黨亞龍 徐永勝 張純

干性年齡相關(guān)性黃斑變性的干細(xì)胞治療
——現(xiàn)狀及展望

黨亞龍 徐永勝 張純

年齡相關(guān)性黃斑變性(AMD)不僅是發(fā)達(dá)國家，而且也是發(fā)展中國家主要的、不可逆的致盲眼病。過去的十多年中，新生血管性AMD（濕性AMD）的治療方法已經(jīng)發(fā)生了巨大變化。然而，以黃斑地圖樣萎縮為特征的非新生血管性AMD（干性AMD）仍然無有效的治療方法。干細(xì)胞科學(xué)最新進(jìn)展證實(shí)：RPE細(xì)胞能夠以共培養(yǎng)或特定誘導(dǎo)因子從干細(xì)胞分化獲得。同時，研究顯示RPE細(xì)胞移植能夠維持模型動物的視功能。更重要的是，美國FDA已經(jīng)批準(zhǔn)一些基于干細(xì)胞的RPE移植臨床試驗(yàn)，而且得到了令人鼓舞的成果。這篇綜述將重點(diǎn)從RPE細(xì)胞誘導(dǎo)方法、細(xì)胞活體移植研究、臨床試驗(yàn)及存在的問題等方面加以綜述。

黃斑變性；干細(xì)胞；色素上皮，眼

年齡相關(guān)性黃斑變性(age-related macular degeneration,AMD)是我國65歲以上人群的主要致盲眼病之一，發(fā)病率逐年上升[1-4]。AMD按照有無脈絡(luò)膜新生血管(choroidal neovascularization,CNV)存在，分為干性AMD和濕性AMD。隨著對CNV發(fā)生機(jī)制的深入研究，抗血管內(nèi)皮細(xì)胞生長因子類藥物、光動力療法、激光光凝及玻璃體切除手術(shù)在不同時機(jī)的應(yīng)用，濕性AMD得到了一定程度的控制[5-7]。

干性AMD主要是由于氧自由基和脂質(zhì)過氧化物等在視網(wǎng)膜內(nèi)蓄積，局部慢性炎癥活化，導(dǎo)致視網(wǎng)膜色素上皮(retinal pigment epithelium,RPE)細(xì)胞凋亡及繼發(fā)的光感受器細(xì)胞損傷，目前尚無確切的藥物療法[8]。因此，細(xì)胞替代及視網(wǎng)膜微環(huán)境調(diào)控成為治療干性AMD的新思路。

干細(xì)胞具有多向分化潛能，在不同誘導(dǎo)條件下，能分化為RPE細(xì)胞和光感受器細(xì)胞，是細(xì)胞替代的來源之一。另外，干細(xì)胞，特別是間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)還有免疫調(diào)節(jié)、抑制神經(jīng)細(xì)胞凋亡的作用，能夠維持和調(diào)節(jié)視網(wǎng)膜微環(huán)境。近年來，大量基礎(chǔ)研究及I/II期臨床試驗(yàn)對干性AMD的干細(xì)胞治療進(jìn)行報(bào)道，本文將從干細(xì)胞來源的RPE細(xì)胞替代、干細(xì)胞對視網(wǎng)膜微環(huán)境的調(diào)控兩個方面加以綜述。

一、干細(xì)胞來源的RPE細(xì)胞替代

健康、有活力的RPE細(xì)胞是干性AMD患者理想的替代細(xì)胞。按照細(xì)胞來源分為：干細(xì)胞來源的RPE細(xì)胞、胎兒/成體RPE細(xì)胞、虹膜色素上皮細(xì)胞、自體RPE細(xì)胞等[9-11]。由于后三種細(xì)胞來源有限、分離純化困難、增殖能力差等，應(yīng)用受到了限制。

胚胎干細(xì)胞(embryonic stem cells,ESC)、誘導(dǎo)多潛能干細(xì)胞(induced pluripotent stem cells,iPS)及成體干細(xì)胞均可以在一定的條件下分化為成熟的有功能的RPE細(xì)胞。

(一)ESC來源的RPE細(xì)胞替代

ESC來源的RPE細(xì)胞替代治療是目前研究的熱點(diǎn)。按照獲取RPE細(xì)胞的方法不同，分為7類：自然分化發(fā)法、基質(zhì)細(xì)胞誘導(dǎo)法(stromal cell derived inducing activity,SDIA)、無血清擬胚體懸浮培養(yǎng)法(serum-free embryoid body-like,SFEB)、小分子誘導(dǎo)法、視網(wǎng)膜決定法(retinal determination,RD)、神經(jīng)球團(tuán)分選法(spherical neural masses,SNMs)和3D視網(wǎng)膜誘導(dǎo)法。

1.自然分化法：約1﹪的ESC可自動分化為RPE細(xì)胞[12]，這些細(xì)胞具備成熟RPE的標(biāo)記，將這些細(xì)胞移植到RCS大鼠(royal college of surgeons rat,RCS rat)的視網(wǎng)膜下，發(fā)現(xiàn)移植細(xì)胞具有極性，能與宿主的光感受器整合，能吞噬脫落的光感受器外節(jié)，維持大鼠的視功能[12-13]。對免疫抑制模型動物的視網(wǎng)膜下移植還發(fā)現(xiàn)了無畸胎瘤及其他病理變化[14]。

雖然該方法分化效率很低，但沒有誘導(dǎo)劑及導(dǎo)入潛在的致病基因等，美國FDA批準(zhǔn)其為Good Manufacturing Practices(GMP)標(biāo)準(zhǔn)[14]。2011年，美國Advanced Cell Technology(Santa Monica,California,USA)應(yīng)用該技術(shù)開展了hESC來源的RPE移植I/II期臨床試驗(yàn)（注冊號：NCT01345006、NCT01344993），治療對象：干性AMD、Stargardt’s病[14]。2012年，Schwartz等[15]報(bào)道了初步研究成果：兩位患者（AMD、Stargardt’s病各1例）通過玻璃體切除手術(shù)在視網(wǎng)膜下腔特定部位植入了5 × 104個RPE細(xì)胞。有效性：（1）在隨訪的4個月內(nèi)，移植物均存在；（2）兩例患者的視力均得到一定程度的提高：干性AMD患者的視力從21個字母提高到28個字母，Stargardt’s病患者的視力從0個字母提高到5個字母（均為early treatment diabetic retinopathy study,ETDRS視力表）。安全性：兩例患者均未發(fā)現(xiàn)細(xì)胞異常增殖、免疫排斥等現(xiàn)象。研究還發(fā)現(xiàn)RPE分化的狀態(tài)與細(xì)胞貼附和生存相關(guān)，輕度色素脫失的RPE細(xì)胞具有更好的增殖和貼附能力。

盡管初步研究顯示了RPE細(xì)胞移植的良好應(yīng)用前景，但仍有一些問題有待克服：(1)RPE成熟度狀態(tài)決定了移植后增殖和存活能力，因此選擇合適分化程度的RPE細(xì)胞是治療成功的關(guān)鍵；(2)用作誘導(dǎo)的hESC不能含致病基因；(3)如何得到極高純度的RPE也是一個重要的課題。

2.SDIA法：2000年，Kawasaki等[16]命名了SDIA法。2002年，他用SDIA法從靈長類動物的ESC誘導(dǎo)出了(8 ± 4)﹪RPE細(xì)胞[17]。后來，他對這些RPE細(xì)胞進(jìn)行了蛋白標(biāo)記、吞噬功能及RCS大鼠的視網(wǎng)膜下腔移植實(shí)驗(yàn)，證實(shí)這些細(xì)胞能夠促進(jìn)宿主光感受器細(xì)胞的生存[18]。該法的優(yōu)點(diǎn)是沒有添加外源性的誘導(dǎo)劑，但是存在PA6基質(zhì)細(xì)胞污染的可能。另外，SDIA法未報(bào)道能誘導(dǎo)出光感受器樣細(xì)胞，因此臨床應(yīng)用前景有限。

3.SFEB法：2005年，Ikeda等[19]應(yīng)用mESC無血清擬胚體(serum-free embryoid body-like,SFEB)懸浮培養(yǎng)，能夠誘導(dǎo)mESC分化為Rx+/Pax+的視網(wǎng)膜前體細(xì)胞。經(jīng)過四年的探索，該團(tuán)隊(duì)成功的獲得了(23.8 ± 2.7)﹪的RPE前體細(xì)胞、(11.5 ± 2.0)﹪紅綠視蛋白陽性的視錐細(xì)胞、(10.7 ± 1.6)﹪藍(lán)色視蛋白陽性的視錐細(xì)胞和(17.2 ± 1.8)﹪視桿細(xì)胞[20]。遺憾的是，SFEB法誘導(dǎo)產(chǎn)生的細(xì)胞整合到宿主視網(wǎng)膜的能力較低[21]，主要原因：（1）SFEB法誘導(dǎo)產(chǎn)生的視網(wǎng)膜前體細(xì)胞比例低；（2）雖然SFEB法模擬了視網(wǎng)膜發(fā)育的過程，但所得細(xì)胞發(fā)育較為成熟，自身的整合能力較差[22]。

4.小分子誘導(dǎo)法：Osakada等[23]在外源性添加CKI-7(Wnt抑制劑)和SB-431542(Nodal抑制劑)能夠獲得了(18.1 ± 1.9)﹪的RPE細(xì)胞，這些細(xì)胞具備成熟RPE的形態(tài)、蛋白標(biāo)記和吞噬能力。小分子誘導(dǎo)具有以下優(yōu)點(diǎn)：（1）誘導(dǎo)劑屬于化學(xué)制品，不同批次和廠家之間差異較??；（2）避免了生物制品的誘導(dǎo)劑可能造成的污染和交叉反應(yīng)；（3）價(jià)格相對低廉，有助于普及應(yīng)用。但是，該方法沒有經(jīng)過動物實(shí)驗(yàn)，其安全性、有效性尚待深入研究。

5.RD法：Lamba等[24]應(yīng)用Noggin(BMP通路抑制劑)、DKK1(Wnt/β-catenin通路抑制劑)和IGF-1，得到高達(dá)(82 ± 23)﹪ Pax+的視網(wǎng)膜前體細(xì)胞，其中86﹪的細(xì)胞也表達(dá)Chx10。將這些細(xì)胞移植到Crx-模型鼠的視網(wǎng)膜下腔可以改善視功能[25]。RD法最突出的優(yōu)點(diǎn)在于能夠在短時間內(nèi)收獲大量的視網(wǎng)膜前體細(xì)胞。

6.SNMs分選法：2008年，Cho等[26]發(fā)現(xiàn)擬胚體形成后經(jīng)過神經(jīng)前體細(xì)胞選擇及擴(kuò)增，可以得到SNMs。其中，大約有5﹪囊泡樣結(jié)構(gòu)最終分化為RPE細(xì)胞[27]。SNMs法具有以下優(yōu)點(diǎn)：（1）沒有外源性添加誘導(dǎo)劑，避免了污染和免疫反應(yīng)的可能；（2）SNMs來源RPE細(xì)胞更接近人體內(nèi)RPE產(chǎn)生過程；（3）SNMs縮短了從ESC轉(zhuǎn)化為RPE的時間。但迄今為止，該方法沒有進(jìn)行嚴(yán)格的動物實(shí)驗(yàn)來驗(yàn)證所得到的RPE是否具有功能。

7.3D視網(wǎng)膜誘導(dǎo)法：2011年，Eiraku等[28]應(yīng)用SFEB法在matrigel構(gòu)建的3D培養(yǎng)體系內(nèi)成功模擬了視網(wǎng)膜發(fā)育的過程。隨后，Zhu等[29]利用matrigel構(gòu)架的3D體系在Activin A存在的情況下30 d之內(nèi)分化為RPE細(xì)胞并能有效整合在模型動物的RPE層。

（二）iPS來源的RPE細(xì)胞替代

2006年，Yamanaka等[30]報(bào)道小鼠成纖維細(xì)胞可以誘導(dǎo)成為ESC樣細(xì)胞，命名為誘導(dǎo)多能干細(xì)胞。iPS具有ESC類似的形態(tài)和分化能力，在不同的誘導(dǎo)條件下能夠分化為3個胚層的細(xì)胞。iPS還具備獨(dú)特的優(yōu)點(diǎn)：(1)來源廣泛；(2)理論上無免疫原性，由于iPS來源于自身的成熟體細(xì)胞，利用iPS分化而來的細(xì)胞進(jìn)行細(xì)胞移植時可以避免種屬或者個體間的排斥反應(yīng)；(3)無倫理學(xué)爭議；(4)iPS還可以構(gòu)建疾病模型及測試藥物。

與ESC類似，iPS具備分化為RPE及光感受器細(xì)胞的能力。iPS來源的RPE表達(dá)成熟RPE的蛋白標(biāo)記，具備吞噬能力，iPS來源的RPE已經(jīng)成功的移植到模型動物并發(fā)揮功能[31-36]。

盡管用于誘導(dǎo)ESC的各種方法大多適合iPS的誘導(dǎo)，但不同的iPS細(xì)胞系間仍存在較大差異。Hirami等報(bào)道[20]，在完全相同的誘導(dǎo)條件（SFEB/ DL）下，201B7細(xì)胞系和253G1細(xì)胞系可以誘導(dǎo)分化為RPE細(xì)胞，而201B6細(xì)胞系則不能。在蛋白表達(dá)上，在向RPE分化的第6天mESC即可發(fā)現(xiàn)Rx+/ Pax+細(xì)胞，但部分iPS細(xì)胞系則需要15 d。這可能與iPS本身基因組特性有關(guān)，但也可能與培養(yǎng)環(huán)境及分化程度有關(guān)。

iPS雖然有各種優(yōu)點(diǎn)，但缺點(diǎn)同樣不可忽視：(1)iPS來源于患者，所以可能攜帶致病基因，只有當(dāng)致病基因被修復(fù)后，iPS誘導(dǎo)所得的細(xì)胞才能安全的移植入受體[37]；(2)iPS潛在的致瘤風(fēng)險(xiǎn)。Hirami等[20]發(fā)現(xiàn)，在iPS分化的第15天，仍有(0.60 ± 0.04)﹪的細(xì)胞NANOG+。

（三）MSCs來源的RPE細(xì)胞替代

盡管RPE和光感受器細(xì)胞來源于神經(jīng)外胚層，但MSCs具備跨胚層分化的能力。Huang等[38]報(bào)道，應(yīng)用光感受器外節(jié)和RPE細(xì)胞條件培養(yǎng)基能夠誘導(dǎo)間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)分化為具備形態(tài)和吞噬功能的RPE細(xì)胞，但這些RPE細(xì)胞也未經(jīng)過嚴(yán)格的動物實(shí)驗(yàn)。

另外，在某些特定條件下，MSCs還可在受損的視網(wǎng)膜內(nèi)進(jìn)一步分化，發(fā)揮細(xì)胞替代作用。Gong等[39]對碘酸鈉誘導(dǎo)的RPE損傷大鼠視網(wǎng)膜下注射BM-MSC，5周后發(fā)現(xiàn)BM-MSC可以轉(zhuǎn)化為RPE、光感受器及膠質(zhì)細(xì)胞。Tomita等[40]發(fā)現(xiàn)MSCs能夠遷徙至機(jī)械損傷大鼠的視網(wǎng)膜內(nèi)（主要是內(nèi)核層），轉(zhuǎn)化為表達(dá)GFAP、Calbindin、Rhodopsin、Vimentin的視網(wǎng)膜細(xì)胞。Castanheira等[41]對激光損傷模型大鼠的玻璃體腔內(nèi)注射MSCs，經(jīng)過8周，大多數(shù)MSCs已經(jīng)遷徙至神經(jīng)節(jié)細(xì)胞層、內(nèi)核層和外核層。這些細(xì)胞表達(dá)光感受器細(xì)胞、雙極細(xì)胞、無長突細(xì)胞、Müller細(xì)胞的標(biāo)記。

（四）視網(wǎng)膜干細(xì)胞(retinal stem cells,RSCs)來源的RPE細(xì)胞替代

魚類和兩棲類動物的RSCs存在睫狀體邊緣帶（ciliary marginal zone，CMZ），當(dāng)視網(wǎng)膜受損時，CMZ能夠不斷產(chǎn)生新的神經(jīng)元。成熟的哺乳類動物的視網(wǎng)膜缺乏再生能力，但Tropepe等[42]發(fā)現(xiàn)成熟小鼠的CMZ細(xì)胞具備增殖及分化為視網(wǎng)膜神經(jīng)元（視桿細(xì)胞、雙極細(xì)胞）及神經(jīng)膠質(zhì)細(xì)胞的能力，他認(rèn)為這類細(xì)胞是RSCs。Aruta等[43]在分離RSCs的基礎(chǔ)上，添加亞油酸、亞硒酸、胰島素、轉(zhuǎn)鐵蛋白和甲狀腺素等誘導(dǎo)因子成功將RSCs誘導(dǎo)分化為具有極性和吞噬功能的RPE樣細(xì)胞。與MSCs類似，該方法得到的RPE樣細(xì)胞也未經(jīng)過動物實(shí)驗(yàn)驗(yàn)證其安全性和有效性。然而，哺乳類RSCs存在與否仍備受爭議。Cicero等[44]認(rèn)為來源于CMZ的RSCs實(shí)際上是睫狀體上皮細(xì)胞。他從分子、細(xì)胞及形態(tài)學(xué)特征上證實(shí)這些細(xì)胞與分化的睫狀體上皮細(xì)胞無明顯差異。他還認(rèn)為已分化的細(xì)胞也可以形成克隆球、自我更新、表達(dá)前體細(xì)胞的標(biāo)記等。Gualdoni等[45]發(fā)現(xiàn)所謂的RSCs在光感受器細(xì)胞分化培養(yǎng)基內(nèi)并不能活化Nrl（光感受器細(xì)胞分化的關(guān)鍵基因）。

另外，Müller細(xì)胞曾被視為RSCs。Bernardos等[46]報(bào)道，斑馬魚的Müller細(xì)胞能夠低水平的表達(dá)PAX6（視網(wǎng)膜前體細(xì)胞的標(biāo)記）和Crx(光感受器細(xì)胞的標(biāo)記)。Song等[47]發(fā)現(xiàn)：Atoh7（Notch通路抑制劑）能夠促使Müller細(xì)胞轉(zhuǎn)化為視網(wǎng)膜神經(jīng)節(jié)細(xì)胞。Müller細(xì)胞由神經(jīng)視網(wǎng)膜前體細(xì)胞發(fā)育而來，而且分化的最晚（神經(jīng)視網(wǎng)膜發(fā)育順序依次是：視網(wǎng)膜神經(jīng)節(jié)細(xì)胞、視錐細(xì)胞、無長突細(xì)胞、水平細(xì)胞、視桿細(xì)胞、雙極細(xì)胞和Müller細(xì)胞），而RPE前體細(xì)胞與神經(jīng)視網(wǎng)膜前體細(xì)胞分層發(fā)育發(fā)生在胚胎早期。因此，Müller發(fā)育為RPE細(xì)胞的難度很大。

二、干細(xì)胞對視網(wǎng)膜微環(huán)境調(diào)控

氧化應(yīng)激損傷、炎性因子活化和視網(wǎng)膜營養(yǎng)缺乏是干性AMD的發(fā)病機(jī)制之一[8]。干細(xì)胞，特別是MSCs具有多種生物學(xué)作用：分泌營養(yǎng)因子、促進(jìn)血管生成、調(diào)節(jié)免疫反應(yīng)、抗凋亡、促進(jìn)細(xì)胞外基質(zhì)的重塑及活化相鄰的宿主干細(xì)胞[48]。另外，免疫原性低的MSCs，也是一種良好的載體：通過外源性的導(dǎo)入神經(jīng)營養(yǎng)因子，也可以在宿主體內(nèi)表達(dá)，發(fā)揮生物學(xué)作用。因此，MSCs也可用于治療干性AMD。

根據(jù)來源不同，MSCs可分為骨髓間充質(zhì)干細(xì)胞（bone marrow mesenchymal stem cells,BM-MSCs）、臍血間充質(zhì)干細(xì)胞(umbilical cord blood derived mesenchymal stem cells,UCB-MSCs)、臍帶間充質(zhì)干 細(xì) 胞(umbilical cord derived mesenchymal stem cells,UC-MSCs)、胎盤間充質(zhì)干細(xì)胞(placenta derived mesenchymal stem cells,PD-MSCs)和脂肪間充質(zhì)干細(xì)胞(adipose tissue derived stromal cells,ASCs)等。BM-MSCs是研究最廣泛、最深入的一類MSCs，本文將重點(diǎn)綜述BM-MSCs對干性AMD的研究及應(yīng)用現(xiàn)狀。

（一）MSCs對視網(wǎng)膜微環(huán)境的調(diào)控

1.MSCs能夠分泌神經(jīng)營養(yǎng)因子：Inoue等[49]發(fā)現(xiàn)BM-MSCs條件培養(yǎng)基可以延緩光感受器細(xì)胞的凋亡，BM-MSCs注射入RCS大鼠的玻璃體腔后，光感受器退化延緩，視網(wǎng)膜功能得到一定保護(hù)。這提示：BM-MSCs可能分泌一些因子抑制光感受器細(xì)胞凋亡。Zhang等[50]發(fā)現(xiàn)光損傷模型中，玻璃體腔內(nèi)注射的BM-MSCs能夠表達(dá)BDNF，保護(hù)外核層視網(wǎng)膜細(xì)胞。Xu等[51-52]的研究提示MSCs能夠表達(dá)bFGF促進(jìn)光損傷模型大鼠的神經(jīng)細(xì)胞保護(hù)。Wang等[53]還對RCS大鼠尾靜脈注射BM-MSCs 1 × 106，結(jié)果顯示：注射組的外核層細(xì)胞存活率顯著高于對照組；視功能和電生理得到明顯改善，血管滲漏減輕；RT-PCR及免疫組化顯示：生長因子及視網(wǎng)膜營養(yǎng)因子表達(dá)上調(diào)。

2.MSCs能夠抑制局部炎癥：Xu等[51-52]發(fā)現(xiàn)玻璃體腔注射BM-MSCs能夠抑制小膠質(zhì)細(xì)胞活化，減輕視網(wǎng)膜損傷。

3.MSCs能抑制神經(jīng)細(xì)胞凋亡：Otani等[54]研究發(fā)現(xiàn)玻璃體腔注射BM-MSCs后，視網(wǎng)膜抑制凋亡基因表達(dá)顯著上調(diào)，包括一些小分子的熱休克蛋白和轉(zhuǎn)錄因子。

4.MSCs可整合入宿主的視網(wǎng)膜：Arnhold等[55]對rhodopsin敲除的視網(wǎng)膜色素變性（Retinitis Pigmentosa,RP）模型小鼠玻璃體腔內(nèi)注射mBM-MSCs，發(fā)現(xiàn)mBM-MSCs不僅整合入宿主的RPE層和神經(jīng)上皮層，而且顯著的保護(hù)了光感受器細(xì)胞。

值得注意的是：（1）不同來源的MSCs在宿主眼內(nèi)的存活及整合能力不同，玻璃體腔注射的UCB-MSCs很少遷徙至宿主視網(wǎng)膜，而且其生存期也僅僅3周[56]，而BM-MSCs存活時間可達(dá)20周并有良好的整合能力[57]；（2）不同種屬和類型的MSCs對視網(wǎng)膜神經(jīng)細(xì)胞的保護(hù)作用也不相同，Levkovitch-Verbin等[58]發(fā)現(xiàn)人源的BM-MSCs都能夠保護(hù)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞，但大鼠來源的BMMSCs無保護(hù)功能；Huang等[59]研究還提示：表達(dá)CX3CL1的MSCs促進(jìn)光損傷視網(wǎng)膜修復(fù)的能力最強(qiáng)；（3）在不同的移植方式下，MSCs對視網(wǎng)膜的保護(hù)作用也不相同，Tzameret等[57]對比了玻璃體腔注射和視網(wǎng)膜下薄膜植片的移植效果：兩者作用持續(xù)時間分別為12周、20周，ERG b波振幅：玻璃體腔注射56.4 μV，視網(wǎng)膜下薄膜植片是66.2 μV；（4）不同的視網(wǎng)膜微環(huán)境也影響MSCs在宿主眼內(nèi)功能的發(fā)揮。

基于成功的動物實(shí)驗(yàn)，一些眼科學(xué)者審慎的開展了MSCs的I/II臨床試驗(yàn)。2005年Kumar等[60]對25例干性AMD和視網(wǎng)膜色素變性(retinitis pigmentosa,RP)患者的玻璃體腔注射自體BM-MSCs，注射后1個月及3個月患者的視力得到了輕度改善。2010年Jonas等[61]（注冊號：NCT01068561）報(bào)道了3例接受玻璃體腔注射BMMSCs的患者（干性AMD 1例）。患者初始視力：光感（光定位差），接受BM-MSCs注射后，12個月隨訪視力并無明顯改善，但無嚴(yán)重并發(fā)癥存在，僅在治療后4周眼壓有所波動（15 ～ 30 mmHg）。Siqueira等[62]對3例RP患者和2例錐桿細(xì)胞營養(yǎng)不良患者的玻璃體腔注射BM-MSCs 1×107/眼，結(jié)果顯示：1周后，4例患者視力提高1行并維持到隨訪結(jié)束。2例患者的電生理有輕度改善，眼底血管造影、光相干斷層掃描及視野等無明顯變化，在隨訪期間無并發(fā)癥。雖然目前僅有的少數(shù)幾個臨床試驗(yàn)的結(jié)果并不令人振奮，但我們需考慮到以下影響因素：（1）入組患者的年齡均較大，自體BM-MSCs增殖能力及活力有限；（2）入組患者的均處于該疾病的晚期，視力極差，恢復(fù)困難。

（二）基因修飾的MSCs對視網(wǎng)膜細(xì)胞的作用

隨著細(xì)胞工程的發(fā)展，MSCs逐漸成為一種有前景的載體細(xì)胞。Guan等[63]將MSCs注射入碘酸鈉損傷模型大鼠視網(wǎng)膜下腔，發(fā)現(xiàn)經(jīng)EPO修飾的MSCs的大鼠玻璃體腔內(nèi)EPO的含量上升，神經(jīng)細(xì)胞保護(hù)作用強(qiáng)于普通MSCs。Machalinska等[64]也發(fā)現(xiàn)轉(zhuǎn)入NT-4基因的MSCs能夠遷徙至視網(wǎng)膜損傷區(qū)域，保護(hù)受損的視網(wǎng)膜細(xì)胞。更重要的是，NT-4修飾的MSCs能夠上調(diào)與細(xì)胞生存相關(guān)的信號及轉(zhuǎn)錄因子，如crystallin β-γ超家族。另外，還能上調(diào)與視覺感知、視覺信號接收及眼發(fā)育等相關(guān)的蛋白。Park等[65]觀察了BDNF修飾的rBM-MSCs視網(wǎng)膜下和玻璃體腔移植效果，發(fā)現(xiàn)4周后，15.7﹪的rBM-MSCs整合入模型大鼠的視網(wǎng)膜，并且視網(wǎng)膜BDNF mRNA和蛋白水平上調(diào)。

基因修飾的MSCs除具備基本的視網(wǎng)膜微環(huán)境調(diào)控作用外，還被賦予了與導(dǎo)入基因相匹配的特殊功能，因此具有較好的應(yīng)用前景。但對于干性AMD來說，導(dǎo)入基因的種類及途徑等都需要詳細(xì)研究，同時其安全性、有效性也需要進(jìn)一步評估。

三、展望

人類對干細(xì)胞生物學(xué)特性、誘導(dǎo)方法、移植手段等不斷深入的研究促使細(xì)胞治療逐漸由夢想變?yōu)楝F(xiàn)實(shí)，但真正的使干細(xì)胞應(yīng)用于臨床實(shí)踐還有很多困難：（1）現(xiàn)有的臨床試驗(yàn)樣本量非常小，其安全性還有待大樣本、多中心研究；（2）雖然眼內(nèi)被認(rèn)為是免疫赦免區(qū)域，但研究表明[66]：移植細(xì)胞在宿主體內(nèi)長期生存仍需要免疫抑制，因此，免疫抑制持續(xù)的時間、推薦劑量等也需要詳細(xì)探討；（3）具有不同發(fā)病機(jī)制及病理過程的疾病可能均表現(xiàn)為RPE或光感受器細(xì)胞的喪失。不同疾病所需細(xì)胞移植的種類、分化程度、移植量、移植方式等都需要進(jìn)一步探討。

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Stem cells-based therapies for dry type of age related macular degeneration: current status and future prospects

Dang Yalong,Xu Yongsheng,Zhang Chun.Department of Ophthalmology,Peking University Third Hospital

Age related macular degeneration (AMD) is one of the leading causes of irreversible visual impairment in the developed and developing countries.The management of neovascular AMD (wet AMD) showed remarkable progression in the past decade.However,nonneovascular AMD (dry AMD) characterized by geographic macular atrophy still cannot be cured.Recently,it is demonstrated that retinal pigment cells may be generated from the stem cells by defined factors or cell co-culturing.Studies also showed cell transplantation may restore visual function in vivo.Moreover,several clinical trails approved by the FDA have showed the promising prospect in stem cells-based therapies in dry AMD.This review will focus on recent advances in stem cell-based RPE differentiation,cell transplantation,clinical trials and the obstacles that must be overcomed for stem cell therapy in dry AMD.

Macular degeneration；Stem cells；Pigment epithelium of eye

2014-02-10)

（本文編輯：李少婷）

10.3877/cma.j.issn.2095-1221.2014.02.008

教育部高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金（編號：20100001120100）

100191 北京，北京大學(xué)第三醫(yī)院眼科(黨亞龍、張純)，臨床干細(xì)胞研究中心（徐永勝）

張純，Email：zhangc1@yahoo.com

Correspondence: Zhang Chun,Email:zhangc1@yahoo.com

黨亞龍,徐永勝,張純.干性年齡相關(guān)性黃斑變性的干細(xì)胞治療——現(xiàn)狀及展望[J/CD].中華細(xì)胞與干細(xì)胞雜志:電子版,2014,4(2):122-129.