨骼系統(tǒng)再生和修復(fù)的重要動力，骨髓間充質(zhì)干細(xì)胞的缺失很可能導(dǎo)致骨細(xì)胞凋亡、骨壞死及骨自我修復(fù)障礙。骨髓間充質(zhì)干細(xì)胞成脂分化與成骨分化在一定水平上保持平衡，打破這種平衡會導(dǎo)致骨髓間充質(zhì)干細(xì)胞增殖能力下降，成骨細(xì)胞分化減少，破骨細(xì)胞數(shù)量上升及生存周期延長，成脂能力增加，成血管能力下降，組織學(xué)上表現(xiàn)為骨小梁吸收、股骨頭塌陷及微骨折形成[23]，股骨頭靜脈壓力增高，組織缺血。

骨壞死患者體內(nèi)骨髓間充質(zhì)干細(xì)胞數(shù)量下降，成脂分化增多，成骨分化減少，造成再生動力不足，再生方向改變，兩者結(jié)合導(dǎo)致骨壞死發(fā)生?；诖?，有研究[24-26]嘗試將外源性骨髓間充質(zhì)干細(xì)胞移植到股骨頭壞死或骨不連部位，以補(bǔ)充病變處骨髓間充質(zhì)干細(xì)胞數(shù)量。然而，外源性骨髓間充質(zhì)干細(xì)胞移植治療只能在確診相應(yīng)疾病的情況下進(jìn)行，治療時機(jī)相對滯后，且該方法需涉及骨髓間充質(zhì)干細(xì)胞體外分離和培養(yǎng)，手術(shù)所帶來的并發(fā)癥也無法避免。目前臨床上外源性骨髓間充質(zhì)干細(xì)胞移植治療骨壞死尚缺乏安全性及有效性全面評估，因此如何通過合理有效的手段激活內(nèi)源性骨髓間充質(zhì)干細(xì)胞，針對致病因素合理調(diào)控骨髓間充質(zhì)干細(xì)胞增殖及分化，達(dá)到對抗外界不利因素及促進(jìn)局部細(xì)胞、組織結(jié)構(gòu)再生及功能重建的目的，是目前干細(xì)胞治療研究領(lǐng)域的新理念[27]。

鋰劑是一種治療躁狂、雙向情感障礙等精神疾病的常用藥物，大多數(shù)患者經(jīng)治療有顯著改善，早在1974年就有學(xué)者對鋰劑的藥理學(xué)展開研究[28]。研究發(fā)現(xiàn)，抑郁癥患者局部神經(jīng)干細(xì)胞增殖能力下降，神經(jīng)元分化減少。而鋰劑可明顯提高該區(qū)域神經(jīng)干細(xì)胞級神經(jīng)元數(shù)量。隨后的體外實(shí)驗(yàn)[29]證實(shí)，鋰劑對神經(jīng)干細(xì)胞增殖及分化都有顯著的調(diào)控作用。那么鋰劑是否對骨壞死區(qū)域骨髓間充質(zhì)干細(xì)胞也能發(fā)揮相同作用，值得進(jìn)一步研究。

Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路在骨骼系統(tǒng)發(fā)育及成年后骨代謝平衡調(diào)節(jié)中發(fā)揮重要作用。該信號通路可促進(jìn)骨髓間充質(zhì)干細(xì)胞增殖及成骨分化和成軟骨分化，減少破骨細(xì)胞生成。研究[30]表明，糖皮質(zhì)激素通過激活GSK-3β抑制Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路，此為激素性股骨頭壞死可能的分子學(xué)機(jī)制之一。基于此機(jī)制，鋰劑可拮抗激素作用并預(yù)防激素性股骨頭壞死[31]。眾多研究表明，低濃度鋰劑可通過激活Wnt/β-catenin信號轉(zhuǎn)導(dǎo)通路增加骨髓間充質(zhì)干細(xì)胞增殖能力，并促進(jìn)其數(shù)量明顯上升，從而使骨再生動力增加；鋰劑可明顯影響骨髓間充質(zhì)干細(xì)胞分化，促進(jìn)其成骨分化，抑制其成脂分化，從而造成骨再生方向改變，其綜合作用有助于修復(fù)骨缺損。然而，目前臨床上尚缺乏使用鋰劑治療股骨頭壞死、骨不連等報道。使用鋰劑治療骨壞死是否有效，仍需大量動物實(shí)驗(yàn)及臨床數(shù)據(jù)支持。

8　結(jié)語

近年來有許多動物實(shí)驗(yàn)使用鋰劑促進(jìn)小鼠骨形成[32]、改善骨質(zhì)疏松[33]，增加大鼠骨密度[34]，為鋰劑治療骨壞死提供了良好的理論基礎(chǔ)。長期服用鋰劑是否引起骨骼系統(tǒng)不良反應(yīng)如可能致癌[35]并引起多器官病變[36]，使用鋰劑治療骨科疾病的有效劑量，如何控制鋰劑局部濃度，鋰劑導(dǎo)致的骨量增加、骨密度上升、骨髓間充質(zhì)干細(xì)胞增殖能力增強(qiáng)及分化方向改變是否可逆，停藥后是否會復(fù)發(fā)，都需要大量研究進(jìn)行探索。

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(收稿：2015-12-18; 修回：2016-03-19)

(本文編輯：盧千語)

國家自然科學(xué)基金(81472066)

200233，上海交通大學(xué)附屬第六人民醫(yī)院骨科

10.3969/j.issn.1673-7083.2016.03.012