脂肪干細胞的研究及應用進展

王賢，張培華

(廣東醫學院附屬醫院整形外科研究所，廣東 湛江 524023)

脂肪干細胞的研究及應用進展

王賢，張培華

(廣東醫學院附屬醫院整形外科研究所，廣東 湛江 524023)

干細胞在治療各種疾病方面擁有巨大的潛能。在細胞治療方面，脂肪干細胞是最有前景的種子細胞之一，其他的還包括胚胎干細胞和誘導的多能干細胞。胚胎干細胞和誘導的多能干細胞都具有多向分化潛能，因而起著舉足輕重的作用。但是，胚胎干細胞和誘導的多能干細胞分別受限于倫理學問題及細胞表型的鑒定。脂肪干細胞則不受這些限制，不僅易于獲取，而且方便擴增。在不同的誘導條件下，脂肪干細胞能分化為脂肪細胞、成骨細胞、軟骨細胞、肌肉細胞、內皮細胞和神經細胞，這些分化潛能可應用于再生醫學領域，如：皮膚重建，骨和軟骨修復等。本篇綜述著重討論目前脂肪干細胞的分離、分化和治療應用。

脂肪干細胞；分離；分化；應用

干細胞包括胚胎干細胞、誘導的多能干細胞和成體干細胞。胚胎干細胞能自我更新，并且能分化為人體的任何一種細胞。但由于涉及倫理問題，胚胎干細胞應用于臨床研究存在很大的爭議，而誘導的多能干細胞和成體干細胞則沒有這方面的問題。自從Zuk等[1]發現脂肪組織中含有多分化潛能的干細胞，脂肪干細胞就逐漸成為了成體干細胞中最有前景的種子細胞之一。脂肪組織通過脂肪抽吸術或者外科手術易于獲取，對供體的創傷相對較小，而且不涉及倫理問題。在體外，脂肪干細胞能大量擴增，操作簡便，并能在相應的誘導條件下分化為脂肪細胞、成骨細胞、軟骨細胞、肌肉細胞、內皮細胞、神經細胞等[1-10]。因此，同其他種子細胞相比，脂肪干細胞所受限制小，具有更加廣闊的臨床應用前景。

1 脂肪干細胞的分離和培養

同其他干細胞相比，來源于脂肪組織的干細胞體內含量更大。目前，從脂肪組中分離脂肪干細胞不僅可以人工分離，也可以采用儀器自動離心分離。為了大量獲得脂肪干細胞，目前大多是在脂肪組織離心分離之后用膠原酶消化[3-10]。Zeng等[11]報道了另一種方法，即組織塊貼壁培養法，是一種更加高效而且快速的脂肪干細胞分離培養方法。上述兩種方法所獲得的脂肪干細胞，在倒置相差顯微鏡下觀察，外形都呈成纖維細胞樣，可融合成魚群狀或者旋渦狀。

2 脂肪干細胞的多向分化潛能

脂肪干細胞具有多向分化潛能，在一定的誘導條件下，可以分化為脂肪細胞、成骨細胞、軟骨細胞、肌肉細胞、內皮細胞和神經細胞[1-10]。但目前對于脂肪干細胞分化的信號通路尚不十分清楚，有待進一步研究[12-15]。分化為脂肪細胞[2-3]：需要額外添加地塞米松、胰島素、3-異丁基-1-甲基黃嘌呤(IBMX)、吲哚美辛作為誘導條件，成脂分化可以通過油紅染色驗證。分化為成骨細胞[2-3]：需要額外添加地塞米松、維生素C、β-磷酸甘油作為誘導條件，成骨分化可以通過茜素紅染色驗證。分化為軟骨細胞[2]：需要額外添加胰島素、轉化生長因子β1(TGF β1)和維生素C作為誘導條件，成軟骨分化可以通過阿爾辛蘭染色驗證。分化為肌肉細胞[3-4]：需要額外添加5-氮雜胞苷、馬血清作為誘導條件，成肌肉分化可通過檢測肌蛋白的生成以及體外肌小管形成實驗來驗證。分化為內皮細胞[5-6]：需要在含有多種因子的內皮細胞生長培養基(EGM-2)中添加大劑量的血管內皮生長因子(VEGF)，成內皮分化可以通過檢測內皮細胞的細胞表面標志CD31、CD105、vWF以及體外血管形成實驗來驗證。分化為神經細胞[7-10]需要額外添加β-硫基乙醇、維甲酸、血小板源性生長因子(PDGF)、堿性成纖維細胞生長因子(bFGF)、毛喉素、神經膠質生長因子2(GGF-2β)，成神經分化可以通過檢測神經細胞標志如GFAP、S100等來驗證。

3 脂肪干細胞的臨床應用

脂肪干細胞儲量大、增殖快，而且具有自我更新和多向分化能力，因此臨床應用前景廣闊[16]。脂肪干細胞分化形成的脂肪細胞可用于軟組織缺損、隆乳、脂肪性營養不良以及軟組織填充美容。在軟組織重建方面，自體脂肪移植已廣泛應用，但是其局限性依舊存在，其中最明顯的就是組織吸收，導致移植的脂肪量減少20%～90%[17]。解決這一問題的關鍵，是形成新生血管，從而給移植組織提供營養支持。脂肪干細胞可以分泌多種血管生長因子，包括血管內皮生長因子(VEGF)[18]，從而刺激新血管形成。

脂肪干細胞在體內和體外都能夠分化為成骨細胞[2-3，19]，成骨細胞可做為細胞材料。脂肪干細胞能與多種生物支架結合，促進新骨生成，用于治療骨缺損[20-22]。一定條件下，脂肪干細胞在體內和體外都可以分化為軟骨組織[2，23]。這就給利用細胞治療來修復關節軟骨的缺陷提供了可能，如：修復骨性關節炎。脂肪干細胞能夠增強肌肉細胞的分化，提高肌肉的修復能力[24]。移植的脂肪干細胞，可以在受損的心肌中，促進血管生成、神經出芽、心肌細胞再生，并保護心功能[25-26]。脂肪干細胞在肌肉重建方面已經取得了一些可喜的成績，進一步的研究有待深入[27]。脂肪干細胞能夠分化為內皮細胞，并呈現為小管樣結構[5-6]，脂肪干細胞還可以促進血管的形成[28-29]，這給缺血性疾病的治療提供了新的方向。脂肪干細胞能夠分化為神經細胞和神經膠質細胞[7-10]。脂肪干細胞通過分泌一些神經生長因子，還能促進周圍神經損傷的修復[30-31]。脂肪干細胞還有望應用于神經退行性病變的治療，如：帕金森病、阿爾茨海默病、亨廷頓氏舞蹈癥等[32-35]。

4 總 結

干細胞將成為細胞治療的有力武器，應用于各種疾病，但仍存在許多挑戰，尤其是干細胞臨床應用的安全性和有效性問題，還有同種異體的移植免疫排斥問題等。脂肪干細胞易于大量獲取，損傷相對較小，并且安全有效，其臨床應用正與日俱增。脂肪干細胞的分離培養和分化誘導方法正在不斷改進。脂肪干細胞尚未發現特異性的細胞表面標志物，通過細胞表面標志物純化脂肪干細胞的方法有待改善。

目前關于脂肪干細胞各方面的研究正在不斷深入，各種臨床應用問題也在不斷被克服，脂肪干細胞的臨床應用具有廣闊的前景。

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Research and application progress of adipose-derived stem cells.

WANG Xian,ZHANG Pei-hua.Plastic Surgery Institute,the Affiliated Hospital of Guangdong Medical College,Zhanjiang 524023,Guangdong,CHINA

Stem cells have a great potential to treat various kinds of diseases.For these cell-based therapies,adipose-derived stem cells(ADSCs)are one of the most promising seed cells,with others including embryonic stem cells (ESCs)and induced pluripotent stem cells(iPSCs).ESCs and iPSCs play important roles due to their pluripotency.However,ESCs and iPSCs have limitations in ethical issues and in identification of cell phenotypes,respectively.Unlike ESCs and iPSCs,ADSCs do not have such limitations,which are not only easily obtained but also uniquely expandable. ADSCs can differentiate into adipocytes,osteoblasts,chondrocytes,myocytes,endotheliocytes and neurons under different differentiation conditions,and these kinds of differentiation potential ofADSCs could be applied in regenerative medicine e.g.,skin reconstruction,bone and cartilage repair.In this review,the current status of ADSC isolation,differentiation and their therapeutic applications are discussed.

Adipose-derived stem cells;Isolation;Differentiation;Application

R329.2+1

A

1003—6350（2016）06—0965—03

10.3969/j.issn.1003-6350.2016.06.037

2015-10-13)

廣東省自然科學基金(編號：2014A030313535)

張培華。E-mail：zhangph1128@126.com