付寅生，張怡，張婷婷，蘆慧穎，孟慶雪

（1老年性疾病干細胞技術(shù)國家地方聯(lián)合工程技術(shù)研究中心，哈爾濱 150028；2黑龍江天晴干細胞股份有限公司，哈爾濱 150028；3哈爾濱富爾斯特生物工程有限責(zé)任公司，哈爾濱 150431）

綜述

羊膜上皮細胞旁分泌特點及其在皮膚損傷修復(fù)中的作用

付寅生2,3，張怡1,2*，張婷婷1,2，蘆慧穎1,2，孟慶雪1,2

（1老年性疾病干細胞技術(shù)國家地方聯(lián)合工程技術(shù)研究中心，哈爾濱 150028；2黑龍江天晴干細胞股份有限公司，哈爾濱 150028；3哈爾濱富爾斯特生物工程有限責(zé)任公司，哈爾濱 150431）

皮膚損傷修復(fù)是一個復(fù)雜而又高度協(xié)同的生物學(xué)過程，多種生長因子及白介素、趨化因子等細胞因子參與該過程，并調(diào)控皮膚愈合過程中創(chuàng)面的再上皮化、新血管生成以及細胞外基質(zhì)沉積與重塑3個重要環(huán)節(jié)。人羊膜上皮細胞是一種類胚胎干細胞，廣泛應(yīng)用于創(chuàng)面損傷修復(fù)研究中。許多研究表明，人羊膜上皮細胞能夠通過旁分泌作用促進皮膚創(chuàng)面的愈合。這些旁分泌因子不僅能夠抑制創(chuàng)面微環(huán)境中細胞的凋亡，還能促進新血管生成和上皮再生。在此，本文綜述人羊膜上皮細胞旁分泌特點，探討人羊膜上皮細胞來源的旁分泌因子在創(chuàng)面愈合中的作用機制，并針對人羊膜上皮細胞或人羊膜上皮細胞來源細胞因子溶液應(yīng)用于皮膚損傷治療的未來可行性進行詳細闡述。

羊膜上皮細胞；細胞因子；旁分泌；創(chuàng)面修復(fù)

人羊膜上皮細胞（human amniotic epithelial cell, hAECs）是人羊膜組織中最主要的干細胞之一，因其具有抗炎、抗纖維化、免疫原性低等特點，在再生醫(yī)學(xué)研究領(lǐng)域獲得廣泛的關(guān)注[1,2]。作為一種多潛能干細胞，hAECs不僅能夠表達胚胎干細胞相關(guān)的表面標(biāo)記和多種多潛能干細胞轉(zhuǎn)錄因子（SSEA-3、SSEA-4、Oct-4、Sox-2、Nanog、Rex-1、FGF-4、CFC-1、DPPA-3等）[2,3]，還具有向3個胚層分化的潛能[4-6]。此外，hAECs能夠通過旁分泌作用釋放多種細胞因子，包括表皮生長因子（EGF）、成纖維生長因子（FGF）、神經(jīng)生長因子（NGF）、血管內(nèi)皮生長因子（VEGF）、胰島素樣生長因子（IGF）等生長因子；粒細胞集落刺激因子（G-CSF）、粒細胞-巨噬細胞集落刺激因子（GM-CSF）等集落刺激因子；IL-6、CCL2、CXCL8等白介素和趨化因子[7-9]。這些細胞因子具有不同的亞型，可通過與靶細胞表面受體的特異性結(jié)合激活相關(guān)的信號通路，調(diào)節(jié)細胞的增殖、分化、遷移以及炎癥反應(yīng)和細胞外基質(zhì)（ECM）的產(chǎn)生，在皮膚創(chuàng)傷、角膜燒傷、肺損傷、腦脊髓炎、卵巢損傷、腦出血等組織損傷修復(fù)研究中顯示了重要的作用[10]。

皮膚創(chuàng)面愈合是一個復(fù)雜的生理過程，包括細胞遷移、炎癥反應(yīng)、血管生成、肉芽組織形成、上皮組織再生和細胞外基質(zhì)重構(gòu)。這些生理過程由多個信號通路交錯形成極其復(fù)雜的信號網(wǎng)絡(luò)調(diào)控機制完成，涉及組織、細胞、分子、基因等多個層面。正常皮膚損傷愈合過程一旦受到供血不足、糖尿病、腎病、重創(chuàng)、衰老等因素的影響，其關(guān)鍵愈合機制會被阻斷，進而發(fā)展成為慢性皮膚創(chuàng)面[11]。目前干細胞技術(shù)是治療慢性皮膚創(chuàng)面最有潛力的方法之一。本文將綜述hAECs的旁分泌特點及其在皮膚創(chuàng)面修復(fù)中的作用機制。

1 hAECs旁分泌特點

大量研究表明，hAECs能夠分泌多種生物活性蛋白，包括生長因子、白介素、趨化因子等（表1），這些蛋白在體內(nèi)局部形成復(fù)雜的分泌蛋白網(wǎng)絡(luò)，并通過調(diào)控分子與分子、分子與細胞、細胞與細胞間的相互作用，激活或阻斷相關(guān)信號通路，獲得免疫調(diào)節(jié)、調(diào)控細胞增殖、分化、遷移以及抑制細胞凋亡等功能[9,37]。早期研究發(fā)現(xiàn)，羊膜或羊膜細胞來源的細胞培養(yǎng)液中含有多種促進細胞增殖和組織再生的細胞因子，如EGF、VEGF、bFGF、血管生成素（ANG）、PDGF、TGFβ（1,2）、組織金屬蛋白酶抑制劑-1,2（TIMP-1,2）[8,12]，并且在動物體內(nèi)移植后能夠顯著提高急、慢性大鼠創(chuàng)面皮膚的愈合能力[8,39]。隨后Grzywocz等[7]利用抗體陣列技術(shù)檢測出羊膜細胞條件培養(yǎng)基中含有表皮生長因子、成纖維生長因子、造血生長因子等生長因子家族中的41種生長因子及受體，并發(fā)現(xiàn)了7種未報道過的羊膜細胞來源生長因子，包括FGF-6、IGFBP-6、IGFBP-4、VEGFR3、M-CSF-R、PDGF-AB、神經(jīng)營養(yǎng)因子（NT4）。我們近期的研究也發(fā)現(xiàn)，培養(yǎng)條件對hAECs旁分泌能力有較大影響，培養(yǎng)體系中額外加入10ng/ml EGF、顯著提高了hAECs的增殖能力的同時造成了PDGF、VEGF的分泌量明顯降低和TGFβ1的分泌量明顯增高（未發(fā)表的數(shù)據(jù)）。由此分析，采用不同制備工藝獲得的hAECs將對炎癥細胞浸潤、新血管生成、上皮再生以及肉芽組織形成等皮膚愈合過程產(chǎn)生不同影響。

表1 羊膜上皮細胞旁分泌因子Table1 Cytokine secretory profiles of hAECs

白介素也是hAECs旁分泌因子之一，通過ELISA和蛋白芯片檢測發(fā)現(xiàn)， hAECs在體外培養(yǎng)過程中向無血清培養(yǎng)體系中釋放白介素1受體拮抗劑（IL-1Ra）和白介素6（IL-6）[14,15]。IL-1Ra是IL-1家族成員， hAECs可通過分泌IL-1Ra抑制角膜上皮細胞IL-1與IL-1R的結(jié)合，從而抑制角膜炎癥反應(yīng)[14]。IL-6與趨化因子CCL2、CXCL8是Th2相關(guān)細胞因子，能夠介導(dǎo)抗炎癥機制[9]、刺激細胞增殖和分化、促血管生成作用[15]、參與表皮角朊細胞的增殖和創(chuàng)面的修復(fù)[40]。趨化因子CXCL5、CXCL12同樣是能夠在hAECs來源的條件培養(yǎng)基中檢測出的炎癥相關(guān)因子[16]，可誘導(dǎo)中性粒細胞、巨噬細胞等炎癥細胞的募集，并能有效刺激角朊細胞的遷移[36]。此外Song等[15]研究發(fā)現(xiàn)，低氧環(huán)境能夠刺激hAECs在體外培養(yǎng)中對ANG、EGF、IL-6、MCP-1的釋放，這些細胞因子分別在損傷組織愈合、免疫因子釋放、新血管生成中有重要作用。該團隊后續(xù)研究結(jié)果證實，hAECs注射到心肌梗死大鼠的病灶位置后會持續(xù)分泌ANG、EGF、IL-6、MCP-1促進新血管的生成[15]。炎癥反應(yīng)是皮膚損傷愈合的關(guān)鍵時期，在創(chuàng)傷刺激下炎癥細胞向傷口床募集，并促進炎癥相關(guān)因子的分泌促進創(chuàng)面的愈合。hAECs分泌的炎癥因子不僅含有炎癥促進因子，還包括炎癥抑制因子，但在皮膚創(chuàng)面微環(huán)境中hAECs的炎癥因子分泌特點以及對炎癥反應(yīng)的調(diào)節(jié)機制仍不十分明確。

2 hAECs旁分泌因子在促進皮膚創(chuàng)面愈合中的作用

皮膚創(chuàng)面愈合過程是一個復(fù)雜而又高度協(xié)調(diào)的生物學(xué)過程。創(chuàng)面上皮形成、新血管生成以及細胞外基質(zhì)的產(chǎn)生與重塑是皮膚創(chuàng)面愈合過程中重要的3個環(huán)節(jié)，因此與這些生物學(xué)過程相關(guān)的表皮角朊細胞、真皮成纖維細胞以及血管內(nèi)皮細胞的增殖和遷移作用是皮膚創(chuàng)面快速愈合的關(guān)鍵。皮膚受損會刺激創(chuàng)周細胞遷移與分化，并釋放大量細胞因子調(diào)控炎癥的發(fā)生、細胞外基質(zhì)的沉積、血管的生成、表皮的再生以及組織重塑[41,42]。研究表明，干細胞或干細胞細胞因子溶液移植到皮膚損傷位置能夠通過旁分泌因子激活局部信號網(wǎng)絡(luò)，提高細胞有絲分裂能力，刺激靶細胞遷移，調(diào)控細胞外基質(zhì)的沉積與降解，促進皮膚創(chuàng)面的愈合[43,44]。體外和體內(nèi)實驗研究表明，hAECs能夠通過旁分泌作用調(diào)控皮膚損傷部位多種細胞的生物學(xué)功能，在提高創(chuàng)面愈合能力以及皮膚附屬器的再生中具有顯著作用[16]。皮膚創(chuàng)面的再上皮化是皮膚愈合的重要環(huán)節(jié)之一，細胞因子及相關(guān)信號網(wǎng)絡(luò)參與上皮形成的完整過程。皮膚發(fā)生損傷時會造成皮膚表層屏障的缺失，此時中性粒細胞、單核細胞和巨噬細胞會募集于傷口床與創(chuàng)面鄰近組織中細胞，如角朊細胞、成纖維細胞、內(nèi)皮細胞、間質(zhì)細胞、黑素細胞等大量釋放并交換細胞因子，同時角朊細胞在多種細胞因子調(diào)控下被激活。激活后的角朊細胞的細胞骨架和表面受體發(fā)生改變，引起細胞與細胞、細胞與細胞外基質(zhì)間連接（橋粒、半橋粒）發(fā)生解離，刺激角朊細胞向傷口床遷移及分化，促進再上皮化過程（圖1）[37,45]。表皮角朊細胞的增殖、分化和遷移能力直接關(guān)系著皮膚創(chuàng)面再上皮化能力。在hAECs的旁分泌因子中，EGF、HB-EGF、TGF-α、IGF-1、FGF-2（bFGF）、FGF-7（KGF）、HGF、VEGF-A、GM-CSF、TGFβ1以及轉(zhuǎn)錄因子CXCL8等細胞因子在調(diào)控角朊細胞增殖和遷移中具有重要的促進作用[36,46]，并通過刺激細胞外基質(zhì)的合成與重塑以及新血管生成過程協(xié)同提高表皮再生能力[46,47]。最新的一項研究結(jié)果發(fā)現(xiàn)，hAECs來源條件培養(yǎng)基能夠活化下游信號通路中ERK、JNK以及AKT的磷酸化作用，通過調(diào)節(jié)MAPK、AKT信號途徑促進角朊細胞的體外遷移能力[16]。此外，在共培養(yǎng)條件下，hAECs能夠誘導(dǎo)角朊細胞中細胞周期蛋白（cyclin D1和cyclin D1）以及Mdm2表達水平的增加，并通過AKT途徑促進角朊細胞DNA的合成（S期細胞數(shù)增加），提高角朊細胞的存活與增殖能力[16]。隨后的體內(nèi)實驗進一步發(fā)現(xiàn)，hAECs來源條件培養(yǎng)基能夠顯著促進ERK、JNK、AKT抑制因子作用下C57BL/5小鼠的皮膚創(chuàng)面愈合速度，證明hAECs來源旁分泌因子能夠通過調(diào)控MAPK、AKT途徑促進上皮形成，加快創(chuàng)面的閉合[16]。

圖1 全層皮膚創(chuàng)面愈合示意圖。a，小鼠皮膚瘡面愈合HE染色觀察；b，模式圖[48]；HE, 增生表皮; E, 表皮; HF, 毛囊; D, 真皮; A, 脂肪組織; M, 肌肉; Es, 血痂; G, 肉芽組織; KC, 角朊細胞; FB, 成纖維細胞; MP, 巨噬細胞; PL, 血小板; LK, 白細胞; PNS, 周圍神經(jīng)系統(tǒng)細胞; PB, 胰島β細胞; HP, 肝細胞; ML, 黑素細胞; MS, 間充質(zhì)干細胞Fig. 1 Histologic (a) and schematic (b) representations of a healing full-thickness excisional wound in mouse skin. HE, hyperproliferative epidermis; E, epidermis; HF, hair follicle; D, dermis; A, adipose tissue; M, muscle; Es, eschar; G, granulation tissue; KC, keratinocytes; FB, fbroblasts; MP, macrophages; PL, platelets; LK, leukocytes; PNS, peripheral nervous system cells; PB, pancreatic β-cells; HP, hepatocytes; ML, melanocytes; MS, mesenchymal stem cells.

圖2 VEGFR信號通路調(diào)控血管內(nèi)皮細胞增殖與遷移[49]Fig. 2 VEGFR signaling regulates the proliferation and migration of vascular endothelial cells

新血管的生成同樣在創(chuàng)面愈合中具有重要作用。這一過程在慢性、難愈合性皮膚創(chuàng)面中新血管的生長尤為重要，血管化能力的降低不僅影響肉芽組織的形成，還影響著上皮的再生[48-50]。而新血管形成過程是通過多種生長因子、趨化因子、細胞亞群之間相互復(fù)雜的作用關(guān)系調(diào)控的[61]。EGF、PDGF、ANG、VEGF、IL-6、bFGF等細胞因子是hAECs主要的旁分泌因子[51,15,49,52]，具有提高血管內(nèi)皮細胞增殖和遷移能力、促進新生血管的生成作用。其中VEGF是皮膚創(chuàng)面新血管生成過程中核心調(diào)控因子之一。VEGF家族通過結(jié)合并激活細胞表面的酪氨酸激酶受體VEGFR2，刺激下游信號通路級聯(lián)反應(yīng)的發(fā)生，以此調(diào)控血管內(nèi)皮細胞增殖與遷移（圖2）[49]。經(jīng)體外培養(yǎng)后，hAECs培養(yǎng)液中VEGF含量顯著高于原培養(yǎng)液，并隨培養(yǎng)時間的延長而累積（未發(fā)表的數(shù)據(jù)）。近期研究也發(fā)現(xiàn)，炎性因子TNFα、IFNβ的刺激下hAECs能夠進一步提高促血管生成因子VEGFA、PDGFB、ANGPT1以及轉(zhuǎn)錄因子FOXC1的表達，促進新血管的生成，加快創(chuàng)傷組織的修復(fù)[51]。此外，在患有Ⅲ期壓瘡的大鼠模型研究中進一步證實，hAECs體內(nèi)移植能夠通過上調(diào)VEGF的表達，促進皮膚創(chuàng)面的愈合[53]。因此，hAECs分泌的VEGF可能是早期創(chuàng)面加快愈合的重要因子，并通過VEGF途徑促進皮膚創(chuàng)面內(nèi)新血管的形成。

細胞外基質(zhì)形成與組織重塑是皮膚創(chuàng)面完整愈合進程中的重要生物學(xué)過程，在組織損傷炎癥期、增生期完成細胞外基質(zhì)的合成、分泌與沉積，并于皮膚創(chuàng)面修復(fù)后期完成細胞外基質(zhì)的重塑[54]。創(chuàng)面損傷初期嗜中性粒細胞、單核細胞、淋巴細胞由周圍血管向創(chuàng)面浸潤，其中嗜中性粒細胞清除病原體及病變組織，并通過TGF-β、PDGF、IL-1等細胞因子調(diào)控淋巴細胞的遷移和巨噬細胞的生成[55,56]。巨噬細胞釋放大量細胞因子，如FGF、TGF-β、PDGF、EGF等調(diào)控成纖維細胞、血管內(nèi)皮細胞的增殖、遷移以及細胞外基質(zhì)的合成，誘導(dǎo)肉芽組織形成[57,58]。TGFβ是創(chuàng)面愈合過程中調(diào)控細胞外基質(zhì)沉積與重塑的關(guān)鍵因子，是hAECs可分泌的生長因子之一[7]。TGF-β家族包括TGF-β1、TGF-β2和TGF-β3，在皮膚組織內(nèi)由巨噬細胞、成纖維細胞、角朊細胞和血小板合成、分泌[55]。TGFβ1和TGFβ2在創(chuàng)面早期愈合過程中募集成纖維細胞和炎癥細胞進入傷口床，可通過抑制由成纖維細胞、巨噬細胞和嗜中性粒細胞所釋放的金屬蛋白酶（MMPs），防止細胞外基質(zhì)的降解，促進肉芽組織形成，血管生成和膠原的合成和產(chǎn)生[54,55]。TGFβ3與TGFβ1和TGFβ2作用相反，在疤痕形成中對TGFβ1有拮抗作用[59]。在小疤痕或無疤痕組織中，如口腔黏膜，TGFβ1的水平下降，同時TGFβ3/TGFβ1比率顯著上升[60]。本實驗室體外研究曾發(fā)現(xiàn)，生長狀態(tài)下hAECs培養(yǎng)液中TGFβ1的含量隨培養(yǎng)時間的延長而增加，而TGFβ3含量無明顯變化，表明hAECs在營養(yǎng)交換過程中能夠增加TGFβ1在周圍環(huán)境中的釋放（未發(fā)表的數(shù)據(jù)）。另一項最新的研究結(jié)果發(fā)現(xiàn)TGFβ1誘導(dǎo)活化的真皮成纖維細胞在hAECs條件培養(yǎng)基處理下降低了向成肌纖維轉(zhuǎn)化能力，并通過降低TGFβ1/Smad3信號通路中Smad2和Smad3磷酸化作用抑制成纖維細胞中膠原蛋白的生成，同時，hAECs培養(yǎng)液中的活性因子對活化狀態(tài)下真皮成纖維細胞中纖維化相關(guān)蛋白的表達具有負反饋調(diào)節(jié)作用，以此抑制瘢痕的形成[61]。以上研究表明hAECs的旁分泌因子可能參與調(diào)節(jié)皮膚創(chuàng)面愈合過程中膠原的沉積與重塑。

3 展望

最近的基礎(chǔ)研究表明，hAECs主要通過旁分泌作用影響皮膚創(chuàng)面的愈合速度，主要體現(xiàn)為促新血管生成以及表皮再生[51,16]。這些研究雖然在一定程度上闡述了hAECs旁分泌作用對皮膚損傷修復(fù)中的作用機制，但仍然缺少充足的證據(jù)揭示hAECs在皮膚創(chuàng)面微環(huán)境中的具體作用機理。EGF、TGFβ、CXCL8、VEGF、TIMPs等因子雖然在創(chuàng)面愈合過程中對血管生成、細胞外基質(zhì)沉積具有重要的促進作用，但需要注意的是在不同愈合階段，這些關(guān)鍵蛋白分泌量的異常仍存在降低創(chuàng)面愈合質(zhì)量的可能性[55,66-69]，如膠原蛋白過度增生導(dǎo)致肥厚性疤痕的產(chǎn)生[69]，炎癥期延長以及血管、肉芽組織過度增殖引起上皮化減慢[56,66,67]，而每個蛋白家族中不同亞型也同樣存在著雙向調(diào)控作用（如TGFβ）[59]，這些因素為hAECs在創(chuàng)面愈合中的具體作用機制的探索增加了較大的困難。體外培養(yǎng)研究發(fā)現(xiàn)，hAECs具有較低的增殖能力以及培養(yǎng)過程中易發(fā)生上皮-間質(zhì)轉(zhuǎn)化現(xiàn)象[70]，對未來大規(guī)模臨床轉(zhuǎn)化有一定限制，這些關(guān)鍵性問題給hAECs的臨床轉(zhuǎn)化研究帶來了新的課題，如體內(nèi)移植的hAECs處于皮膚創(chuàng)面愈合不同階段微環(huán)境下的細胞因子分泌特點及分子調(diào)控機制；hAECs和hAECs來源細胞因子溶液在規(guī)?；a(chǎn)、質(zhì)量控制以及有效性、安全性等方面相對比，哪種更適合作為終端產(chǎn)品用于創(chuàng)傷治療。

雖然hAECs或hAECs來源細胞因子溶液在皮膚組織損傷修復(fù)中的具體作用機制仍不十分明確，但hAECs旁分泌作用具有明顯的促組織修復(fù)、再生以及抗炎癥作用已得到證實[37,38,48,62,63]，表明hAECs或hAECs來源細胞因子溶液是未來可應(yīng)用于難愈合性皮膚損傷的潛在治療方法之一。目前clinicaltrials.gov上登記的hAECs或羊膜細胞來源細胞因子溶液（amnion-derived cell cytokine solution，ACCS）相關(guān)臨床研究項目包括了眼表損傷、支氣管瘺、原發(fā)性卵巢功能障礙、難愈合性糖尿病足等多種難治性疾病[65]。皮膚損傷修復(fù)臨床研究用的hAECs主要以hAECs懸液、hAECs來源細胞因子溶液以及負載hAECs的羊膜組織產(chǎn)品形式存在，其中已有多款富含活性hAECs的羊膜產(chǎn)品在歐美批準(zhǔn)上市，用于難治性皮膚損傷及眼科疾病的治療，如AmnioGenix公司的AmnioMTMTM；BioTissue公司的AmnioGraft?；Osiric Therapeutics公司的Grafx?等，這些成功的產(chǎn)品化案例證實了hAECs在皮膚創(chuàng)面治療中的應(yīng)用前景。

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The paracrine effect of human amniotic epithelial cells and their roles in wound healing

Fu Yinsheng2,3, Zhang Yi1,2*, Zhang Tingting1,2, Lu Huiying1,2, Meng Qingxue1,2,
(1National and local joint stem cell research & engineering center for aging diseases, Harbin 150028, China ;2Heilongjiang Tian Qing Stem Cell Co., Ltd, Harbin 150028, China ;3Harbin First Bio-Engineering Co., Ltd, Harbin 150431, China)

Human skin wound healing is a complex and highly coordinated biological process in which growth factors, interleukins and chemokines play a part and regulate three critical steps namely re-epithelialization, neoangiogenesis, and extracellular matrix (ECM) deposition and remodeling. As primitive stem cells, human amniotic epithelial cells (hAECs) are widely applied in wound healing research. Recent evidence reveals that hAECs facilitate wound healing by secreting soluble bioactive factors via paracrine to protect recipient cells from apoptosis, stimulate neovascularization and promote re-epithelialization. This review will focus on the secretory profles of hAECs and their roles in wound healing. The therapeutic potential of hAECs or hAECs-derived cytokine solution in wound healing is also discussed.

Human amniotic epithelial cells; cytokine; paracrine; wound healing

R458

A

10.16705/ j. cnki. 1004-1850.2017.02.015

2016-12-13

2017-03-30

黑龍江省自然科學(xué)基金項目（C201430）

付寅生，男（1986年），漢族，工程師

*通訊作者(To whom correspondence should be addressed)：neo_yi_zhang@163.com