白小紅,張德雙 綜述,陳 娟審校
(四川大學(xué)華西第二醫(yī)院新生兒科,四川 成都 610041)
新生兒缺氧缺血性腦病(Hypoxic-Ischemia Encephalopathy,HIE)是新生兒期常見且危害嚴(yán)重的疾病。重度HIE可以導(dǎo)致新生兒死亡及兒童腦癱、智力落后、癲癇等神經(jīng)系統(tǒng)后遺癥[1]。HIE發(fā)病機(jī)制復(fù)雜,其治療及防治遠(yuǎn)期后遺癥已成為臨床一大難題。近年干細(xì)胞治療神經(jīng)系統(tǒng)疾病方面取得的進(jìn)展,使其可以成為HIE的新的治療模式[2]。臍血間充質(zhì)干細(xì)胞(Human umbilical cord blood-mesenchymal stem cells,HUCB-MSCs)在一定條件下可以分化為神經(jīng)元,利用HUCB-MSCs移植對已經(jīng)壞死的神經(jīng)元進(jìn)行替代,最大程度減少重度HIE患兒的遠(yuǎn)期后遺癥。本文對 HUCB-MSCs的基礎(chǔ)研究以及HUCB-MSCs治療HIE方面的進(jìn)展進(jìn)行綜述。
間充質(zhì)干細(xì)胞是一類具有多向分化潛能的組織干細(xì)胞,存在于骨髓、臍血、脂肪等組織。MSCs在體外誘導(dǎo)條件下不僅可以分化為中胚層來源的細(xì)胞如成骨細(xì)胞、脂肪細(xì)胞等,而且可以跨胚層分化為神經(jīng)細(xì)胞[3,4]。Erics等[5]于 2000 年報道,從臍帶血中分離出兩種貼壁細(xì)胞,一種為破骨細(xì)胞樣,另一種為成纖維細(xì)胞樣,后者表達(dá)細(xì)胞表面抗原 SH2、SH3、SH4、ASMA、MAB1470、CD13、CD29 和 CD49e,與骨髓來源的MSCs相同,因此這種細(xì)胞也屬于間充質(zhì)干細(xì)胞。實驗證明,臍血MSCs以及臍帶MSCs都可以分化為骨細(xì)胞、軟骨細(xì)胞、脂肪細(xì)胞、神經(jīng)細(xì)胞、肝細(xì)胞骨骼肌細(xì)胞[6,7]。骨髓和臍血是 MSCs的兩個主要來源組織。臍血又有以下優(yōu)點(diǎn):臍血的采集方便,采集臍帶血對產(chǎn)婦及新生兒不會造成危害;臍血中的干/祖細(xì)胞較成人骨髓中的干/祖細(xì)胞增殖分化能力更強(qiáng)[8];臍血中的淋巴細(xì)胞幼稚,免疫功能不完全,移植物抗宿主病的發(fā)生率低[9];臍血中不會有腫瘤細(xì)胞;臍帶血中潛伏性病毒和病原微生物的感染及傳播概率相對較低[10]。因此,臍血是較骨髓更為理想的MSCs的來源組織。
1.1 HUCB-MSCs的分離 目前,HUCB-MSCs分離常使用的方法有以下幾種:①密度梯度離心法及貼壁細(xì)胞分離法:首先從臍帶血中分理處單個核的細(xì)胞,再根據(jù)其體外培養(yǎng)中貼壁生長的特性,使用貼壁篩選法將其從造血系統(tǒng)中分離出來。此法簡單有效,成本低廉,對細(xì)胞的損傷小,能較好的保持細(xì)胞活性,其缺點(diǎn)則是分離出來的細(xì)胞成分復(fù)雜,純度不高。②免疫磁珠分離法:其原理是利用表面附有特異性抗體的磁珠與間充質(zhì)干細(xì)胞結(jié)合,再用永久磁鐵吸引出間充質(zhì)干細(xì)胞,這樣分離出來的細(xì)胞純度較高,而且分離過程中不會影響細(xì)胞的活性。③流式細(xì)胞儀法:根據(jù)干細(xì)胞體積大小的不同或是細(xì)胞表面的特殊標(biāo)志將臍血中的各種細(xì)胞分離,采用此法分離,可以獲得純度很高的干細(xì)胞,但是對干細(xì)胞活性影響很大,并且實驗條件要求高,所需的標(biāo)本量大這也限制了此種方法的廣泛運(yùn)用于干細(xì)胞的分離,但是其對間充質(zhì)干細(xì)胞的鑒定有很大的作用。國外還有人運(yùn)用單克隆的方法獲得純的MSCs細(xì)胞株,但是該方法不便于大量獲得MSCs[11]。
1.2 HUCB-MSCs的培養(yǎng) HUCB-MSCs的培養(yǎng)具有一定的難度,培養(yǎng)成功率僅在25%左右[12],且在培養(yǎng)方面目前還有很多爭議,尚無統(tǒng)一的方案。影響臍血MSCs培養(yǎng)效率的因素很多,除了臍血中所含MSCs數(shù)量較少的原因外,不同的培養(yǎng)基、pH值、胎物血清濃度和種植濃度都會影響MSCs的生長。Romanov等[13]用含10% 胎牛血清的LG-DMEM 培養(yǎng)基養(yǎng)出優(yōu)質(zhì)生長良好的間充質(zhì)干細(xì)胞。Lee[6]用含20%牛血清、L-谷氨酰胺的IMDM培養(yǎng)基,并加入堿性成纖維生長因子(bFGF),也培養(yǎng)出生長良好的干細(xì)胞。Ramasamy等也發(fā)現(xiàn)在培養(yǎng)基中加入bFGF可以提高干細(xì)胞的增殖,使更多的細(xì)胞停留在S期,并且不會改變臍血MSCs的免疫表型及免疫調(diào)節(jié)蛋白的功能[14]。有研究發(fā)現(xiàn),運(yùn)用人自體血小板裂解物(humanplatelet lysates,HPL)培養(yǎng)的MSCs的免疫表型以及所在生物特性與用10%胎牛血清所培養(yǎng)的一致,7.5%HPL可以促進(jìn) MSCs的增殖[15,16]。Zhu等對比不同培養(yǎng)基對 MSCs增殖分化的影響,發(fā)現(xiàn)低濃度的胎牛血清(2%)加上EGF,血小板衍生生長因子(PDGF)所構(gòu)成的培養(yǎng)基可促使MSCs增殖,而15%的胎牛血清加上β-巰基乙醇則可以增加MSCs向成骨細(xì)胞的分化[17]。
1.3 HUCB-MSCs誘導(dǎo)分化成為神經(jīng)細(xì)胞 大量實驗證明,在不同誘導(dǎo)條件之下,臍血MSCs可以向神經(jīng)細(xì)胞分化。Lim等[18]證實,用腦源性神經(jīng)營養(yǎng)因子(BDNF)可以刺激人臍血MSCs向神經(jīng)元分化,且分化的細(xì)胞依賴MAPK/ERK和P13k/Akt的信號途徑生存。有人發(fā)現(xiàn),激活蛋白激酶(PKA)信號途徑增加環(huán)磷酸腺苷(cAMP)的含量,可以增加臍血MSCs神經(jīng)突樣結(jié)構(gòu)和神經(jīng)標(biāo)志基因的表達(dá)[19]。也有實驗[20]證明,HUCB-MSCs經(jīng)誘導(dǎo)可以在形態(tài)學(xué)、表型及功能上都能分化進(jìn)入到施萬細(xì)胞。侯玲玲等[21]采用抗氧化劑誘導(dǎo)發(fā)現(xiàn),MSCs經(jīng)經(jīng)誘導(dǎo)后可以表達(dá)神經(jīng)元特異性烯醇化酶(NSE)和神經(jīng)絲蛋白(NF)。Kang等[22]發(fā)現(xiàn),雌激素也具有誘導(dǎo)臍血MSCs向神經(jīng)元分化的功能。Ma等發(fā)現(xiàn)運(yùn)用β-巰基乙醇或者丹參都可以體外誘導(dǎo)人臍帶Wharton's Jelly來源的MSCs像神經(jīng)元分化,并且所分化的細(xì)胞表達(dá)β-tubulinⅢ、NF和GFAP等成熟神經(jīng)元標(biāo)志蛋白[23]。雖然臍血MSCs可以向神經(jīng)細(xì)胞分化,但是所分化的神經(jīng)細(xì)胞是否具有正常神經(jīng)元一樣的生理生化功能以及分化成的神經(jīng)細(xì)胞是否能夠長期存活,這些問題仍需要進(jìn)一步研究。
缺氧缺血所致的腦損傷分為兩個階段:原發(fā)性損傷階段(4~6 h)和繼發(fā)性損傷階段(6~72 h)。原發(fā)性損傷階段主要是由于缺氧,細(xì)胞能量代謝失衡,酸中毒,谷氨酸鹽釋放,細(xì)胞內(nèi)Ca積累,脂質(zhì)過氧化作用和NO的神經(jīng)毒性破壞細(xì)胞的平衡,最終導(dǎo)致的細(xì)胞死亡。繼發(fā)性損傷則是由于興奮性中毒、神經(jīng)元的凋亡、以及星形膠質(zhì)細(xì)胞的反應(yīng)性激活所引起。據(jù)其發(fā)病的特點(diǎn),目前主張在“三項支持,三項對癥”的基礎(chǔ)上加亞低溫療法,可以降低大腦和機(jī)體的代謝率,節(jié)省大腦能量消耗,以挽救瀕死神經(jīng)元,減少神經(jīng)元的凋亡。此外,還有運(yùn)用氧自由基清除劑、鈣通道阻滯劑以及重組人促紅細(xì)胞生成素進(jìn)行治療[24~25],但這些治療措施卻對促進(jìn)神經(jīng)元的再生無效,HUCB-MSCs的移植治療則能使所移植的細(xì)胞向腦組織移植并定向誘導(dǎo)分化為神經(jīng)細(xì)胞,抑制或取代壞死和凋亡神經(jīng)細(xì)胞,激發(fā)內(nèi)源性神經(jīng)組織修復(fù)系統(tǒng),實現(xiàn)HIE后腦功能的康復(fù)。
2.1 HUCB-MSCs移植—有效促進(jìn)神經(jīng)元再生大量運(yùn)用臍血移植治療神經(jīng)系統(tǒng)疾病的研究結(jié)果表明其治療效果良好。Chen等[26]從尾靜脈給予卒中模型的大鼠輸入人的臍血,觀察到移植入大鼠體內(nèi)的臍血MSCs可在體內(nèi)存活并遷移到損傷大腦半球,并整合到損傷的腦組織,促進(jìn)損傷腦組織的恢復(fù)減輕大鼠的運(yùn)動功能障礙。Borlongan等[27]在大鼠卒中模型建立后1小時即給大鼠臍血MSCs聯(lián)合甘露醇移植,發(fā)現(xiàn)移植后第3天,在損傷的大腦的區(qū)域沒有查見所移植干細(xì)胞,但移植組動物的腦組織損傷減輕以及神經(jīng)行為功能較對照組有改善。他們推測是移植入體內(nèi)的MSCs所釋放的神經(jīng)營養(yǎng)因子通過血腦屏障到達(dá)損傷腦區(qū)域促進(jìn)了腦功能恢復(fù)。Pedro等[28]在新生大鼠缺氧缺血腦損傷后3小時,通過腹腔注射的方式將臍血MSCs移植入大鼠的體內(nèi),他們發(fā)現(xiàn)接受了干細(xì)胞移植的動物一周后的運(yùn)動感覺反射明顯改善,同時在移植后第3天,移植組的腦紋狀體和皮質(zhì)的壞死及凋亡的神經(jīng)元數(shù)量較對照組明顯減少。Xia等[29]也通過腦室內(nèi)注射的途徑對缺氧缺血性腦損傷(HIBI)新生大鼠進(jìn)行HUBCMSCs的移植,他們發(fā)現(xiàn)移植組的mNSS評分較對照組的明顯增高,而且腦組織的損傷也有所減輕,所移植的MSCs可以分化成為星形膠質(zhì)細(xì)胞。
2.2 HUCB-MSCs移植治療腦損傷的神經(jīng)修復(fù)機(jī)制 目前對于MSCs移植對于腦損傷的神經(jīng)修復(fù)和重塑的機(jī)制仍然不清楚,目前主要是傾向于以下四種機(jī)制:
2.2.1 直接細(xì)胞替代機(jī)制 所移植的干細(xì)胞向受損組織細(xì)胞分化并替代受損細(xì)胞,與周圍細(xì)胞建立聯(lián)系,促進(jìn)神經(jīng)功能恢復(fù)。Lu等[30]將HUCB-MSCs于外傷性腦損傷后24小時經(jīng)鼠尾靜脈注入大鼠體內(nèi),發(fā)現(xiàn)所移植的細(xì)胞可以向腦損傷區(qū)域遷移,并且表達(dá)神經(jīng)元表面標(biāo)志蛋白、微管結(jié)合蛋白-2(microtubule associated protein-2,MAP-2)和膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)。更多的實驗則是證明了只有很小一部分移植入體內(nèi)的干細(xì)胞能向神經(jīng)細(xì)胞分化,根本不足以達(dá)到改善神經(jīng)系統(tǒng)功能的作用[31~33]。因此,推測直接的細(xì)胞替代可能不是主要機(jī)制。
2.2.2 參與血管的形成 有研究[32]運(yùn)用人臍血MSCs移植治療大鼠局灶性腦缺血,結(jié)果發(fā)現(xiàn)腦缺血灶周邊區(qū)域的微血管分布較對照組密集,證明了人臍血MSCs可以促進(jìn)缺血灶邊緣區(qū)的微血管的增生。Chen等[33]用局灶性腦缺血大鼠的缺血腦組織提取液培養(yǎng)骨髓間充質(zhì)干細(xì)胞,發(fā)現(xiàn)其表達(dá)血管內(nèi)皮生長因子(vascular endothelial cell growth factor,VEGF)明顯增加。Hess等[34]還發(fā)現(xiàn) MSCs可以分化為血管內(nèi)皮細(xì)胞,參與腦血管的重建。腦損傷后,在腦損傷區(qū)域的趨化因子SDF-1(stromal cell-derived factor-1)的表達(dá)水平增高,臍血MSCs表面有CXCR4(CXC chemokine receptor-4)的表達(dá),可以使移植的MSCs向受損腦組織定植并且接受臍血MSCs移植后,腦組織的SDF-1的表達(dá)水平較對照組明顯增高[35],這些證據(jù)都提示所移植的MSCs對腦血管再生有重要作用。
2.2.3 提高多種神經(jīng)營養(yǎng)因子的水平 體外實驗和體內(nèi)實驗[36,37]均觀察到MSCs可以增加多種營養(yǎng)因子的產(chǎn)生。Zhang[38]和 Chen 等[39]均發(fā)現(xiàn) MSCs移植治療腦損傷,可以提高神經(jīng)生長因子(nerve growth factor,NGF)和腦源性神經(jīng)營養(yǎng)因(brain-derived neurotrophic factor,BDNF)的水平,這些細(xì)胞因子可以調(diào)節(jié)細(xì)胞的增殖分化,對神經(jīng)細(xì)胞起支持營養(yǎng)作用,減少神經(jīng)損傷,并促進(jìn)自體神經(jīng)干細(xì)胞的分化從而恢復(fù)神經(jīng)功能。
2.2.4 控制炎癥反應(yīng) Vendrame等[40]的研究顯示卒中的大鼠模型接受臍血MSCs移植后,體內(nèi)炎癥反應(yīng)介質(zhì)TNF-α和 IL-1β的水平比對照組低,而具有抗炎作用的IL-10的水平則較對照組高,同時減少了脾釋放炎癥反應(yīng)細(xì)胞(如巨噬細(xì)胞),調(diào)節(jié)腦缺氧缺血損傷之后的瀑布式的炎癥反應(yīng)。
將臍血MSCs移植作為臨床上治療新生兒HIE的常規(guī)手段之一,還有許多問題亟需解決。①臍血MSCs分離的效率仍然很低,且缺乏特異性手段鑒定何種細(xì)胞分化為神經(jīng)元的潛能更大。②移植的時間窗和移植的途徑:關(guān)于移植時間窗目前有兩個觀點(diǎn),有人認(rèn)為在缺氧缺血腦損傷之后的24~72小時是炎癥反應(yīng)的高峰期,腦的微環(huán)境不適于移植的MSCs的存活,應(yīng)該在缺氧缺血損傷后72小時之后進(jìn)行移植;另一觀點(diǎn)則認(rèn)為移植MSCs可以控制損傷腦組織炎癥介質(zhì)釋放,減少腦損傷程度,應(yīng)該盡早進(jìn)行MSCs移植。實驗中運(yùn)用腦室內(nèi)注射、腹膜內(nèi)注射,以及經(jīng)大鼠尾靜脈注射的途徑均有治療效果,但是何種移植途徑治療效果更好目前仍不確定。③促進(jìn)神經(jīng)修復(fù)的機(jī)制:臍血MSCs移植后可以從損傷部位觀察到MSCs分化的神經(jīng)細(xì)胞,且大鼠的癥狀有所緩解,但其作用的具體機(jī)制,以及何種機(jī)制起著主要作用,這都需要進(jìn)一步的研究。
臍血MSCs目前已初步應(yīng)用于臨床。在國內(nèi)外兒科領(lǐng)域,有學(xué)者[41,42]運(yùn)用臍血 MSCs治療腦性癱瘓患兒,結(jié)果顯示臍血MSCs移植能有效的改善腦癱患兒的運(yùn)動功能。有個案報道[43]運(yùn)用臍血MSCs治療1例兒童型脊肌萎縮癥之后,該患兒的肌力提高,神經(jīng)功能改善明顯。隨著研究的進(jìn)一步深入,臍血MSCs移植將有望成為新生兒HIE的常規(guī)治療方法之一,給危重HIE患兒的康復(fù)帶來希望。
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