袁 洋 綜述，王思涵，郭小凱，劉 暢 審校

(吉林大學(xué)口腔醫(yī)院正畸科，吉林 長(zhǎng)春 130021)

滑膜間充質(zhì)干細(xì)胞的生物學(xué)特性及其在軟骨組織工程中應(yīng)用的研究進(jìn)展

袁 洋 綜述，王思涵，郭小凱，劉 暢 審校

(吉林大學(xué)口腔醫(yī)院正畸科，吉林 長(zhǎng)春 130021)

滑膜間充質(zhì)干細(xì)胞(Synovium-derived mesenchymal stem cells，SMCSs)的增殖能力強(qiáng)，分離方法簡(jiǎn)便，具有多向分化潛能。其中SMSCs的成軟骨分化能力尤為突出，被廣泛應(yīng)用于軟骨缺損修復(fù)的實(shí)驗(yàn)中，因此SMSCs可能成為軟骨組織工程領(lǐng)域中新的種子細(xì)胞，為關(guān)節(jié)軟骨疾病治療提供了新的可能性。本文對(duì)SMSCs的組織來(lái)源、生物學(xué)特性以及在軟骨組織工程中的應(yīng)用展開(kāi)綜述。

滑膜間充質(zhì)干細(xì)胞；種子細(xì)胞；軟骨組織工程

干細(xì)胞是一類具有高度自我更新能力和多向分化潛能的細(xì)胞，可向不同胚層來(lái)源的組織分化。間充質(zhì)干細(xì)胞首先在骨髓組織中被分離出，隨后，在其他多種組織中都發(fā)現(xiàn)了間充質(zhì)干細(xì)胞，如脂肪、肌肉等。De等[1]首先在人體滑膜組織中分離出一種具有高度增殖能力，并且可以向多種細(xì)胞分化的細(xì)胞，經(jīng)鑒定后命名為滑膜間充質(zhì)干細(xì)胞(Synovium-derived mesenchymal stem cells，SMSCs)。SMSCs在表現(xiàn)出一般成體干細(xì)胞生物學(xué)行為的同時(shí)，還具有高度成軟骨分化能力[2-3]，在體內(nèi)體外實(shí)驗(yàn)中均表現(xiàn)出了其優(yōu)越的軟骨缺損修復(fù)的能力[4-5]，為關(guān)節(jié)軟骨損傷疾病提供了新的治療方向。

1 SMSCs的組織來(lái)源

自從人體干細(xì)胞于1999年在骨髓組織中被分離出后，隨后也陸續(xù)在其他組織中被分離出來(lái)[6]。同時(shí)，干細(xì)胞也可在滑膜、骨周膜和關(guān)節(jié)軟骨等各種結(jié)締組織中分離出來(lái)[2]?；じ鶕?jù)在關(guān)節(jié)囊內(nèi)的解剖位置可分為兩層：表層的內(nèi)膜和內(nèi)膜下方的內(nèi)膜下層，即纖維性滑膜和脂肪性滑膜。內(nèi)膜較為疏松，沒(méi)有血管和基底膜支持，內(nèi)膜下層是由散亂的細(xì)胞和血管組成的網(wǎng)狀結(jié)締組織[7]。Futami等[8]學(xué)者發(fā)現(xiàn)兩種滑膜來(lái)源的SMSCs在細(xì)胞形態(tài)、表面分子、集落形成效率、成軟骨、成骨、成脂能力等方面具有相似的特點(diǎn)。

2 SMSCs生物學(xué)特性

2.1 SMSCs表型特點(diǎn) SMSCs表面分子與骨髓間充質(zhì)干細(xì)胞（Bone marrow-derived mesenchymal stem cells，BMSCs）相似。通過(guò)流式細(xì)胞儀分析，來(lái)源于健康人和類風(fēng)濕性關(guān)節(jié)炎患者的兩種SMSCs表面CD14、CD35、CD45為陰性，而CD44、CD90、CD105和CD166為陽(yáng)性[9]，此結(jié)論與Harvanová等[7]研究結(jié)果相符合。此外，還有一些分子如CD44、CD73、CD271等存在于SMSCs表面[10-11]，但是在這些研究報(bào)道中，SMSCs表面的分子有著一定的差異。Gullo等[2]通過(guò)流式細(xì)胞儀鑒定出SMSCs表面存在CD271、CD81、CD73、CD166、CD146、CD13，CD45和CD90。而也有學(xué)者的研究顯示SMSCs表面的CD14、CD34、CD45為陰性，CD44、CD73、CD90和CD105為陽(yáng)性，結(jié)果與BMSCs相似。以上論述也說(shuō)明在SMSCs表面分子的研究中，尚存在一些小的爭(zhēng)議。Van Landuyt學(xué)者等[12]發(fā)現(xiàn)在SMSCs亞群中CD271在經(jīng)過(guò)培養(yǎng)和未經(jīng)過(guò)培養(yǎng)的SMSCs中的表達(dá)有著一定的區(qū)別，而且CD34在健康人來(lái)源SMSCs中的表達(dá)要高于骨關(guān)節(jié)炎患者。因此，本文認(rèn)為SMSCs表面的分子表型可能會(huì)隨著周圍環(huán)境條件變化而發(fā)生輕微改變，具體機(jī)制尚不清楚，有待于進(jìn)一步研究。

2.2 SMSCs的增殖特點(diǎn) 干細(xì)胞鑒別的重要條件是集落形成效率和成纖維細(xì)胞樣形態(tài)，主要是通過(guò)觀察細(xì)胞的克隆能力和細(xì)胞的梭形形態(tài)[13]。Futami等[8]發(fā)現(xiàn)鼠來(lái)源SMSCs的集落形成數(shù)目要明顯高于骨髓和肌肉來(lái)源的間充質(zhì)干細(xì)胞。研究表明單克隆細(xì)胞群的平均傳代次數(shù)為20次，甚至一部分細(xì)胞群傳代次數(shù)超過(guò)50次，表現(xiàn)出了高水平的克隆能力[14]。并且SMSCs的集落形成能力隨著傳代次數(shù)的增加而逐漸減小，此現(xiàn)象與選取樣本的方法無(wú)關(guān)[4]。

SMSCs作為一種新興的種子細(xì)胞，和傳統(tǒng)的間充質(zhì)干細(xì)胞相比，具有更強(qiáng)的增殖能力[15]，因此受到組織工程學(xué)的廣泛關(guān)注。SMSCs用單層細(xì)胞培養(yǎng)方法可達(dá)到廣泛擴(kuò)增的目的，而且細(xì)胞衰老現(xiàn)象很少。而且，適宜低密度的擴(kuò)增可以使SMSCs得到最大程度的增殖，這樣高水平的增殖能力使得在臨床應(yīng)用中，小樣本的滑膜組織即可獲得足量的SMSCs[16]。除了SMSCs自身的高度增殖能力以外，Kim等[17]發(fā)現(xiàn)堿性成纖維細(xì)胞生長(zhǎng)因子（Basic fibroblast growth factor，bFGF）能改變SMSCs在單層細(xì)胞培養(yǎng)過(guò)程中的形態(tài)，并且提高SMSCs的增殖效率。以上結(jié)果表明與傳統(tǒng)間充質(zhì)干細(xì)胞培養(yǎng)的產(chǎn)量相比，SMSCs的增殖效果更好，并且可以通過(guò)細(xì)胞因子處理使其增殖效率達(dá)到更高的水平。

2.3 SMSCs的多向分化潛能 SMSCs在適宜的誘導(dǎo)條件下可以分化為軟骨細(xì)胞、骨細(xì)胞和脂肪細(xì)胞[1]。此外，Peck等[18]將SMSCs應(yīng)用到骨骼肌修復(fù)的研究中，證實(shí)SMSCs可向肌細(xì)胞分化，而Liu等[19]提取顳下頜關(guān)節(jié)來(lái)源SMSCs，通過(guò)用bFGF誘導(dǎo)使SMSCs向神經(jīng)細(xì)胞分化，在鏡下觀察到SMSCs表現(xiàn)出神經(jīng)細(xì)胞的形態(tài)，并且發(fā)現(xiàn)SMSCs不僅分化為神經(jīng)細(xì)胞，還表達(dá)了特異性標(biāo)記分子。綜上可知，SMSCs具有多向分化潛能。

而另一方面，與其他組織來(lái)源的間充質(zhì)干細(xì)胞相比，SMSCs也表現(xiàn)出了更好的分化潛能。與脂肪來(lái)源間充質(zhì)干細(xì)胞（Adipose-derived stem cells，ADSCs）相比，SMSCs在成骨分化、成軟骨分化兩方面均要優(yōu)于ADSCs，二者在成脂分化能力上相似[20]。與骨髓來(lái)源干細(xì)胞相比，SMSCs在成軟骨分化和成脂分化能力上都要強(qiáng)于BMSCs，而成骨分化能力較BMSCs稍弱[21]。這三種來(lái)源的間充質(zhì)干細(xì)胞在成骨分化能力的比較上存在爭(zhēng)議，因文獻(xiàn)較少尚無(wú)定論，需要進(jìn)一步明確。

3 SMSCs在軟骨分化組織工程中的應(yīng)用

SMSCs作為一種軟骨分化能力極強(qiáng)的間充質(zhì)干細(xì)胞，為組織工程學(xué)提供了新的細(xì)胞來(lái)源[22]。如前文所述，SMSCs與骨髓、脂肪、肌肉等組織來(lái)源的間充質(zhì)干細(xì)胞相比，具有更高水平的軟骨分化能力[23]，在實(shí)驗(yàn)中也表現(xiàn)出了較好的成軟骨效果，修復(fù)了缺損的軟骨模型，因此更適合于軟骨組織工程修復(fù)。

鑒于骨髓間充質(zhì)干細(xì)胞共培養(yǎng)研究取得較好結(jié)果[24]，SMSCs與關(guān)節(jié)軟骨細(xì)胞共培養(yǎng)的策略也被設(shè)計(jì)出來(lái)。Zhang等[25]將軟骨細(xì)胞用腺病毒載體轉(zhuǎn)染TGF-β3基因，和SMSCs共培養(yǎng)，發(fā)現(xiàn)不但能有效的誘導(dǎo)SMSCs向軟骨細(xì)胞分化，而且分化得到的軟骨細(xì)胞表面具有典型的軟骨細(xì)胞表型分子。在修復(fù)軟骨的過(guò)程中通常是采用微球培養(yǎng)方法增強(qiáng)誘導(dǎo)分化的效果[26]。SMSCs向軟骨分化的機(jī)制尚未研究清楚，有研究表明SMSCs在生長(zhǎng)因子誘導(dǎo)下向軟骨分化可能與RhoA/Rho激酶信號(hào)通路的調(diào)節(jié)有關(guān)[27]。

三維支架在干細(xì)胞培養(yǎng)中的應(yīng)用成為近些年的研究熱點(diǎn)[28]。干細(xì)胞的增殖及其分化方向取決于其所處的微環(huán)境。因此，成熟的軟骨細(xì)胞和SMSCs被廣泛的用于三維支架培養(yǎng)，以促進(jìn)關(guān)節(jié)軟骨的再生[29]。研究證明了SMSCs在水凝膠三維支架中，用TGF-β3和BMP-2進(jìn)行誘導(dǎo)，可促進(jìn)SMSCs在三維培養(yǎng)中更好地向軟骨方向分化，來(lái)完成關(guān)節(jié)軟骨損傷的修復(fù)[30]。

此外，研究表明大部分SMSCs在10 min內(nèi)粘附到軟骨缺損表面，促進(jìn)了關(guān)節(jié)軟骨的修復(fù)[31]，而基于這種局部粘附技術(shù)，一種新的移植方法被提出來(lái)，即制作SMSCs的共聚物[32]。這種方法能夠使SMSCs變得肉眼可見(jiàn)，并且重量更大，使操作變得簡(jiǎn)單，而且，與分散的間充質(zhì)干細(xì)胞相比，SMSCs的共聚物在懸液中下沉的速度更快。使用SMSCs的共聚物進(jìn)行研究能減少軟骨缺損表面的干細(xì)胞流失，還能改善SMSCs移植的手術(shù)過(guò)程。Suzuki等[33]對(duì)SMSCs共聚物的性質(zhì)和用途進(jìn)行研究，發(fā)現(xiàn)體外培養(yǎng)過(guò)程中SMSCs共聚物產(chǎn)生的軟骨基質(zhì)比單層細(xì)胞培養(yǎng)多，體內(nèi)培養(yǎng)過(guò)程中SMSCs共聚物通過(guò)表面張力迅速粘附到軟骨缺損表面，幾乎沒(méi)有干細(xì)胞的丟失，從而證明了SMSCs共聚物移植的方法具有操作簡(jiǎn)易、軟骨分化水平更高且粘附效率更高等優(yōu)點(diǎn)，為軟骨修復(fù)提供了一種新的可能性。

4 展望

SMSCs因取材方法簡(jiǎn)單、增殖能力強(qiáng)、粘附效率高、成軟骨分化水平高等特點(diǎn)而被看作是軟骨組織工程中新興的種子細(xì)胞，并且為關(guān)節(jié)軟骨損傷的修復(fù)提供了新的研究方向[34-36]。SMSCs的高成軟骨分化能力被認(rèn)為可能與其局部生理環(huán)境有關(guān)，原滑膜組織分泌的營(yíng)養(yǎng)因子和滑膜液的刺激，使其周圍的生理環(huán)境適合于SMSCs向成軟骨分化[13]，但是具體機(jī)制還未清楚，有待進(jìn)一步研究。

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Biological characteristics and application of synovium derived mesenchymal stem cells in cartilage tissue engineering.

YUAN Yang,WANG Si-han,GUO Xiao-kai,LIU Chang.
Department of Orthodontics,School of Stomatology,Jilin University,Changchun 130021,Jilin,CHINA

Besides the general multipotency in common with other MSCs,synovium-derived mesenchymal stem cells exhibit easy access to cell resources and higher ability of proliferation and superiority in chondrogenesis. Under appropriate culture conditions,SMSCs were induced to differentiate into chondrocyte,osteocyte,and adipocyte lineages.This provides a new MSC-based therapeutic strategy for the repair of cartilage injury.This article expands overview of the tissue origin,biological characteristics of SMSCs and application in cartilage tissue engineering.

Synovium-derived mesenchymal stem cells;Seed cell;Cartilage tissue engineering

R329.2+5

A

1003—6350（2015）02—0214—04

10.3969/j.issn.1003-6350.2015.02.0075

2014-07-23)

袁 洋。E-mail：328354630@qq.com