去分化脂肪細(xì)胞多向分化潛能與應(yīng)用

張洋，肖麗玲

(暨南大學(xué)附屬第一醫(yī)院整形外科，廣東 廣州 510632)

去分化脂肪細(xì)胞多向分化潛能與應(yīng)用

張洋，肖麗玲

(暨南大學(xué)附屬第一醫(yī)院整形外科，廣東 廣州 510632)

去分化脂肪細(xì)胞(DFAT cells)來源于成熟脂肪細(xì)胞，且具有多向分化潛能，其特征類似于干細(xì)胞的一種細(xì)胞。在體外可通過天花板培養(yǎng)法提取出DFAT細(xì)胞，經(jīng)過誘導(dǎo)后，DFAT細(xì)胞可向脂肪細(xì)胞、骨細(xì)胞、軟骨細(xì)胞、內(nèi)皮細(xì)胞、肌細(xì)胞、神經(jīng)細(xì)胞等分化。因此，DFAT細(xì)胞是一種在組織工程和干細(xì)胞治療中很好的潛在資源，從而廣泛應(yīng)用于多種疾病及組織損傷修復(fù)的研究當(dāng)中。

去分化脂肪細(xì)胞；分化；脂肪干細(xì)胞；臨床應(yīng)用

作為干細(xì)胞移植治療的代表，骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells，BMSCs)與脂肪來源干細(xì)胞(adipose-derived stem cells，ADSCs)的多向分化潛能在轉(zhuǎn)化醫(yī)學(xué)的應(yīng)用中已被大量研究及體內(nèi)外實(shí)驗(yàn)所證明[1-5]。然而，某些客觀因素卻限制了其在臨床中的廣泛使用，比如BMSCs來源的局限性、取材的有創(chuàng)性、對供體年齡的限制、提取細(xì)胞的均一性差，以及傳代后的老化現(xiàn)象；ADSCs雖然來源廣泛，取材創(chuàng)傷性小，但同樣對供體的年齡有所限制，且細(xì)胞的均一性較差。

相較于BMSCs與ADSCs，來源于皮下脂肪組織的去分化脂肪細(xì)胞(dedifferentiated fat cells，DFAT cells)，除了具有類似ADSCs來源廣、取材易、低免疫性等特征之外，還具有對供體年齡限制低、具有較高的細(xì)胞均一性等特點(diǎn)。此外，DFAT細(xì)胞同樣擁有與BMSCs和ADSCs相類似的多向分化潛能[6-7]。因此，DFAT細(xì)胞在組織工程和干細(xì)胞治療中是一個(gè)很好的潛在資源。

1 DFAT細(xì)胞的分離與鑒定

1.1 DFAT細(xì)胞的提取與去分化機(jī)制 成熟脂肪細(xì)胞經(jīng)天花板培養(yǎng)法可自發(fā)去分化為不含脂滴的類成纖維細(xì)胞，即DFAT細(xì)胞。相對于傳統(tǒng)的(培養(yǎng)瓶)天花板培養(yǎng)法，宋子儀等[8]首次采用培養(yǎng)皿加細(xì)胞載玻片組合的培養(yǎng)法，既保證了去分化的效果，又極大地節(jié)省了實(shí)驗(yàn)材料；Wei等[9]也采用了培養(yǎng)皿細(xì)胞培養(yǎng)法，并對提取方法進(jìn)行了改良，使其更有利于脂肪細(xì)胞內(nèi)脂滴的去除。然而，對于成熟脂肪細(xì)胞去分化的機(jī)制，國內(nèi)外學(xué)者雖然對此做了大量研究，但至今尚未定論。宋子儀等[8]發(fā)現(xiàn)在成熟脂肪細(xì)胞去分化過程的中后期，脂肪分解的關(guān)鍵基因激素敏感脂酶(HSL)和脂肪組織甘油三酯脂肪酶(ATGL)的mRNA水平分別上調(diào)了40倍和10倍，而成脂關(guān)鍵基因過氧化物酶體增殖物激活受體γ(PPARγ)、脂肪細(xì)胞型脂肪酸結(jié)合蛋白(aP2)和脂蛋白酯酶(LPL)的mRNA水平分別上調(diào)了8倍、3倍和7.5倍，證明了脂肪去分化是一個(gè)以脂解為主并伴有一定水平成脂的脂代謝過程。Peng等[10]和Lessard等[11]在最近的研究中也指出，在成熟脂肪細(xì)胞去分化的過程中，其脂肪細(xì)胞標(biāo)志物PPARγ、aP2、LPL和脂聯(lián)素均有顯著下降。Lessard等[11]還發(fā)現(xiàn)，在脂肪細(xì)胞去分化的過程中FAP、DPP4、MMP1和TGFβ1均明顯升高，且發(fā)現(xiàn)FAP和DPP4對脂肪組織的重塑和細(xì)胞可塑性有所關(guān)聯(lián)。此外，Ono等[12]則首次對DFAT細(xì)胞進(jìn)行了基因表達(dá)譜的研究，發(fā)現(xiàn)在脂肪細(xì)胞去分化的過程中，其功能表型相關(guān)基因表達(dá)減少，而細(xì)胞增殖、細(xì)胞形態(tài)學(xué)的改變和相關(guān)基因的分化調(diào)控則相對增加。

1.2 DFAT細(xì)胞的鑒定 至今DFAT細(xì)胞的表型和細(xì)胞性質(zhì)仍不清楚，但是近年來對DFAT細(xì)胞表面標(biāo)志的各項(xiàng)研究表明，其CD29、CD44、CD73、CD90、CD105顯示為陽性，CD14、CD34、CD45、CD117、CD133、CD271、CD309、HLA-DR顯示為陰性，與BMSCs、ADSCs的表面標(biāo)志基本相同[13-17]。

此外，Gao等[18]發(fā)現(xiàn)DFAT細(xì)胞表達(dá)若干胚胎干細(xì)胞的表面標(biāo)志，如Oct4、Sox2、c-Myc、Nanog等。在最近的相關(guān)研究中，Song等[19]發(fā)現(xiàn)DFAT細(xì)胞還表達(dá)與血管周圍細(xì)胞高度相關(guān)的標(biāo)志物，如CD140b、NG2和肌間線蛋白等，且顯示了良好的血管網(wǎng)形成能力；但是常見的血管內(nèi)皮細(xì)胞標(biāo)記物，如CD31、CD34和cd309則為陰性。

2 DFAT細(xì)胞多向分化潛能的研究與應(yīng)用

2.1 向脂肪細(xì)胞分化 成熟的脂肪細(xì)胞在去分化的過程后形成DFAT細(xì)胞，而DFAT細(xì)胞經(jīng)體外誘導(dǎo)培養(yǎng)后，鏡下可見顯著性脂滴聚集。Kou等[16]研究發(fā)現(xiàn)DFAT細(xì)胞的成脂能力要優(yōu)于ADSCs。此外，陳曉煒等[20]將DFAT細(xì)胞與纖維蛋白膠混合后注射于裸鼠皮下，并構(gòu)建出了脂肪組織；而Nobusue等[21]則將DFAT細(xì)胞移植到小鼠皮下，14 d后即可發(fā)現(xiàn)有高度血管化的脂肪墊生成。目前的各項(xiàng)體內(nèi)外實(shí)驗(yàn)均證實(shí)了DFAT細(xì)胞具有強(qiáng)大的成脂分化能力。此外，Guo等[22]發(fā)現(xiàn)DFAT細(xì)胞在分化的過程中，受胰島素受體底物1(insulin receptorsubstrate 1，IRS1)介導(dǎo)，上調(diào)了miR-145，使脂肪的生成受到抑制。最近，Hu等[23]發(fā)現(xiàn)在DFAT細(xì)胞成脂過程中視網(wǎng)膜母細(xì)胞瘤-1(Rb1)基因同樣起到了負(fù)調(diào)節(jié)作用。

2.2 向骨細(xì)胞、軟骨細(xì)胞分化 相對于傳統(tǒng)的使用地塞米松成骨誘導(dǎo)方法，Nakamura等[24]發(fā)現(xiàn)將骨形態(tài)發(fā)生蛋白-9(bone morphogenetic protein，BMP-9)與FK506聯(lián)合使用可有效的誘導(dǎo)DFAT細(xì)胞成骨分化。Oki等[25]使用全反式維甲酸即可在體內(nèi)外完成對DFAT細(xì)胞成骨分化的誘導(dǎo)。對于其成骨分化能力研究，Kishimoto等[26]將從頰脂墊中分離出的DFAT細(xì)胞與ADSCs誘導(dǎo)成骨后比較，發(fā)現(xiàn)DFAT細(xì)胞在堿性磷酸酶(BAP)、骨鈣素(OCN)、鈣沉積和茜素紅染法的評估均優(yōu)于ADSCs。相似的，Sakamoto等[27]發(fā)現(xiàn)，DFAT細(xì)胞在成骨誘導(dǎo)后，Runx2基因表達(dá)、堿性磷酸酶(ALP)活性以及骨鈣素(OCN)和鈣含量的測定均優(yōu)于hMSCs。此外，Tansriratanawong等[28]將DFAT細(xì)胞與牙周韌帶干細(xì)胞體外共培養(yǎng)后，增強(qiáng)了RUNX2基因的表達(dá)。在新型生物復(fù)合材料的研究中，Sakamoto等[27]將DFAT細(xì)胞種植到α磷酸三鈣/膠原海綿(α-TCP/CS)上，14 d后電鏡下可見眾多球形細(xì)胞幾乎完全將α-TCP/CS覆蓋，形成培養(yǎng)骨的礦化細(xì)胞外基質(zhì)沉積；Kishimoto等[29]則將DFAT細(xì)胞與剛性支架組成的鈦纖維網(wǎng)(TFM)結(jié)合制成新型生物材料；Shirakata等[30]則將DFAT細(xì)胞與聚乳酸-羥基乙酸/羥基磷灰石(PLGA/HA)復(fù)合材料使用在了大鼠顱骨缺損的模型上，并取得了明顯效果。這些生物復(fù)合材料的誕生對于骨組織工程來說是一種不錯(cuò)的選擇。此外，將DFAT細(xì)胞運(yùn)用到骨疾病的治療中也逐漸被重視起來[31]，Kikuta等[32]在卵巢摘除后誘導(dǎo)的骨質(zhì)疏松模型上，將DFAT細(xì)胞進(jìn)行骨髓內(nèi)注射，可使其骨密度明顯增加。然而在誘導(dǎo)DFAT細(xì)胞向軟骨分化的過程中至今并沒有有效的誘導(dǎo)方法。Okita等[33]發(fā)現(xiàn)，將適量鍶離子(Sr)添加到誘導(dǎo)軟骨生成的培養(yǎng)基中，明顯促進(jìn)DFAT細(xì)胞早期向軟骨細(xì)胞的分化。這對提高DFAT細(xì)胞向軟骨分化及用于軟骨再生治療或許是一種可行的方法。

2.3 向內(nèi)皮細(xì)胞分化 Jumabay等[34]的研究發(fā)現(xiàn)DFAT細(xì)胞可以在體外自發(fā)進(jìn)行內(nèi)皮細(xì)胞分化，且使用BMP4和BMP9可促進(jìn)分化的進(jìn)行。Kou等[16]卻提出在DFAT細(xì)胞經(jīng)過常見的血管生成因子誘導(dǎo)的前后，均無表達(dá)內(nèi)皮細(xì)胞關(guān)鍵標(biāo)志物CD31、CD34、CD309和vWF等。這與Matsumoto等[35]和Poloni等[15]的報(bào)道是一致的。而Shimizu等[36]將DFAT細(xì)胞培養(yǎng)于微血管內(nèi)皮細(xì)胞生長基-2(microvascularendothelial cell growth medium-2，EGM-2MV)即可檢測到DFAT細(xì)胞表達(dá)內(nèi)皮細(xì)胞的表面標(biāo)記；而將DFAT細(xì)胞與人齒齦內(nèi)皮細(xì)胞(human gingival endothelial cells，HGECs)共培養(yǎng)后，則可在12 h內(nèi)形成豐富的毛細(xì)血管樣結(jié)構(gòu)，并可保持管狀結(jié)構(gòu)24 h以上不被分解；另外，共培養(yǎng)的DFAT細(xì)胞明顯增強(qiáng)周細(xì)胞表面標(biāo)記的表達(dá)，促進(jìn)了微血管的成熟與穩(wěn)定。此外，Soejim等[37]和Asami等[38]均通過實(shí)驗(yàn)發(fā)現(xiàn)，將DFAT細(xì)胞與堿性成纖維細(xì)胞生長因子(bFGF)混合使用于人工真皮移植后，明顯縮短皮膚再生和血管形成所需要的時(shí)間，甚至在移植后第2天即可觀察到真皮內(nèi)已有毛細(xì)血管的滲透。Kashimura等[39]也通過實(shí)驗(yàn)證實(shí)局部注射DFAT細(xì)胞后，可顯著增加皮瓣移植區(qū)新生血管的生成，促進(jìn)皮瓣的存活。對于牙周組織的再生，Sugawara等[40]將DFAT細(xì)胞與膠原支架結(jié)合構(gòu)成的生物支架運(yùn)用于牙周組織缺損模型，證明了DFAT細(xì)胞用于細(xì)胞移植治療的可行性。

2.4 向肌肉細(xì)胞分化 在向心肌分化的研究中，楊華等[41]通過使用催產(chǎn)素對DFAT細(xì)胞進(jìn)行誘導(dǎo)分化，3周后DFAT細(xì)胞在基因及蛋白水平上即可檢測到心臟特異性標(biāo)記GATA4、Nkx2.5及cTnT的表達(dá)，但未發(fā)現(xiàn)自主搏動(dòng)現(xiàn)象。而Jumabay等[42]則通過抑制骨形態(tài)發(fā)生蛋白(bone morphogenetic proteins，BMP)和Wnt信號(hào)通路，可增強(qiáng)DFAT細(xì)胞向心肌樣細(xì)胞的分化，且檢測到此細(xì)胞具有自主收縮性。此外，Jumabay等[43]則將DFAT細(xì)胞移植于大鼠急性心肌梗死模型，并檢測到DFAT細(xì)胞可有效的聚集于心肌梗死區(qū)，表達(dá)心臟橫紋肌肌動(dòng)蛋白，且梗死區(qū)的毛細(xì)血管密度也得以顯著增加。近期，李福海等[44]首次證明了維生素C可誘導(dǎo)DFAT細(xì)胞向心肌分化，并且在心臟細(xì)胞裂解液體外模擬心肌微環(huán)境的條件下，可進(jìn)一步提高細(xì)胞的心肌分化效率。在向平滑肌分化的研究中，Sakuma等[45]在小鼠膀胱壁冷凍傷模型中注射DFAT細(xì)胞后，在受傷的膀胱組織中監(jiān)測到平滑肌肌動(dòng)蛋白-α陽性區(qū)顯著大于對照組，證明了DFAT細(xì)胞可有助于膀胱平滑肌組織的再生。Obinata等[46]則將DFAT細(xì)胞注射入小鼠尿道擴(kuò)張模型中，即可在受損平滑肌層觀察到移植的DFAT細(xì)胞，且平滑肌肌動(dòng)蛋白-α呈陽性染色，其肌肉層厚度增加明顯。Hsiao等[47]利用細(xì)胞纖維技術(shù)制作出一種螺旋彈簧狀三維細(xì)胞結(jié)構(gòu)模型，其中含有的DFAT細(xì)胞經(jīng)誘導(dǎo)后生成平滑肌細(xì)胞，這種新型結(jié)構(gòu)可精確控制平滑肌細(xì)胞的排列與方向，可作為組織工程的構(gòu)建模塊，使用于器官或細(xì)胞移植等臨床治療。在向骨骼肌分化的研究中，Kazama等[48]通過肌源性誘導(dǎo)后，可導(dǎo)致DFAT細(xì)胞MyoD和肌細(xì)胞生成素的表達(dá)，這些結(jié)果表明，DFAT細(xì)胞可以在體外培養(yǎng)中誘導(dǎo)分化為骨骼肌細(xì)胞。

2.5 向神經(jīng)細(xì)胞分化 Ohta等[49]發(fā)現(xiàn)，DFAT細(xì)胞可表達(dá)如巢蛋白、β微管蛋白和膠質(zhì)纖維酸性蛋白等神經(jīng)標(biāo)志物，并可明顯改善脊髓損傷大鼠模型的后肢運(yùn)動(dòng)功能。Yamada等[50]也通過相似實(shí)驗(yàn)證實(shí)，并指出DFAT細(xì)胞在改善脊髓損傷后肢體運(yùn)動(dòng)能力的同時(shí)，可促進(jìn)髓鞘再生和減少膠質(zhì)瘢痕生成。此外，Matsumine等[51]將DFAT細(xì)胞移植入大鼠面神經(jīng)缺損模型，證明了DFAT細(xì)胞可以促進(jìn)再生神經(jīng)的成熟。

3 DFAT細(xì)胞的臨床應(yīng)用潛力

相關(guān)實(shí)驗(yàn)證明DFAT細(xì)胞即使體外傳代22代后仍保有增殖和分化的能力[21]，且Poloni等[52]證實(shí)了DFAT細(xì)胞經(jīng)過去分化過程后并無基因?qū)W改變和致瘤傾向。因此DFAT細(xì)胞在臨床應(yīng)用領(lǐng)域?qū)碛袕V闊的前景。上文中已綜合敘述了DFAT細(xì)胞移植療法用于牙周組織再生[28，36，40]、皮膚移植[37-38]、脊髓損傷[49-50]、骨質(zhì)疏松[32]、尿道、膀胱平滑肌損傷[45-46]等方面的治療，并取得了顯著的效果。此外，在慢性腎功能不全、腎小球腎炎的治療中，DFAT細(xì)胞移植療法也取得了不錯(cuò)的效果[53-54]。

DFAT細(xì)胞移植治療在慢性創(chuàng)面修復(fù)中也備受關(guān)注，近些年來諸如糖尿病足、壓瘡、血管源性疾病等多因素、多系統(tǒng)病變所致慢性潰瘍的發(fā)病率逐年升高。而糖尿病已經(jīng)成為第三大威脅人類健康的慢性疾病，糖尿病微循環(huán)改變損傷組織內(nèi)血管重建過程[55]是導(dǎo)致潰瘍難愈的原因之一。BMSCs與ADSCs是干細(xì)胞移植治療的代表，雖然可以從糖尿病患者脂肪中成功分離到ADSCs，且已證明了其多向分化的潛能[56]，但是相關(guān)研究發(fā)現(xiàn)糖尿病可使ADSCs的血管生成能力減弱，從而影響了新生血管形成及傷口愈合[57]，而且對干細(xì)胞的自我修復(fù)能力與自體干細(xì)胞治療的效果也會(huì)產(chǎn)生影響[58]。而Jumabay等[59]實(shí)驗(yàn)發(fā)現(xiàn)，與ADSCs相比，糖尿病可增強(qiáng)DFAT細(xì)胞的增殖能力，且其向脂肪細(xì)胞和內(nèi)皮細(xì)胞分化的能力也相應(yīng)的增強(qiáng)。此外，Watson等[17]通過比較了來源自同一糖尿病患者體內(nèi)脂肪的DFAT細(xì)胞與ADSCs，發(fā)現(xiàn)DFAT細(xì)胞的端粒酶水平是ADSCs的2.5倍，其細(xì)胞衰老過程也因此較之緩慢，而且DFAT細(xì)胞上清液與ADSCs上清液對人皮膚成纖維細(xì)胞(human dermalfibroblasts，HDFs)的遷移具有相似的作用，各種相關(guān)研究都表明DFAT細(xì)胞對于慢性創(chuàng)面治療具有巨大潛力。

4 展 望

DFAT細(xì)胞較之ADSCs，其均一性高、增殖能力強(qiáng)、成脂分化能力強(qiáng)及對供者的年齡要求低，并且具有與ADSCs相似的體內(nèi)及體外成脂、成骨、成軟骨、成肌肉、成神經(jīng)等多向分化潛能，這些特點(diǎn)使DFAT細(xì)胞具有更為廣泛的臨床應(yīng)用價(jià)值。如何建立一種DFAT細(xì)胞高效的提取、擴(kuò)增方法對其在轉(zhuǎn)化醫(yī)學(xué)應(yīng)用方面是很有必要的。對于DFAT細(xì)胞的研究，目前仍未有用于臨床治療的報(bào)道。

隨著社會(huì)老齡化的進(jìn)程，難愈性創(chuàng)面患者數(shù)量的逐漸增多，結(jié)合DFAT細(xì)胞的各種臨床潛在應(yīng)用優(yōu)勢，推測DFAT細(xì)胞在轉(zhuǎn)化醫(yī)學(xué)領(lǐng)域?qū)l(fā)揮其巨大潛力。因此對DFAT細(xì)胞在細(xì)胞治療與醫(yī)學(xué)轉(zhuǎn)化應(yīng)用中應(yīng)予以足夠的重視。相信隨著進(jìn)一步的深入研究，DFAT細(xì)胞的臨床應(yīng)用前景將會(huì)更加廣闊。

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Multilineage differentiation potential and application of dedifferentiated fat cells.

ZHANG Yang,XIAO Li-ling. Departmentof Plastic Surgery,the FirstAffiliated Hospitalof Jinan University,Guangzhou 510632,Guangdong,CHINA

Dedifferentiated fatcells(DFAT cells)derived from matured adipocytes,similar to stem cells,have multilineage differentiation potential.DFAT cells can be collected by ceiling culture in vitro.Under appropriate culture conditions for inducing differentiation,DFAT cells can transdifferentiate into adipocytes,osteoblasts,chondrocytes,endothelial cells,muscle cells,nerve cells and so on.Therefore,DFAT cells are considered as a potential resource for tissue engineering and stem celltherapy,which are widely used in various diseases and tissue damage repair studies.

Dedifferentiated fatcells(DFAT);Differentiation;Adipose-derived stem cells;Clinicalapplication

R329.2+8

A

1003—6350（2017）09—1458—05

10.3969/j.issn.1003-6350.2017.09.029

2016-07-27)

廣東省自然科學(xué)基金（編號(hào)：S2013010015264）

肖麗玲。E-mail：xlilin@live.cn