汪正財(cái) 顧子春 李奕潤(rùn) 李華

基質(zhì)血管片段促進(jìn)脂肪移植后再血管化的機(jī)制研究進(jìn)展

汪正財(cái) 顧子春 李奕潤(rùn) 李華

基質(zhì)血管片段（Stromal vascular fraction，SVF）是移植的脂肪組織中去除成熟脂肪細(xì)胞后所剩余的細(xì)胞成分，除含有一定數(shù)量的脂肪來(lái)源干細(xì)胞（Adipose Derived Stem Cells，ADSC）外，還有許多其他細(xì)胞，均具有促進(jìn)血管生成的作用。本文通過(guò)對(duì)近年來(lái)相關(guān)研究文獻(xiàn)進(jìn)行綜述，以闡明SVF促進(jìn)脂肪移植后再血管化的機(jī)制。綜合文獻(xiàn)結(jié)果，血管的再生與形成受多種因素調(diào)控，SVF細(xì)胞不僅可分泌多種因子，協(xié)同促進(jìn)血管再生，還具有周細(xì)胞形態(tài)可穩(wěn)定內(nèi)皮網(wǎng)絡(luò)。而SVF包含的ADSC具有多向分化潛能，包含的脂肪巨噬細(xì)胞在缺氧環(huán)境下可促進(jìn)局部血管生成，有望為臨床上提高自體脂肪移植的存活率提供方向。

基質(zhì)血管片段 再血管化 脂肪移植 脂肪來(lái)源干細(xì)胞 巨噬細(xì)胞

自體脂肪移植來(lái)源廣泛、取材容易、微創(chuàng)、充填效果好，且不存在免疫排斥反應(yīng)，已廣泛應(yīng)用于臨床。但是，存在術(shù)后移植脂肪存活率不確定，影響了臨床應(yīng)用的效果[1]。

研究表明ADSCs輔助下的自體脂肪移植，能夠增加脂肪存活率，術(shù)后4個(gè)月時(shí)脂肪體積可超過(guò)原始體積的80%[2]，因?yàn)锳DSCs能夠在早期促進(jìn)移植區(qū)的血管化[3-4]。實(shí)驗(yàn)證明，血供良好的供區(qū)脂肪更容易存活[5]。因此，研究脂肪移植后的血運(yùn)重建分子機(jī)制，可為提高自體脂肪移植存活率提供思路。

SVF輔助自體脂肪移植的臨床前期動(dòng)物模型實(shí)驗(yàn)表明，與傳統(tǒng)的脂肪移植相比，SVF輔助移植能明顯增加移植受區(qū)毛細(xì)血管密度、提高移植脂肪的存活率[6]。有研究顯示，脂肪移植術(shù)后，SVF細(xì)胞與脂肪來(lái)源間葉干細(xì)胞相比，其表皮生長(zhǎng)因子（EGF）、趨化因子（SDF-1 或 CXCL12，NAP-2 或 CXCL7）、趨化因子受體（CXCR1，CCR2 和 CCR3）相關(guān)基因表達(dá)均明顯上調(diào)[7]。另外，SVF能通過(guò)下調(diào)炎癥相關(guān)基因IL-6和趨化因子配體2（CXCL2）的表達(dá)，來(lái)調(diào)節(jié)炎癥反應(yīng)，減少中性粒細(xì)胞的浸潤(rùn)，促進(jìn)血管再生[8-9]。上述結(jié)果表明，SVF可能通過(guò)分泌相關(guān)活化因子調(diào)節(jié)血管再生。

但是，SVF通過(guò)何種機(jī)制促進(jìn)再血管化目前仍不明確，我們將對(duì)SVF的組成細(xì)胞成分，及其包含細(xì)胞促進(jìn)再血管化機(jī)制的研究進(jìn)展進(jìn)行綜述。

1 SVF組成

SVF是脂肪組織經(jīng)膠原酶消化后提取的細(xì)胞成分總和，是一組混雜的細(xì)胞群，包含ADSCs、血管內(nèi)皮祖細(xì)胞、單核細(xì)胞、周細(xì)胞、成纖維細(xì)胞、紅細(xì)胞、造血干細(xì)胞、淋巴細(xì)胞、巨噬細(xì)胞，以及細(xì)胞外基質(zhì)等[10-13]。

2 SVF在脂肪移植中促進(jìn)再血管化的機(jī)制

2.1 SVF直接參與血管重建

研究表明，在脂肪移植的早期，SVF中已分解細(xì)胞成分的再循環(huán)利用和血管內(nèi)皮細(xì)胞之間的動(dòng)態(tài)重組，能夠促使SVF快速形成血管網(wǎng)[14]。

2.1.1 ADSC通過(guò)分化直接參與血管再生

ADSC是多能干細(xì)胞，可以直接分化為血管內(nèi)皮細(xì)胞、平滑肌細(xì)胞和周細(xì)胞[15-16]。而內(nèi)皮細(xì)胞和血管壁細(xì)胞（平滑肌細(xì)胞和周細(xì)胞）可通過(guò) TGF-β、血管生成素-2、PDGF-B/PDGFR-β、Notch、S1P/Edg信號(hào)通路的激活來(lái)調(diào)節(jié)血管生長(zhǎng)、穩(wěn)定、成熟[17-18]。同時(shí)，周細(xì)胞還可促使血管內(nèi)皮祖細(xì)胞的出現(xiàn)，維持血管完整性，并協(xié)同形成血管網(wǎng)[19-20]。

2.1.2 基質(zhì)細(xì)胞穩(wěn)定內(nèi)皮網(wǎng)絡(luò)并參與形成血管網(wǎng)絡(luò)系統(tǒng)

Traktuev等[21]的研究表明，脂肪基質(zhì)細(xì)胞可協(xié)同內(nèi)皮細(xì)胞共同參與形成新的微血管，構(gòu)成穩(wěn)定的血管網(wǎng)絡(luò)系統(tǒng)。該研究將含有脂肪基質(zhì)細(xì)胞和內(nèi)皮細(xì)胞混合物的膠原基質(zhì)移植到小鼠皮下，2周后發(fā)現(xiàn)基質(zhì)細(xì)胞和內(nèi)皮細(xì)胞混合移植區(qū)的血管網(wǎng)的密度和成熟程度，要明顯高于單純基質(zhì)細(xì)胞或內(nèi)皮細(xì)胞移植區(qū)。

2.2 SVF通過(guò)旁分泌促進(jìn)顆粒脂肪移植術(shù)后血管再生

在SVF移植組織中，其血管密度、血流量，以及分泌的肝細(xì)胞生長(zhǎng)因子（Hepatocyte growth factor，HGF）、血管內(nèi)皮生長(zhǎng)因子（Vascular endothelial growth factor，VEGF）和堿性成纖維細(xì)胞生長(zhǎng)因子（Basic fibroblast growth factor，bFGF）等，均比對(duì)照組明顯增多[22-23]。若抑制HGF的合成，可觀察到SVF細(xì)胞促缺血組織血管化的能力明顯減弱[24]；且經(jīng)VEGF抗體處理過(guò)的干細(xì)胞在缺血組織中喪失了促血管再生能力[25]。

SVF在脂肪移植后可分泌抗炎癥因子，如IL-6、8、11、17，細(xì)胞趨化因子，單核細(xì)胞趨化蛋白1和2，巨噬細(xì)胞集落刺激因子等；也可分泌免疫調(diào)節(jié)因子如IL-1Ra；還可抑制促炎癥因子干擾素γ和IL-12的分泌[26-27]，促進(jìn)組織炎癥的修復(fù)，對(duì)血管生成起著輔助協(xié)同作用[28]。SVF同時(shí)也可分泌角質(zhì)形成細(xì)胞生長(zhǎng)因子、VEGF、成纖維細(xì)胞生長(zhǎng)因子2和內(nèi)皮細(xì)胞生長(zhǎng)因子，促進(jìn)傷口上皮化；還可分泌血管生成素、瘦素，調(diào)節(jié)血管的形成[29]。

2.2.1 ADSC分泌眾多生物活性因子以促進(jìn)血管生成

Procházka等[30]通過(guò)分離ADSC分泌的因子，制成濃縮治療因子，注射到兔缺血肢體中，發(fā)現(xiàn)實(shí)驗(yàn)組缺血組織血流灌注是對(duì)照組的2倍；免疫組化顯示實(shí)驗(yàn)組毛細(xì)血管密度明顯多于對(duì)照組。這表明ADSC分泌的細(xì)胞因子可促進(jìn)血管再生。

事實(shí)上，ADSC在脂肪移植后，可有效地分泌大量促血管生成因子和抗凋亡因子，如HGF、bFGF、VEGF、PDGF-B和TGF-β等[31-32]。其中，VEGF可活化內(nèi)皮祖細(xì)胞；誘導(dǎo)內(nèi)皮細(xì)胞表達(dá)整合素1、αv、β3、β5及其配體，分泌多種組織蛋白酶，降解細(xì)胞外基質(zhì)；共同促進(jìn)內(nèi)皮細(xì)胞的增殖、遷移和新生血管的融合，抑制內(nèi)皮細(xì)胞凋亡[33-35]。HGF則與其受體結(jié)合后，通過(guò)激活Grb2/Sos-Ras-Raf-MAPK信號(hào)途徑，以促進(jìn)血管內(nèi)皮細(xì)胞的增殖[36-37]。bFGF可通過(guò)FGFR1（成纖維細(xì)胞生長(zhǎng)因子受體1）/c-Src/p38/NF-κB （核因子-κB）信號(hào)途徑，誘導(dǎo)VEGF的表達(dá)[38]；同時(shí)核因子-κB的活化可促進(jìn)內(nèi)皮細(xì)胞DNA合成、細(xì)胞分裂增生[39]，促進(jìn)血管的再生。TGF-β有助于細(xì)胞外基質(zhì)的產(chǎn)生，并能促進(jìn)內(nèi)皮細(xì)胞和壁細(xì)胞之間的相互作用[40]，有利于血管的生成。

血小板源性生長(zhǎng)因子 （Platelet Derived Growth Factor，PDGF）促進(jìn)再血管化的可能機(jī)制包括：①PDGF-C促進(jìn)內(nèi)皮細(xì)胞、周細(xì)胞和平滑肌細(xì)胞的遷移、增殖；②PDGF-C招募成纖維細(xì)胞，促進(jìn)其增殖遷移，這對(duì)于細(xì)胞骨架的形成繼而促進(jìn)新生血管的生成具有重要作用；③PDGF-C通過(guò)調(diào)節(jié)巨噬細(xì)胞的遷移增殖和基因表達(dá)來(lái)促進(jìn)血管化[41]；④PDGF促使ADSC的VEGF基因表達(dá)上調(diào)，分泌VEGF增多，促進(jìn)血管的再生[21]；⑤PDGF刺激 ADSC分泌細(xì)胞外囊泡（EV），EV包含一系列促血管再生因子，如MFG-E8、ANGPTL1、血小板生成素和基質(zhì)金屬蛋白酶（MMP），促進(jìn)內(nèi)皮細(xì)胞遷移并激活血管再生因子和其他信號(hào)分子，從而加快血管的重建[42]。PDGF刺激ADSC產(chǎn)生的EV還合成表達(dá)C-Kit和SCF蛋白；C-Kit是一種酪氨酸激酶受體，在祖細(xì)胞分化為血管內(nèi)皮細(xì)胞時(shí)表達(dá)，是內(nèi)皮祖細(xì)胞增殖、動(dòng)員的關(guān)鍵因素[43]，因而EV在脂肪移植后可聚集更多的內(nèi)皮祖細(xì)胞，有利于血管再生。SCF是C-Kit配體，具有促進(jìn)內(nèi)皮細(xì)胞類血管形成、遷移和存活的作用[44]；若阻斷C-Kit與SCF蛋白可觀察到EV促血管生成效應(yīng)明顯減弱[45]。

由于脂肪移植后組織處于缺氧狀態(tài)，刺激ADSC激活缺氧誘導(dǎo)因子 HIF-1α的表達(dá)，而 HIF-1α可使VEGF、血小板生長(zhǎng)因子、血管生成素、HGF、bFGF等促血管生成因子基因的表達(dá)上調(diào)[46-48]。這些細(xì)胞因子與血管內(nèi)皮細(xì)胞或相應(yīng)細(xì)胞上的受體結(jié)合后，可發(fā)揮促血管生成效應(yīng)[48]。

近期研究發(fā)現(xiàn)，ADSC可通過(guò)分泌微泡促進(jìn)血管再生，其潛在機(jī)制可能是微小RNA-31通過(guò)微泡從ADSC遷移到血管內(nèi)皮細(xì)胞內(nèi)，標(biāo)記并抑制缺氧誘導(dǎo)因子抑制因子（一種抗血管生成基因），從而促進(jìn)新生血管的形成[49]。

2.2.2 脂肪巨噬細(xì)胞的旁分泌作用

Koh等[14]在動(dòng)物實(shí)驗(yàn)中，將去除了巨噬細(xì)胞的SVF移植到去除了巨噬細(xì)胞的小鼠中，發(fā)現(xiàn)其在中央和周圍的移植區(qū)血管數(shù)量比對(duì)照組明顯減少，且周邊形成的血管末端是鈍性而不連續(xù)的。這表明巨噬細(xì)胞對(duì)于移植后血管網(wǎng)的形成具有重要作用；而去巨噬細(xì)胞的SVF移植到正常的小鼠中形成的血管在移植中央?yún)^(qū)域較多，周邊區(qū)域較少，加入VEGF-A后血管網(wǎng)的密度可部分恢復(fù)正常，表明脂肪巨噬細(xì)胞可能通過(guò)分泌VEGF-A和其他血管生成因子促進(jìn)新血管網(wǎng)形成。研究表明，缺氧可誘導(dǎo)巨噬細(xì)胞分泌VEGF、bFGF等血管再生因子，促進(jìn)新生血管的形成[50]。

脂肪巨噬細(xì)胞根據(jù)其活化狀態(tài)不同可分為M1型巨噬細(xì)胞和M2型巨噬細(xì)胞。在SVF中，90%以上的脂肪巨噬細(xì)胞都是M2型[51]。M1型巨噬細(xì)胞是一種促炎癥型巨噬細(xì)胞，可被促炎癥介質(zhì)（如 IFNγ）激活，而大量分泌 TNF-α、IL-6、IL-12等促炎癥因子；M2型巨噬細(xì)胞則是一種抗炎癥型巨噬細(xì)胞，ADSC分泌的PGE2通過(guò)PGE2-EP2/4途徑促進(jìn)M2巨噬細(xì)胞分化，分化成熟的M2巨噬細(xì)胞能夠分泌IL-4、IL-10、TGF-β等抗炎因子，以及bFGF、VEGF等促血管生成因子，抑制炎癥反應(yīng)、促血管網(wǎng)生成[33，52]，增加SVF輔助自體脂肪移植術(shù)后的脂肪細(xì)胞長(zhǎng)期生存率[53]。而IL-10不僅能夠在缺氧條件下促進(jìn)M2巨噬細(xì)胞分泌VEGF，抑制M1巨噬細(xì)胞增殖[54]，還可修復(fù)內(nèi)皮細(xì)胞衰老性功能損傷，維持動(dòng)脈正常結(jié)構(gòu)[55-56]，改善移植區(qū)域缺血狀態(tài)。

巨噬細(xì)胞可分泌基質(zhì)金屬蛋白酶1（MMP-1），降解血管基底膜及其周圍的細(xì)胞外基質(zhì)[57]，還可分泌MMP-9、MMP-12、MMP-7，促進(jìn)相鄰血管內(nèi)皮頂端細(xì)胞之間的融合，遷移延伸而形成新生管腔，生成血管[58-59]。有實(shí)驗(yàn)表明，巨噬細(xì)胞通過(guò)調(diào)節(jié)TIE-2的表達(dá)，來(lái)參與缺血組織的血管形成[60]。

2.2.3 內(nèi)皮細(xì)胞的分泌作用

內(nèi)皮細(xì)胞可分泌外泌體，相鄰的內(nèi)皮細(xì)胞可作為靶細(xì)胞與外泌體結(jié)合，其中含有的mi-RNA（miR-214）可抑制相鄰內(nèi)皮細(xì)胞的毛細(xì)血管共濟(jì)失調(diào)突變基因的表達(dá)和凋亡，促進(jìn)內(nèi)皮生長(zhǎng)、遷移以及新生血管的形成[61-62]。

內(nèi)皮細(xì)胞也可通過(guò)表達(dá)CXCL-1激活ERK1/2信號(hào)通路，誘導(dǎo)表皮細(xì)胞生長(zhǎng)因子（Epidermal Growth Factor，EGF）的分泌，促進(jìn)血管生成[63]。

脂肪移植后的組織損傷，可誘導(dǎo)受損的內(nèi)皮細(xì)胞、細(xì)胞外基質(zhì)、血小板等滲出液的其他成分，共同釋放大量的促血管化因子，如 bFGF、PDGF、TGF-β 和 EGF，以促進(jìn)移植區(qū)域的血管再生，改善缺血、缺氧情況[64]。

2.2.4 其他細(xì)胞成分的促血管作用

SVF含有的細(xì)胞外基質(zhì)能通過(guò)促進(jìn)血管的出芽延伸、內(nèi)腔的形成和形態(tài)的成熟，從而促進(jìn)新生血管網(wǎng)的生長(zhǎng)[37，65]。基質(zhì)細(xì)胞、成纖維細(xì)胞和平滑肌細(xì)胞均能分泌HGF，調(diào)節(jié)血管再生[37]。

3 結(jié)論與展望

血管生成的基本過(guò)程包括：血管基底膜的降解；內(nèi)皮細(xì)胞的增殖、遷移；血管的融合、重塑；周細(xì)胞的穩(wěn)定。而SVF是一組混雜的細(xì)胞群，包含有各類細(xì)胞成分，可直接參與或間接分泌生物活性因子誘導(dǎo)基底膜的降解，影響內(nèi)皮細(xì)胞的增殖、遷移，促進(jìn)新生血管的融合與重塑，增加周細(xì)胞對(duì)血管網(wǎng)的穩(wěn)定，從而調(diào)節(jié)脂肪移植術(shù)后的血管再生。因此，SVF是一個(gè)整體，各成分之間相互影響，協(xié)同作用于血管生成的整個(gè)過(guò)程，但是各成分之間協(xié)同促血管再生和各細(xì)胞促再血管化的具體機(jī)制仍需要進(jìn)一步探索。

SVF促脂肪移植術(shù)后再血管化，在臨床有著廣闊的應(yīng)用前景，不僅可提高顆粒脂肪移植術(shù)后脂肪細(xì)胞的存活率、促進(jìn)燒傷創(chuàng)面愈合、改善糖尿病足潰瘍與糖尿病視網(wǎng)膜病變的血運(yùn)，還可改善心肌缺血、提高心功能、促放射性潰瘍創(chuàng)面愈合[12]。但有關(guān)SVF的細(xì)胞輔助治療尚處于臨床前期研究或人體實(shí)驗(yàn)研究階段，其有效性和安全性仍需進(jìn)一步探討。

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Mechanism of Promoted Neovascularization by SVF after Fat Grafting

WANG Zhengcai,GU Zichun,LI Yirun,LI Hua．Department of Plastic and Reconstructive Surgery,Sir Run Run Shaw Hospital,Medical College,Zhejiang University,Hangzhou 310016,China.Corresponding author:LI Hua(E-mail:hualihz@sina.com).

【Summary】Stromal vascular fraction(SVF)are the remaining cells of the ingredients after removing mature fat cells in the adipose tissue of transplantation.In addition to containing a certain amount of adipose derived stem cells(ADSC),SVF also includes many other cells,which may all have the potential of promoting angiogenesis.In this paper,the role of SVF in angiogenesis after fat transplantation was summarized by reviewing relative literature in recent years.According to the literature,angiogenesis and fat graft revascularization are regulated by various factors:SVF promotes secretion of a diverse array of cytokines and growth;SVF differentiated to pericytes has the function of stabilizing endothelium vascular network;As components of SVF,adipose derived stem cells (ADSC)have the potential of multi-directional differentiation,and macrophages can promote local angiogenesis in hypoxia environment.These results may provide directions of improving the survival rate of fat cells in clinical autologous fat transplantation.

Stromal vascular fraction;Revascularization;Fat graft;Adipose-derived stem cells;Adipose macrophages

R622+.9

B

1673－0364（2017）06－0349－05

10.3969/j.issn.1673-0364.2017.06.014

浙江省自然科學(xué)基金項(xiàng)目（LY14H150001）。

310003 浙江省杭州市 浙江大學(xué)醫(yī)學(xué)院附屬邵逸夫醫(yī)院整形外科。

李華（E-mail：hualihz@sina.com）。

2017年9月29日；

2017年10月23日）