叢培芳， 柳云恩， 張玉彪， 佟昌慈， 史秀云， 劉 穎， 毛 舜， 佟 周， 施 琳， 金紅旭， 侯明曉

沈陽(yáng)軍區(qū)總醫(yī)院 急診醫(yī)學(xué)部 全軍重癥(戰(zhàn))創(chuàng)傷救治中心實(shí)驗(yàn)室 遼寧省重癥創(chuàng)傷和器官保護(hù)重點(diǎn)實(shí)驗(yàn)室，遼寧 沈陽(yáng) 110016

·干細(xì)胞研究·

脂肪干細(xì)胞移植治療脊髓損傷研究進(jìn)展

叢培芳， 柳云恩， 張玉彪， 佟昌慈， 史秀云， 劉 穎， 毛 舜，
佟 周， 施 琳， 金紅旭， 侯明曉

沈陽(yáng)軍區(qū)總醫(yī)院 急診醫(yī)學(xué)部 全軍重癥(戰(zhàn))創(chuàng)傷救治中心實(shí)驗(yàn)室 遼寧省重癥創(chuàng)傷和器官保護(hù)重點(diǎn)實(shí)驗(yàn)室，遼寧 沈陽(yáng) 110016

脂肪干細(xì)胞； 脊髓損傷； 血脊髓屏障

Adipose-derived stem cells； Spinal cord indury； Blood spinal cord barrier

脊髓損傷(spinal cord injury，SCI)由于其損傷位置的特殊性，常導(dǎo)致?lián)p傷平面以下肢體不可逆的神經(jīng)功能喪失，造成截癱、四肢癱等嚴(yán)重的并發(fā)癥，給工作和生活帶來(lái)毀滅性的打擊，同時(shí)，也為社會(huì)帶來(lái)沉重的經(jīng)濟(jì)負(fù)擔(dān)[1]。目前，我國(guó)經(jīng)濟(jì)的不斷發(fā)展，機(jī)動(dòng)車(chē)事故、跌倒以及暴力行為等造成的SCI患者不斷增加，而治療卻僅限于早期使用大劑量類(lèi)固醇和急性外科干預(yù)，以盡量減少脊髓水腫和隨后的繼發(fā)性或遲發(fā)性損傷[2-4]。因此，尋找治療SCI的有效手段已經(jīng)成為現(xiàn)代醫(yī)學(xué)工作者需要面對(duì)的一大難題。

脂肪組織是一種高度特化的結(jié)締組織，是指成熟的脂肪細(xì)胞在體內(nèi)聚集成團(tuán)，由薄層疏松結(jié)締組織分隔成小葉，不均勻地分布在身體的各個(gè)部位，并有機(jī)械性和審美功能。其在體內(nèi)含量豐富，含有的干祖細(xì)胞能夠分化為成骨細(xì)胞、軟骨細(xì)胞、脂肪細(xì)胞譜系等[5]。近年來(lái)，干細(xì)胞以其向多種組織細(xì)胞分化的能力而成為多種機(jī)體損傷的治療熱點(diǎn)，與骨髓間充質(zhì)干細(xì)胞和臍帶血干細(xì)胞等其他種類(lèi)的干細(xì)胞相比，脂肪干細(xì)胞(adipose-derived stem cells，ADSCs)的獲取方式更為便利，并且不受特定時(shí)間限制，可通過(guò)微創(chuàng)的吸脂術(shù)大量獲取[6]。研究證實(shí)，ADSCs可以通過(guò)分泌各種細(xì)胞因子、調(diào)節(jié)炎癥反應(yīng)、改善缺氧、促進(jìn)血管新生、抑制細(xì)胞凋亡或直接分化替代受損細(xì)胞，協(xié)同治療難愈性創(chuàng)面、放射性膀胱損傷、放射性唾液腺損傷、造血功能障礙以及SCI等[7-12]。本研究就ADSCs治療SCI方面的研究進(jìn)展進(jìn)行簡(jiǎn)單概述。

1 ADSCs的神經(jīng)分化能力

神經(jīng)細(xì)胞由于分化程度高，所以一旦受損，修復(fù)起來(lái)十分緩慢，嚴(yán)重時(shí)還可能造成不可修復(fù)的損傷。藥物對(duì)于修復(fù)神經(jīng)元的效果也不是十分理想，因此干細(xì)胞治療中，其具有的神經(jīng)分化能力對(duì)于SCI的修復(fù)十分重要。ADSCs具有很強(qiáng)的神經(jīng)分化能力，其分化方法方便，分化率高[13-14]，分化后的細(xì)胞可表達(dá)巢蛋白和神經(jīng)元特異性烯醇化酶等神經(jīng)細(xì)胞特異性標(biāo)志物，其形態(tài)也與神經(jīng)細(xì)胞相似。而Ji等[15]的研究更進(jìn)一步表明，在腦源性神經(jīng)營(yíng)養(yǎng)因子(brain-derived neurotrophic factor，BDNF)和神經(jīng)營(yíng)養(yǎng)因子-3(neurotrophin-3，NT-3)的誘導(dǎo)下，ADSCs更容易進(jìn)行神經(jīng)分化。

2 ADSCs與骨髓間質(zhì)干細(xì)胞治療SCI比較

一些研究發(fā)現(xiàn)，神經(jīng)干細(xì)胞的標(biāo)記物在骨髓間質(zhì)干細(xì)胞(bone marrow derived mesenchymal stem cells，BMSCs)與ADSCs中也有存在，表明二者均有神經(jīng)分化的潛能[16-17]。利用化學(xué)藥物，如β-巰基乙醇、二甲基亞砜和叔丁基羥基茴香醚可以使BMSCs分化為神經(jīng)球[18]，但分化中所使用的化學(xué)物質(zhì)的毒性限制了其在臨床試驗(yàn)中的使用[19]。因此，現(xiàn)在更多的使用堿性成纖維細(xì)胞生長(zhǎng)因子和表皮生長(zhǎng)因子進(jìn)行神經(jīng)分化誘導(dǎo)[20]。一直以來(lái)，BMSCs輔助治療SCI的研究更多，在治療中，BMSCs可以釋放神經(jīng)營(yíng)養(yǎng)因子，減少白細(xì)胞介素-1β等炎癥因子的表達(dá)，并減少凋亡反應(yīng)的發(fā)生，行為學(xué)實(shí)驗(yàn)也證明其有助于SCI后功能的恢復(fù)，并且不良反應(yīng)很小[21-25]。

ADSCs進(jìn)行神經(jīng)分化誘導(dǎo)的方法與BMSCs相同，但Chung等[26]研究發(fā)現(xiàn)，ADSCs具有更強(qiáng)的神經(jīng)分化能力，在同等培養(yǎng)條件下，ADSCs可形成約500 μm的神經(jīng)球，而骨髓干細(xì)胞僅能形成不到100 μm直徑的神經(jīng)球，因此，同等量的ADSCs與BMSCs進(jìn)行神經(jīng)誘導(dǎo)分化的效率更高，治療效果也更好。另外，也有研究證實(shí)，相比于BMSCs，ADSCs表達(dá)BDNF、血管內(nèi)皮細(xì)胞生長(zhǎng)因子(vascular endothelial cell growth factor，VEGF)與肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor，HGF)的水平更高，更有增殖速度快、不易老化以及運(yùn)動(dòng)功能恢復(fù)更好等優(yōu)點(diǎn)[27]。

3 ADSCs治療SCI的可能機(jī)制

ADSCs治療SCI的可能機(jī)制中，其對(duì)血脊髓屏障的保護(hù)作用是其中之一。血脊髓屏障(blood spinal cord barrier，BSCB)主要由毛細(xì)血管基底膜、血管內(nèi)皮細(xì)胞、星形膠質(zhì)細(xì)胞等構(gòu)成，對(duì)維持脊髓內(nèi)環(huán)境的穩(wěn)定、維持正常的神經(jīng)功能具有重要作用。其在SCI中的破壞通常被認(rèn)為是脊髓繼發(fā)性損傷加重的重要原因[28- 29]。SCI發(fā)生后，ADSCs移植可能通過(guò)抑制過(guò)氧化物酶的表達(dá)和小膠質(zhì)細(xì)胞的活化，減少繼發(fā)性炎癥反應(yīng)和細(xì)胞凋亡，進(jìn)而減少BSCB的破壞，促進(jìn)大鼠后肢運(yùn)動(dòng)功能的恢復(fù)[30]。

缺氧、炎癥因子、理化損傷和水腫等同樣是造成SCI的原因[31-33]。多個(gè)研究表明，ADSCs可以通過(guò)分泌的前列腺素E2和白細(xì)胞介素-10并且抑制基質(zhì)金屬蛋白酶-9來(lái)進(jìn)行免疫抑制，同時(shí)可以下調(diào)炎癥反應(yīng)，緩解疼痛[34-36]。而由于其神經(jīng)分化的特性，ADSCs還可以分泌BDNF、親膽堿能神經(jīng)元因子(cholinergic neuronotrophic factor，CTNF)等神經(jīng)營(yíng)養(yǎng)因子，進(jìn)一步的修復(fù)神經(jīng)損傷，有助于后續(xù)肢體功能上的恢復(fù)[6,37-39]。

4 經(jīng)神經(jīng)營(yíng)養(yǎng)因子修飾的ADSCs治療脊髓損傷

ADSCs可以分泌神經(jīng)生長(zhǎng)因子幫助損傷修復(fù)，而近年來(lái)，經(jīng)神經(jīng)營(yíng)養(yǎng)因子修飾的ADSCs治療SCI更是研究的熱點(diǎn)[40-42]。多個(gè)研究表明，經(jīng)BDNF、17β-雌二醇和神經(jīng)鈣黏蛋白等修飾的ADSCs能進(jìn)入受損脊髓組織內(nèi)促進(jìn)受損脊髓節(jié)段神經(jīng)元的修復(fù)，促進(jìn)軸突再生，并能有效縮短神經(jīng)元的修復(fù)時(shí)間[43-46]。熊敏等[43]研究發(fā)現(xiàn)，ADSCs對(duì)于SCI大鼠可有顯著的治療效果，而經(jīng)BDNF轉(zhuǎn)染的ADSCs移植于SCI大鼠與單純應(yīng)用ADSCs治療的SCI大鼠相比，其運(yùn)動(dòng)功能評(píng)分(Basso Beattie Bresnahan,BBB)明顯更高，BDNF和神經(jīng)生長(zhǎng)因子的表達(dá)也更多，神經(jīng)損傷標(biāo)志物神經(jīng)膠質(zhì)纖維酸性蛋白的表達(dá)則被抑制，表明經(jīng)BDNF轉(zhuǎn)染的ADSCs治療SCI大鼠，其神經(jīng)結(jié)構(gòu)以及神經(jīng)功能恢復(fù)更好。

綜上所述，ADSCs對(duì)于SCI的恢復(fù)具有積極作用，其易獲得性、神經(jīng)分化率高等特點(diǎn)相比于BMSCs而言，更有利于臨床的應(yīng)用。但其現(xiàn)今研究大都仍停留在實(shí)驗(yàn)室階段，研究結(jié)果缺乏有效的臨床數(shù)據(jù)的支持。有理由相信，隨著研究的不斷深入，ADSCs將在SCI治療中發(fā)揮更大作用，為更多的患者帶來(lái)福音。

[1] Oyinbo CA.Secondary injury mechanisms in traumatic spinal cord injury:a nugget of this multiply cascade[J].Acta Neurobiol Exp(Wars),2011,71(2):281-299.

[2] Bracken MB.Steroids for acute spinal cord injury[J].Cochrane Database Syst Rev,2012,1:CD001046.

[3] Tsao YT,Chen WL,Tsai WC.Steroids for acute spinal cord injury:revealing silent pathology[J].Lancet,2009,374(9688):500.

[4] Vawda R,Fehlings MG.Mesenchymal cells in the treatment of spinal cord injury:current&future perspectives[J].Curr Stem Cell Res Ther,2013,8(1):25-38.

[5] Yamada T,Akamatsu H,Hasegawa S,et al.Age-related changes of p75 neurotrophin receptor-positive adipose-derived stem cells[J].J Dermatol Sci,2010,58(1):36-42.

[6] Arboleda D,Forostyak S,Jendelova P,et al.Transplantation of predifferentiated adipose-derived stromal cells for the treatment of spinal cord injury[J].Cell Mol Neurobiol,2011,31(7):1113-1122.

[7] Qiu X,Zhang S,Zhao X,et al.The therapeutic effect of adipose-derived mesenchymal stem cells for radiation-induced bladder injury[J].Stem Cells Int,2016,2016:3679047.

[8] Krumboeck A,Giovanoli P,Plock JA.Fat grafting and stem cell enhanced fat grafting to the breast under oncological aspects-recommendations for patient selection[J].Breast,2013,22(5):579-584.

[9] Cavarretta IT,Altanerova V,Matuskova M,et al.Adipose tissue-derived mesenchymal stem cells expressing prodrug-converting enzyme inhibit human prostate tumor growth[J].Mol Ther,2010,18(1):223-231.

[10] Ahn JO,Chae JS,Coh YR,et al.Human adipose tissue-derived mesenchymal stem cells inhibit T-cell lymphoma growth in vitro and in vivo[J].Anticancer Res,2014,34(9):4839-4847.

[11] Ahn JO,Coh YR,Lee HW,et al.Human adipose tissue-derived mesenchymal stem cells inhibit melanoma growth in vitro and in vivo[J].Anticancer Res,2015,35(1):159-168.

[12] Haubner F,Muschter D,Pohl F,et al.A co-culture model of fibroblasts and adipose tissue-derived stem cells reveals new insights into impaired wound healing after radiotherapy[J].Int J Mol Sci,2015,16(11):25947-25958.

[13] Huang T,He D,Kleiner G,et al.Neuron-like differentiation of adipose-derived stem cells from infant piglets in vitro[J].J Spinal Cord Med,2007,30(Suppl 1):S35-S40.

[14] Wang Q,Zhou L,Guo Y,et al.Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis[J].Neural Regen Res,2013,8(35):3353-3358.

[15] Ji W,Zhang X,Ji L,et al.Effects of brainderived neurotrophic factor and neurotrophin-3 on the neuronal differentiation of rat adiposederived stem cells[J].Mol Med Rep,2015,12(4):4981-4988.

[16] Buhring HJ,Battula VL,Treml S,et al.Novel markers for the prospective isolation of human MSC[J].Ann N Y Acad Sci,2007,1106:262-271.

[17] Tondreau T,Lagneaux L,Dejeneffe M,et al.Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation[J].Differentiation,2004,72(7):319-326.

[18] Woodbury D,Schwarz EJ,Prockop DJ,et al.Adult rat and human bone marrow stromal cells differentiate into neurons[J].J Neurosci Res,2000,61(4):364-370.

[19] Yang Q,Mu J,Li Q,et al.A simple and efficient method for deriving neurospheres from bone marrow stromal cells[J].Biochem Biophys Res Commun,2008,372(4):520-524.

[20] Kamishina H,Cheeseman JA,Clemmons RM.Nestin-positive spheres derived from canine bone marrow stromal cells generate cells with early neuronal and glial phenotypic characteristics[J].In Vitro Cell Dev Biol Anim,2008,44(5-6):140-144.

[21] Chen YB,Jia QZ,Li DJ,et al.Spinal cord injury in rats treated using bone marrow mesenchymal stem-cell transplantation[J].Int J Clin Exp Med,2015,8(6):9348-9354.

[22] Matyas JJ,Stewart AN,Goldsmith A,et al.Effects of bone-marrow-derived MSC transplantation on functional recovery in a rat model of spinal cord injury:comparisons of transplant locations and cell concentrations[J].Cell Transplant,2017,26(8):1472-1482.

[23] Morita T,Sasaki M,Kataoka-Sasaki Y,et al.Intravenous infusion of mesenchymal stem cells promotes functional recovery in a model of chronic spinal cord injury[J].Neuroscience,2016,335:221-231.

[24] Jiang PC,Xiong WP,Wang G,et al.A clinical trial report of autologous bone marrow-derived mesenchymal stem cell transplantation in patients with spinal cord injury[J].Exp Ther Med,2013,6(1):140-146.

[25] Xiong LL,Liu F,Lu BT,et al.Bone marrow mesenchymal stem-cell transplantation promotes functional improvement associated with CNTF-STAT3 activation after hemi-sectioned spinal cord injury in tree shrews[J].Front Cell Neurosci,2017,11:172.

[26] Chung CS,Fujita N,Kawahara N,et al.A comparison of neurosphere differentiation potential of canine bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells[J].J Vet Med Sci,2013,75(7):879-886.

[27] Zhou Z,Chen Y,Zhang H,et al.Comparison of mesenchymal stromal cells from human bone marrow and adipose tissue for the treatment of spinal cord injury[J].Cytotherapy,2013,15(4):434-448.

[28] Ritz MF,Graumann U,Gutierrez B,et al.Traumatic spinal cord injury alters angiogenic factors and TGF-beta1 that may affect vascular recovery[J].Curr Neurovasc Res,2010,7(4):301-310.

[29] Yang J,Wang G,Gao C,et al.Effects of hyperbaric oxygen on MMP-2 and MMP-9 expressions and spinal cord edema after spinal cord injury[J].Life Sci,2013.[Epub ahead of print]

[30] 李 萌.脂肪間充質(zhì)干細(xì)胞移植對(duì)脊髓損傷大鼠血脊髓屏障的影響及相關(guān)機(jī)制研究[D].廣州:南方醫(yī)科大學(xué),2015.

[31] Jang JW,Lee JK,Kim SH.Activation of matrix metalloproteinases-9 after photothrombotic spinal cord injury model in rats[J].J Korean Neurosurg Soc,2011,50(4):288-292.

[32] Lee JY,Kim HS,Choi HY,et al.Valproic acid attenuates blood-spinal cord barrier disruption by inhibiting matrix metalloprotease-9 activity and improves functional recovery after spinal cord injury[J].J Neurochem,2012,121(5):818-829.

[33] Sanli AM,Serbes G,Sargon MF,et al.Methothrexate attenuates early neutrophil infiltration and the associated lipid peroxidation in the injured spinal cord but does not induce neurotoxicity in the uninjured spinal cord in rats[J].Acta Neurochir(Wien),2012,154(6):1045-1054.

[34] Gonzalez-Rey E,Anderson P,Gonzalez MA,et al.Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis[J].Gut,2009,58(7):929-939.

[35] Chang KA,Lee JH,Suh YH.Therapeutic potential of human adipose-derived stem cells in neurological disorders[J].J Pharmacol Sci,2014,126(4):293-301.

[36] Sacerdote P,Niada S,Franchi S,et al.Systemic administration of human adipose-derived stem cells reverts nociceptive hypersensitivity in an experimental model of neuropathy[J].Stem Cells Dev,2013,22(8):1252-1263.

[37] Aras Y,Sabanci PA,Kabatas S,et al.The effects of adipose tissue-derived mesenchymal stem cell transplantation during the acute and subacute phases following spinal cord injury[J].Turk Neurosurg,2016,26(1):127-139.

[38] Nakajima H,Uchida K,Guerrero AR,et al.Transplantation of mesenchymal stem cells promotes an alternative pathway of macrophage activation and functional recovery after spinal cord injury[J].J Neurotrauma,2012,29(8):1614-1625.

[39] Oh JS,Park IS,Kim KN,et al.Transplantation of an adipose stem cell cluster in a spinal cord injury[J].Neuroreport,2012,23(5):277-282.

[40] Choi EW,Lee JM,Lee HW,et al.Therapeutic effects of CTLA4Ig gene-transduced adipose tissue-derived mesenchymal stem cell transplantation on established autoimmune thyroiditis[J].Cell Transplant,2015,24(11):2221-2236.

[41] Bao XJ,Liu FY,Lu S,et al.Transplantation of Flk-1+ human bone marrow-derived mesenchymal stem cells promotes behavioral recovery and anti-inflammatory and angiogenesis effects in an intracerebral hemorrhage rat model[J].Int J Mol Med,2013,31(5):1087-1096.

[42] Zong X,Wu S,Li F,et al.Transplantation of VEGF-mediated bone marrow mesenchymal stem cells promotes functional improvement in a rat acute cerebral infarction model[J].Brain Res,2017.[Epub ahead of print]

[43] 熊 敏,曾 云,劉志剛,等.經(jīng)腦源性神經(jīng)營(yíng)養(yǎng)因子基因修飾的脂肪間充質(zhì)干細(xì)胞對(duì)大鼠急性脊髓損傷的影響[J].中華實(shí)驗(yàn)外科雜志,2013,30(5):1032-1034.

[44] Zhou J,Lu P,Ren H,et al.17β-estradiol protects human eyelid-derived adipose stem cells against cytotoxicity and increases transplanted cell survival in spinal cord injury[J].J Cell Mol Med,2014,18(2):326-343.

[45] Chen X,Yang Q,Zheng T,et al.Neurotrophic effect of adipose tissue-derived stem cells on erectile function recovery by pigment epithelium-derived factor secretion in a rat model of cavernous nerve injury[J].Stem Cells Int,2016,2016:5161248.

[46] 熊 敏,何 寧,曾 云,等.腦源性神經(jīng)營(yíng)養(yǎng)因子基因修飾脂肪間充質(zhì)干細(xì)胞治療大鼠急性脊髓損傷[J].中華實(shí)驗(yàn)外科雜志,2012,29(11):2271-2273.

全軍十二五面上項(xiàng)目(CSY12J002)；全軍重大新藥創(chuàng)制項(xiàng)目(2013ZX09J13109-02B)；全軍十二五面上項(xiàng)目(CSY13J002)；總后衛(wèi)生部重大新上(ASM14L008)

叢培芳(1987-)，女，遼寧沈陽(yáng)人，藥師，碩士

侯明曉，E-mail：houmingxiao188@163.com

2095-5561(2017)05-0298-04DOI∶10.16048/j.issn.2095-5561.2017.05.10

2017-09-08