間充質(zhì)干細(xì)胞在皮膚損傷修復(fù)中的作用研究

?

·綜述·

間充質(zhì)干細(xì)胞在皮膚損傷修復(fù)中的作用研究

張立王強(qiáng)

(復(fù)旦大學(xué)附屬中山醫(yī)院皮膚科，上海200032)

Effect of Mesenchymal Stem Cells in Repairment of Skin Injury RepairmentZHANGLiWANGQiangDepartmentofDermatology,ZhongshanHospital,FudanUniversity,Shanghai200032,China

皮膚是機(jī)體最大的器官，具有屏障、吸收、感覺、體溫調(diào)節(jié)、代謝、免疫等多種生理功能。大面積深度皮膚燒傷、皮膚良惡性腫瘤、皮膚色素性疾病等引起的皮膚損傷嚴(yán)重影響患者健康[1]。其中，皮膚燒傷不僅破壞皮膚的屏障功能，還改變皮膚的痛覺、溫覺、觸覺等[2]。皮膚損傷修復(fù)需要經(jīng)過皮膚細(xì)胞嚴(yán)密的編排、整合及分化、遷移、增殖和凋亡過程，才能實(shí)現(xiàn)皮膚多層結(jié)構(gòu)的再生[3]。研制含皮膚附屬器的功能性組織工程全層皮膚是解決大面積皮損的有效途徑，具有重要的研究價(jià)值。近年來，隨著組織工程學(xué)的飛速發(fā)展，發(fā)現(xiàn)間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)是構(gòu)建全層皮膚的理想種子細(xì)胞，在治療重癥皮膚創(chuàng)傷方面展現(xiàn)了良好的應(yīng)用前景。

1MSCs的組成和分類

MSCs是中胚層發(fā)育的早期細(xì)胞，是一類體外易培養(yǎng)、增殖能力強(qiáng)，具有自我更新、多向分化潛能及獨(dú)特免疫調(diào)節(jié)特性的異質(zhì)細(xì)胞群。在適當(dāng)條件下，MSCs可定向分化為中胚層、外胚層及內(nèi)胚層細(xì)胞，如骨細(xì)胞、軟骨細(xì)胞、內(nèi)皮細(xì)胞、肌細(xì)胞、脂肪細(xì)胞等，廣泛參與組織損傷修復(fù)的過程。MSCs最初在骨髓中發(fā)現(xiàn)，近年發(fā)現(xiàn)在脂肪組織、牙髓、胎盤、羊水、臍帶血以及妊娠期胎兒的肝、肺、腎中也存在MSCs[4]。

臍帶間充質(zhì)干細(xì)胞(UC-MSCs)易獲取，且不受倫理道德方面的限制，因此可廣泛應(yīng)用于研究。研究[5]發(fā)現(xiàn)，臍帶組織含有豐富的MSCs，并可在體外分化成不同的細(xì)胞系。臍帶由羊膜、臍血管以及沃頓膠(Wharton's jelly，WJ) 組成。從臍帶WJ中可分離獲得大量具有自我復(fù)制、自我更新、高度增殖和多向分化潛能的MSCs(WJ-MSCs)。據(jù)文獻(xiàn)[6]報(bào)告，WJ-MSCs 和人汗腺細(xì)胞共培養(yǎng)后，可表達(dá)細(xì)胞角蛋白7(cytokeratin7，CK7)和 CK19 等汗腺特異性標(biāo)志物，將其移植到裸鼠創(chuàng)面后，可促進(jìn)創(chuàng)面受損汗腺結(jié)構(gòu)與功能的修復(fù)。

脂肪來源干細(xì)胞(ADSCs)是表達(dá)MSCs標(biāo)志的多能細(xì)胞，位于真皮層內(nèi)，在均質(zhì)脂肪組織血管基質(zhì)的細(xì)胞培養(yǎng)過程中大量出現(xiàn)[7]。從抽脂術(shù)中易獲取ADSCs，損傷小，比其他來源MSCs有優(yōu)勢[7]。目前，ADSCs已成功移植到人體，發(fā)現(xiàn)其有向施旺細(xì)胞分化的潛能[8]。施旺細(xì)胞能分泌層粘連蛋白，并可促進(jìn)神經(jīng)再生。誘導(dǎo)自體皮膚干細(xì)胞分化為施旺細(xì)胞開辟了通過組織工程促進(jìn)神經(jīng)再生的新途徑。但是，誘導(dǎo)多能干細(xì)胞技術(shù)，即從活檢的小塊皮膚中提取成纖維細(xì)胞并將其誘導(dǎo)成干細(xì)胞的技術(shù)，目前無法短期內(nèi)應(yīng)用于臨床[9]。

在體外實(shí)驗(yàn)中[10]實(shí)現(xiàn)了毛囊再生，這使得來源于新生小鼠胚胎的上皮MSCs或來源于成人隆突的干細(xì)胞實(shí)現(xiàn)毛發(fā)再生成為可能。最新研究[11-13]表明，在裸鼠背部注入上皮bulge細(xì)胞和真皮乳頭細(xì)胞的懸浮液，可使裸鼠皮膚毛囊和觸須再生。將毛囊成功移植到新生皮膚來源的前體細(xì)胞后，獲得了含毛囊的組織工程皮膚[14]。皮膚乳頭細(xì)胞和MSCs在體外共培養(yǎng)，再與加入外根鞘來源的角質(zhì)形成細(xì)胞和毛囊黑素細(xì)胞共同構(gòu)成微囊，實(shí)現(xiàn)毛干再生[15]?？傊?，這些研究表明來自隆突部位或真皮乳頭的干細(xì)胞和背部胚胎皮膚懸浮細(xì)胞有形成毛發(fā)的潛能。

成人皮膚含有多種干細(xì)胞群，包括表皮干細(xì)胞和黑素干細(xì)胞[16]。在皮膚毛囊附近的血管周圍含有多能MSCs，表明MSCs有望運(yùn)用于毛囊豐富的頭皮[17]。越來越多的證據(jù)顯示，周細(xì)胞(即結(jié)締組織微毛細(xì)血管管壁細(xì)胞)可能有多能干細(xì)胞的功能[18]。MSCs分布于整個真皮毛細(xì)血管周圍，通過血液循環(huán)促進(jìn)更多組織的再生[19]。

2MSCs相關(guān)細(xì)胞因子

生長因子參與和調(diào)控創(chuàng)傷皮膚愈合的整個過程，在治療慢性難愈合皮膚創(chuàng)口和大面積燒傷創(chuàng)面方面有著廣闊的應(yīng)用前景[20]?；罨?activins)是轉(zhuǎn)化生長因子-β(TGF-β)超家族成員，包括Activin A、Activin B和Activin AB。這些成員分別與相應(yīng)的受體(ACVR)結(jié)合，參與調(diào)節(jié)細(xì)胞增殖、分化、凋亡，從而維持皮膚內(nèi)環(huán)境穩(wěn)態(tài)，參與組織修復(fù)等[21]。其中，Activin A是由皮膚成纖維細(xì)胞分泌，并以旁分泌的形式作用于角質(zhì)形成細(xì)胞，對維持皮膚內(nèi)環(huán)境穩(wěn)態(tài)、傷口愈合和毛發(fā)生長有重要作用[22]。activins和其拮抗劑卵泡抑素(follistatin)共同調(diào)節(jié)毛囊的發(fā)育和周期循環(huán)[23]。在小鼠延遲愈合的皮膚創(chuàng)面中，表皮基底層follistatin的過表達(dá)，而Activin A的過表達(dá)能促進(jìn)肉芽組織及疤痕的形成，這表明Activin A在皮膚愈合過程中起重要作用[24]。

干細(xì)胞遷移到相應(yīng)的組織內(nèi)才能實(shí)現(xiàn)該部位的再生和修復(fù)，而這個過程受趨化因子/趨化因子受體系統(tǒng)的調(diào)控[25]。國外學(xué)者[26]發(fā)現(xiàn)，富含Activin A的培養(yǎng)液可以在無STAT的活化作用下維持胚胎干細(xì)胞的不分化狀態(tài)。文獻(xiàn)[27]報(bào)告，Activin A是維持胚胎干細(xì)胞自我更新和多能性必需的生長因子。外源性activin能通過激活MAPK通路進(jìn)而促進(jìn)角質(zhì)形成細(xì)胞的增殖和遷移[28-29]。在無血清條件下，Activin A能通過FGF/MAPK通路誘導(dǎo)干細(xì)胞分化為定期內(nèi)胚層細(xì)胞[30]。在未分化的MSCs中，activin受體呈高表達(dá)，且TGF-β/activin信號是MSCs分化遷移的重要通路之一[31]。

3MSCs來源的外泌體

MSCs的療效很大程度上取決于其釋放可溶性因子的能力，這些因子(包括生長因子、細(xì)胞因子、趨化因子)在微環(huán)境中相互作用，促進(jìn)組織再生、抑制細(xì)胞凋亡、促進(jìn)細(xì)胞增殖、加速血管生成、調(diào)節(jié)免疫反應(yīng)[32]。除可溶性因子外，MSCs也分泌微球狀細(xì)胞外囊泡(extracellular vesicles，EVs)，在不同細(xì)胞間進(jìn)行信息傳遞，調(diào)節(jié)細(xì)胞間的信號傳導(dǎo)，發(fā)揮多種生物學(xué)功能。由MSCs 分泌的EVs是一種釋放到細(xì)胞外空間的膜性囊泡。根據(jù)體積、浮選密度、脂質(zhì)成分、沉降率、運(yùn)輸?shù)鞍最愋秃蜕镛D(zhuǎn)化途徑的不同，將EVs分為微泡、微粒子、外泌體[33]。這些囊泡釋放到細(xì)胞外空間，但不進(jìn)入血液等體液中，這個過程依賴鈣離子和鈣蛋白酶的協(xié)助[34]。

外泌體是一種由真核細(xì)胞的多泡體(MVBs)與胞膜融合后釋放到胞外的納米級膜性小囊泡[35]。外泌體的出現(xiàn)及其旁分泌理論極大地完善了再生醫(yī)學(xué)中有關(guān)干細(xì)胞的理論。作為信息載體，外泌體通過傳輸?shù)鞍?、活性脂、mRNA、microRNA和改變受體細(xì)胞表型發(fā)揮作用。外泌體在干細(xì)胞和組織損傷細(xì)胞間的信息傳遞是雙向的：損傷細(xì)胞通過外泌體向干細(xì)胞傳遞特異性信息，使干細(xì)胞重新設(shè)定程序并獲得組織特征性的表型；同時，干細(xì)胞亦通過外泌體向損傷細(xì)胞傳遞信息，通過上調(diào)受體細(xì)胞中的抗凋亡基因 BCL2L1、BCL2 和BIRC8 的表達(dá)，及下調(diào)促凋亡基因 CASP1、CASP8 、LTA的表達(dá)，由此抑制損傷細(xì)胞的凋亡，促進(jìn)其再生和修復(fù)[36]。

人類UC-MSCs介導(dǎo)的外泌體(hucMSC-Ex)在小鼠燒傷模型皮膚修復(fù)中起重要作用。在小鼠燒傷模型[37]中，hucMSC-Ex能加速損傷皮膚中上皮細(xì)胞再生，并伴細(xì)胞角蛋白19(CK19)、增殖細(xì)胞核抗原(PCNA)、膠原蛋白1(Collagen I)表達(dá)的增多。在體外實(shí)驗(yàn)[37]中，皮膚受熱受壓后，hucMSC-Ex能促進(jìn)皮膚細(xì)胞增殖并抑制其凋亡。進(jìn)一步研究[37]表明，在小鼠燒傷模型中，hucMSC-Ex釋放Wnt4，促進(jìn)β-catenin 核轉(zhuǎn)位并增強(qiáng)其活性，增加皮膚細(xì)胞的增殖和遷移；在體外試驗(yàn)中敲除hucMSC-Ex內(nèi)的Wnt4則可抑制β-catenin的活性，進(jìn)而抑制細(xì)胞增殖和遷移；而在在體試驗(yàn)中，抑制hucMSC-Ex中Wnt4的表達(dá)后，燒傷的治療效果下降。由hucMSC-Ex激活的AKT通路通過增加小鼠燒傷模型中的細(xì)胞凋亡來降低熱壓[37]。

4MSCs的免疫調(diào)節(jié)

組織內(nèi)穩(wěn)態(tài)受免疫系統(tǒng)的調(diào)節(jié)。MSCs 在自體或同種異體移植中，參與由固有免疫細(xì)胞和適應(yīng)性免疫細(xì)胞介導(dǎo)的免疫抑制和免疫調(diào)節(jié)[38]。MSCs能抑制由絲裂原激活的T細(xì)胞的活性[39]，介導(dǎo)樹突狀細(xì)胞、幼稚和效應(yīng)T細(xì)胞、NK細(xì)胞[40]產(chǎn)生炎性反應(yīng)耐受表型，抑制B細(xì)胞增殖[41]。在免疫性疾病如克隆恩病[42]和I型糖尿病[43]中，MSCs起免疫調(diào)節(jié)作用。在加拿大和新西蘭，MSCs被批準(zhǔn)用于治療小兒移植物抗宿主病(GVHD)。

最新研究表明，MSC-Ex是一種有免疫調(diào)節(jié)作用的膜性囊泡。MSC-Ex介導(dǎo)NF-κB-SEAP報(bào)告基因陽性的THP1-Xblue和THP-1受體細(xì)胞系的多粘菌素耐藥和依賴髓樣分化因子88(MYD88)的胚胎分泌型堿性磷酸酶(SEAP)的表達(dá)。與脂多糖(LPS)相比，MSC-Ex促進(jìn)抗炎因子白介素-10(IL-10)和轉(zhuǎn)化生長因子-β1(TGF-β1)的轉(zhuǎn)錄，抑制促炎因子IL-1B、IL-6、腫瘤壞死因子-α(TNF-α)和IL-12p40的轉(zhuǎn)錄。人源和鼠源性單核細(xì)胞中，MSC-Ex介導(dǎo)的細(xì)胞因子轉(zhuǎn)錄譜相似。MSC-Ex介導(dǎo)THP-1細(xì)胞(而不是MyD88缺陷的細(xì)胞)激活CD4+T細(xì)胞并使其向CD4+CD25+FoxP3+調(diào)控性T 細(xì)胞(Tregs)轉(zhuǎn)化(1 THP-1∶1000 CD4+T細(xì)胞)。MSC-Ex能提高小鼠同種異體皮膚移植的成活率，同時增加Tregs[44]。

5展望

關(guān)于組織工程全層皮膚的建立，近些年在種子細(xì)胞和功能方面的研究有很大進(jìn)步。但如何建立皮膚附屬器(包括血管、汗腺、皮脂腺、神經(jīng)、毛囊等)齊全的人造皮膚仍是難點(diǎn)。外泌體特別是MSC-Ex 的發(fā)現(xiàn)，克服了MSC移植后異常分化的缺點(diǎn)，有很好的開發(fā)前景。MSC-Ex有可能替代MSC成為皮膚損傷修復(fù)的新型治療方式。

參考文獻(xiàn)

[1]Iqbal T, Saaiq M, Ali Z. Epidemiology and outcome of burns: early experience at the country's first national burns centre[J]. Burns,2013,39(2):358-362.

[2]Blais M, Parenteau-Bareil R, Cadau S, et al. Concise review: tissue-engineered skin and nerve regeneration in burn treatment[J]. Stem Cells Transl Med,2013,2(7):545-551.

[3]Bielefeld KA, Amini-Nik S, Alman BA. Cutaneous wound healing: recruiting developmental pathways for regeneration[J]. Cell Mol Life Sci,2013,70(12):2059-2081.

[4]Lee OK, Kuo TK, Chen WM, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood[J]. Blood,2004,103(5):1669-1675.

[5]Secco M, Zucconi E, Vieira NM, et al. Multipotent stem cells from umbilical cord: cord is richer than blood![J]. Stem Cells,2008,26(1):146-150.

[6]Li H, Fu X, Ouyang Y, et al. Adult bone-marrow-derived mesenchymal stem cells contribute to wound healing of skin appendages[J]. Cell Tissue Res,2006,326(3):725-736.

[7]Tobita M, Orbay H, Mizuno H. Adipose-derived stem cells: current findings and future perspectives[J]. Discov Med,2011,11(57):160-170.

[8]Kaewkhaw R, Scutt AM, Haycock JW. Anatomical site influences the differentiation of adipose-derived stem cells for Schwann-cell phenotype and function[J]. Glia,2011,59(5):734-749.

[9]Sipp D. Challenges in the clinical application of induced pluripotent stem cells[J]. Stem Cell Res Ther,2010,1(1):9.

[10]Zheng Y, Nace A, Chen W, et al. Mature hair follicles generated from dissociated cells: a universal mechanism of folliculoneogenesis[J]. Dev Dyn,2010,239(10):2619-2626.

[11]Asakawa K, Toyoshima K E, Ishibashi N, et al. Hair organ regeneration via the bioengineered hair follicular unit transplantation[J]. Sci Rep,2012,2:424.

[12]Toyoshima KE, Asakawa K, Ishibashi N, et al. Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches[J]. Nat Commun,2012,3:784.

[13]Sato A, Toyoshima KE, Toki H, et al. Single follicular unit transplantation reconstructs arrector pili muscle and nerve connections and restores functional hair follicle piloerection[J]. J Dermatol,2012,39(8):682-687.

[14]Lee LF, Jiang TX, Garner W, et al. A simplified procedure to reconstitute hair-producing skin[J]. Tissue Eng Part C Methods,2011,17(4):391-400.

[15]Lindner G, Horland R, Wagner I, et al. De novo formation and ultra-structural characterization of a fiber-producing human hair follicle equivalent in vitro[J]. J Biotechnol,2011,152(3):108-112.

[16]Nishimura EK. Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation[J]. Pigment Cell Melanoma Res,2011,24(3):401-410.

[17]Yamanishi H, Fujiwara S, Soma T. Perivascular localization of dermal stem cells in human scalp[J]. Exp Dermatol,2012,21(1):78-80.

[18]Bouacida A, Rosset P, Trichet V, et al. Pericyte-like progenitors show high immaturity and engraftment potential as compared with mesenchymal stem cells[J]. PLoS One,2012,7(11):e48648.

[19]Ema H, Suda T. Two anatomically distinct niches regulate stem cell activity[J]. Blood,2012,120(11):2174-2181.

[20]Werner S, Grose R. Regulation of wound healing by growth factors and cytokines[J]. Physiol Rev,2003,83(3):835-870.

[21]Chen YG, Wang Q, Lin S L, et al. Activin signaling and its role in regulation of cell proliferation, apoptosis, and carcinogenesis[J]. Exp Biol Med (Maywood),2006,231(5):534-544.

[22]Mcdowall M, Edwards NM, Jahoda CA, et al. The role of activins and follistatins in skin and hair follicle development and function[J]. Cytokine Growth Factor Rev,2008,19(5-6):415-426.

[23]Werner S, Alzheimer C. Roles of activin in tissue repair, fibrosis, and inflammatory disease[J]. Cytokine Growth Factor Rev,2006,17(3):157-171.

[24]Munz B, Smola H, Engelhardt F, et al. Overexpression of activin A in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair[J]. EMBO J,1999,18(19):5205-5215.

[25]Zlotnik A. Chemokines and cancer[J]. Int J Cancer,2006,119(9):2026-2029.

[26]Beattie GM, Lopez AD, Bucay N, et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers[J]. Stem Cells,2005,23(4):489-495.

[27]Xiao L, Yuan X, Sharkis SJ. Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells[J]. Stem Cells,2006,24(6):1476-1486.

[28]Zhang L, Wang W, Hayashi Y, et al. A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure[J]. EMBO J,2003,22(17):4443-4454.

[29]Zhang M, Liu NY, Wang XE, et al. Activin B promotes epithelial wound healing in vivo through RhoA-JNK signaling pathway[J]. PLoS One,2011,6(9):e25143.

[30]Sui L, Mfopou JK, Geens M, et al. FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells[J]. Biochem Biophys Res Commun,2012,426(3):380-385.

[31]Ng F, Boucher S, Koh S, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages[J]. Blood,2008,112(2):295-307.

[32]Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators[J]. J Cell Biochem,2006,98(5):1076-1084.

[33]Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses[J]. Nat Rev Immunol,2009,9(8):581-593.

[34]Heijnen HF, Schiel AE, Fijnheer R, et al. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules[J]. Blood,1999,94(11):3791-3799.

[35]Lee Y, El AS, Wood MJ. Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy[J]. Hum Mol Genet,2012,21(R1):R125-R134.

[36]Camussi G, Deregibus MC, Cantaluppi V. Role of stem-cell-derived microvesicles in the paracrine action of stem cells[J]. Biochem Soc Trans,2013,41(1):283-287.

[37]Zhang B, Wang M, Gong A, et al. HucMSC-exosome mediated -Wnt4 signaling is required for cutaneous wound healing[J]. Stem Cells,2015,33(7):2158-2168.

[38]Marigo I, Dazzi F. The immunomodulatory properties of mesenchymal stem cells[J]. Semin Immunopathol,2011,33(6):593-602.

[39]Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli[J]. Blood,2002,99(10):3838-3843.

[40]Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses[J]. Blood,2005,105(4):1815-1822.

[41]Corcione A, Benvenuto F, Ferretti E, et al. Human mesenchymal stem cells modulate B-cell functions[J]. Blood,2006,107(1):367-372.

[42]Newman RE, Yoo D, Leroux MA, et al. Treatment of inflammatory diseases with mesenchymal stem cells[J]. Inflamm Allergy Drug Targets,2009,8(2):110-123.

[43]Wu H, Mahato RI. Mesenchymal stem cell-based therapy for type 1 diabetes[J]. Discov Med,2014,17(93):139-143.

[44]Zhang B, Yin Y, Lai RC, et al. Mesenchymal stem cells secrete immunologically active exosomes[J]. Stem Cells Dev,2014,23(11):1233-1244.

通訊作者王強(qiáng)，Email：wang.qiang@zs-hospital.sh.cn

中圖分類號R 751.05

文獻(xiàn)標(biāo)識碼A