成髓鞘細(xì)胞研究和應(yīng)用進(jìn)展

1、成髓鞘細(xì)胞研究和應(yīng)用進(jìn)展,陳 琳 清華大學(xué)第二附屬醫(yī)院神經(jīng)外科中心chenlin_china@163.com,第一屆全國(guó)兒童神經(jīng)修復(fù)學(xué)術(shù)會(huì)議暨第四屆全國(guó)兒童神經(jīng)康復(fù)論壇. 深圳. 2016.11. 25-27.,,安逸生活 pk 艱苦卓絕創(chuàng)業(yè)高原如此艱苦的環(huán)境：勇于擔(dān)當(dāng)，發(fā)揮引領(lǐng)作用以苦為樂(lè)，人生需要有一股精神,,特朗普：我拒絕政治正確， 我只做正確的事情精英政治,,2016.10.11. 中英脫髓鞘倫敦會(huì)議,要點(diǎn),一些基

2、本概念與影像學(xué)檢查兒童脫髓鞘疾病臨床分類成髓鞘細(xì)胞分類神經(jīng)修復(fù)的路徑各種細(xì)胞近期研究一覽,Year 1977~ 2016 40年 demyelination remyelination Pubmed文獻(xiàn)量趨勢(shì)圖,髓鞘構(gòu)成成分,CNS 髓鞘中60 ~ 70% 為水分固體成分中脂類占70%, 蛋白質(zhì)占30%構(gòu)成髓鞘蛋白質(zhì)主要兩種成分：蛋白脂蛋白( PLP) 和髓鞘堿性蛋白(MBP)髓鞘相關(guān)糖蛋白(MAG) 髓鞘少突膠質(zhì)

3、細(xì)胞糖蛋白(MOG)Wolfram 蛋白脂DM 準(zhǔn)大小蛋白其他微量蛋白,髓鞘功能,提供軸突與周?chē)M織電絕緣，避免干擾相鄰軸突之間軸突與其他結(jié)構(gòu)之間加快動(dòng)作電位傳遞：“跳躍式傳導(dǎo)”機(jī)制引導(dǎo)受損軸突：再生,,Myelin-related cells (Schwann cells and oligodendrocytes) co-operate with the axon in the formation and mainten

4、ance of myelin sheaths.,中樞神經(jīng)系統(tǒng)(CNS)有髓神經(jīng)纖維與周?chē)?PNS)相似結(jié)構(gòu)有髓鞘和郎飛結(jié)髓鞘外無(wú)基膜（神經(jīng)膜）相鄰神經(jīng)纖維有時(shí)融合（箭頭）,兒童發(fā)育,髓鞘化過(guò)程主要集中在：出生后18個(gè)月內(nèi)（1.5年）1歲半接近成人oligodendrocyte少突膠質(zhì)細(xì)胞：大多數(shù)細(xì)胞5-10年成熟，存活>50年。每年減少1-300 OLS,The final stage of oligodendrocy

5、te development is myelination. Unlike the peripheral nervous system where there is a strict size-dependent bias for myelination (only axons >1 um diameter enjoy the privilege), in the CNS axons as small as 300 nm are

6、 sometimes myelinated. Nevertheless, CNS myelination is also influenced by axonal size. Surprisingly, cultured oligodendrocytes will begin myelinating even synthetic axon-like tubes lacking the usual neuron-glia signali

7、ng cues. In this type of ‘blind’ myelination, only tubes with a diameter of ≥ 400 nm were myelinated, suggesting this mode of myelination may be particularly important for larger axons.Moreover, once differentiating, i

8、t appears that oligodendrocytes only have a very narrow window of opportunity to select which adjacent axons to myelinate (~5 h in the developing zebrafish and ~12 h in myelinating cultures of rodent derived cells), irre

9、spective of the total number of sheaths being made. This implies that the signaling pathways mediating this process are likely to be relatively robust. Although in vitro studies have implicated several key pathways, to

10、date no one single molecule has been shown to be indispensable for myelination of CNS axons in vivo, highlighting the great degree of redundancy in the control of this vital process.Despite this, recent in vivo studies

11、have revealed a great deal about how these various signaling pathways converge to control the extent or timing of myelination, if not overall myelination per se.,髓鞘脫失（脫髓鞘）一種very常見(jiàn)的臨床神經(jīng)病理變化,,多指數(shù)T2/磁化轉(zhuǎn)移MRI,provides an imp

12、ortant step for understanding ‘typical’ myelin development as well as providing the ability to identify when and where white matter abnormalities occur in neurodevelopmental disorders.myelin water volume fraction (MVF

13、)髓磷脂體積分?jǐn)?shù),myelin water fraction (MWF)髓磷脂水分,脫髓鞘疾病分類（一）,兒童脫髓鞘?。褐?chē)?和 中樞性周?chē)悦撍枨什。憾嘁噪p下肢或四肢癱瘓為首發(fā)中樞性脫髓鞘?。阂砸暳φ系K、肢體無(wú)力、發(fā)熱、抽搐、頭痛等為首發(fā),脫髓鞘疾病分類（二）,經(jīng)典病種：多發(fā)性硬化、急性播散性腦脊髓炎、慢性炎性脫髓鞘性多神經(jīng)根神經(jīng)病其他病種：缺血缺氧性腦病、腦性癱瘓、脊髓損傷、腦外傷、腦白質(zhì)營(yíng)養(yǎng)不良等神經(jīng)創(chuàng)傷、變性、遺傳和血管

14、性疾病,Remyelination can occur in the damaged central nervous system (CNS),Since the discovery in the 1960s that remyelination can occur in the damaged CNS (Bunge et al. 1961) there has been much progress in understanding

15、the cellular and molecular biology of oligodendroglia and the factors that regulate their propagation, migration, differentiation, maturation, and ability to myelinate nerve axons,脊髓損傷,脊髓挫傷1個(gè)月后，64％的存活纖維發(fā)生脫髓鞘盡管有些軸突保持著解剖上

16、的完整，但已無(wú)生理功能損傷部位遠(yuǎn)端的軸突出現(xiàn)華勒變性Franklin RJ, et al.1997,Spinal cord injury is accompanied by chronic progressive demyelination,Totoiu MO, Keirstead HS. 2005. Extent of demyelination and remyelination up to 450 days followin

17、g contusive spinal cord injury in adult rats1 day post injury: the overall number of demyelinated axons peaked7-14 days post injury: declinedthen progressively increased up to 450 days post injuryOligodendrocyte and

18、Schwann cell remyelinated axons appeared by 14 days post injuryremyelinated axons were present from 14 to 450 days post injury, remyelination was incomplete spinal cord injury is accompanied by chronic progressive demy

19、elination,,oligodendrocyte progenitor cell proliferationgeneration of new oligodendrocytesformation of thinner myelin,Domingues et al.2016.,Domingues et al.2016.,First research on transplantation of myelin-forming cell

20、s into the demyelinated spinal cord The cells survive, migrate, and find axons that need myelin insulation, and remyelinate them thereby restoring ability to conduct impulsesStephen Waxman and Jeffery Kocsis,細(xì)胞移植髓鞘

21、化修復(fù)再生治療脫髓鞘病理的神經(jīng)修復(fù)重要策略和研究方向,嗅鞘細(xì)胞少突膠質(zhì)細(xì)胞雪旺細(xì)胞多能誘導(dǎo)干（iPS）細(xì)胞神經(jīng)干細(xì)胞胚胎干細(xì)胞臍帶血/臍帶間充質(zhì)細(xì)胞骨髓間充質(zhì)細(xì)胞脂肪干細(xì)胞皮膚干細(xì)胞,Olfactory ensheathing cell (OEC) 嗅鞘細(xì)胞,,Remyelination,（1）嗅鞘細(xì)胞髓鞘化修復(fù)類于雪旺細(xì)胞,Pellitteri R, et al. (2010) OECs 能產(chǎn)生多種神經(jīng)營(yíng)養(yǎng)生

22、長(zhǎng)因子體外：能促進(jìn)軸突生長(zhǎng)體內(nèi)：可形成髓鞘，促進(jìn)髓鞘再生刺激軸突再生發(fā)芽?jī)?yōu)于雪旺細(xì)胞：與星形膠質(zhì)細(xì)胞接觸,（1）嗅鞘細(xì)胞髓鞘化修復(fù)類于雪旺細(xì)胞,Babiarz J, et al. (2011)分離幼年和成年大鼠的嗅球，分析表達(dá)GFP的OEC的髓鞘化軸突的能力。OEC：幼鼠OECs能髓鞘化背根神經(jīng)節(jié)（DRG）軸突。嗅鞘細(xì)胞與軸突組裝成束需要1周，如果形成可以檢測(cè)到的軸突髓鞘，需要1周以上SC：大鼠雪旺細(xì)胞不捆束軸突，在

23、1周內(nèi)能形成P0 +和MBP +髓鞘節(jié)段大多數(shù)培養(yǎng)的OEC調(diào)寧蛋白（calponin）染色陽(yáng)性，雪旺細(xì)胞為陰性幾乎所有的OEC和雪旺細(xì)胞P75NTR和GFAP陽(yáng)性兩種細(xì)胞之間只有細(xì)微的免疫標(biāo)記差異,（1）嗅鞘細(xì)胞髓鞘化修復(fù)類于雪旺細(xì)胞,Babiarz J, et al. (2011)The diameter of OEC generated myelin was greater than for Schwann cell mye

24、lin on DRG axons OEC but not Schwann cells myelinated DRG axons in the absence of vitamin C,（2）嗅鞘細(xì)胞修復(fù)腦缺血卒中,Shi X, et al. (2010)修復(fù)大腦中動(dòng)脈閉塞（MCAO）大鼠腦白質(zhì) MCAO后56天結(jié)果：減少梗死體積，降低死亡率，改善神經(jīng)功能缺損LFB髓鞘染色，NF免疫組織化學(xué)，Western blot：OEC移植大

25、鼠髓鞘和軸突再生,（3）嗅鞘細(xì)胞修復(fù)周?chē)窠?jīng)損傷,Radtke C, et al. (2010) 嗅鞘細(xì)胞移植作為輔助治療周?chē)窠?jīng)損傷嗅鞘細(xì)胞移植促進(jìn)再生過(guò)程：介導(dǎo)趨化因子，神經(jīng)營(yíng)養(yǎng)和神經(jīng)保護(hù)作用髓鞘形成：橋接，建立一個(gè)允許軸突再生的環(huán)境,（4）嗅鞘細(xì)胞在體內(nèi)、體外與其他細(xì)胞的相互作用,Chuah MI, et al. (2010) 與其他類型的細(xì)胞：在體外和嗅鞘細(xì)胞移植后的膠質(zhì)疤痕和炎癥環(huán)境下的相互作用嗅鞘細(xì)胞和星形膠質(zhì)細(xì)胞

26、 克服膠質(zhì)瘢痕的有害影響不同脊髓損傷的實(shí)驗(yàn)?zāi)Ｐ停篛EC移植相關(guān)的膠質(zhì)瘢痕的形態(tài)學(xué)改變?cè)隗w外：嗅鞘細(xì)胞和膠質(zhì)瘢痕的細(xì)胞類型組成之間的相互作用嗅鞘細(xì)胞：免疫細(xì)胞特性，移植到中樞神經(jīng)系統(tǒng)損傷部位時(shí)，調(diào)制神經(jīng)炎癥,（5）嗅鞘細(xì)胞移植聯(lián)合瘢痕切除修復(fù)脊髓挫傷,Zhang SX, et al. (2011)疤痕消融+ LP/ OEC移植促進(jìn)大鼠脊髓慢性挫傷解剖恢復(fù)和P0（髓磷脂糖蛋白,P-zero）陽(yáng)性髓鞘孟加拉玫瑰紅光毒性方法 單獨(dú)

27、移植病灶腔：嗅黏膜固有層（LP）或聯(lián)合體外培養(yǎng)的OECs,（6）嗅鞘細(xì)胞移植修復(fù)肌萎縮側(cè)索硬化,Li Y, et al. (2011) OEC移植到脊髓：延長(zhǎng)SOD1（G93A）ALS大鼠生存期神經(jīng)保護(hù)作用和髓鞘化移植的嗅鞘細(xì)胞存活超過(guò)4周，在脊髓內(nèi)遷移4.2毫米,（7）嗅鞘細(xì)胞移植修復(fù)各種脫髓鞘模型,Sasaki M, et al. (2011)不同的脫髓鞘環(huán)境對(duì)OEC髓鞘化修復(fù)的影響OECs的遷移和髓鞘形成：炎癥處于活

28、動(dòng)狀態(tài)炎癥基本平息狀態(tài),（7）嗅鞘細(xì)胞移植修復(fù)各種脫髓鞘模型(2),Azimi Alamouti M, et al. Remyelination of the corpus callosum by olfactory ensheathing cell in an experimental model of multiple sclerosis. (2015),,（8）嗅鞘細(xì)胞移植修復(fù)效果的動(dòng)物種

29、屬特異性,Wewetzer K, et al. (2011) 細(xì)胞增殖控制：種間差異鼠、狗、豬、猴、人Rodent： require mitogens for in vitro expansion a complex response to elevated intracellular cAMP, and undergo spontaneous immortalization upon prolonged mitogen st

30、imulation,（9）胚胎/新生/成年嗅鞘細(xì)胞移植修復(fù)效果存在差異,Coutts DJ. Embryonic-derived olfactory ensheathing cells remyelinate focal areas of spinal cord demyelination more efficiently than neonatal or adult-derived cells. Cell Transplant. 20

31、13 Form myelin sheathsOptimal donor age for OEC associated remyelination p75 purified OEC transplants from three donor agesolfactory bulbs of embryonic, neonatal, and adult rats and purified by immunopanningremyelina

32、ting potential was directly compared by transplantation into the same adult rat toxin-induced model of spinal cord demyelinationRemyelination efficiency 3 weeks after transplantation was assessed morphologically and by

33、immunostainingall donor ages remyelinatethis process is most efficiently achieved by embryonic-derived OECs.,Oligodendrocyte少突膠質(zhì)細(xì)胞,Oligodendrocyte precursor cells (OPCs), a subpopulation that accounts for 5 to 8% of c

34、ells within the central nervous system, are potential sources of oligodendrocyte replacement after SCI. OPCs react rapidly to injuries, proliferate at a high rate, and can differentiate into myelinating oligodendrocytes.

35、 However, posttraumatic endogenous remyelination is rarely complete.,Wang Y, et al. (2011),少突膠質(zhì)祖細(xì)胞移植：在成年大鼠脊髓，趨向炎癥區(qū)域存活分化成：可形成髓鞘的少突膠質(zhì)細(xì)胞,Li H, et al. (2009),兩層少突膠質(zhì)細(xì)胞分化的轉(zhuǎn)錄調(diào)控 “雙管齊下”的方式：創(chuàng)建一個(gè)基因控制的故障安全系統(tǒng)確保在發(fā)育過(guò)程、脫髓鞘病變的修復(fù)過(guò)程中：髓

36、鞘化修復(fù)有序進(jìn)行和有效明確地表達(dá),Ishii A, et al. (2009),人類髓鞘蛋白質(zhì)組學(xué)111 種已確認(rèn)的蛋白質(zhì)/轉(zhuǎn)錄物：在少突膠質(zhì)細(xì)胞表達(dá)，在星形膠質(zhì)細(xì)胞和神經(jīng)元不表達(dá)163 additional proteinscomplexity of this metabolically active membrane,Sun F, et al. (2010),軸突變性對(duì)少突膠質(zhì)細(xì)胞譜系細(xì)胞的影響： 背根切斷術(shù)喚起：修復(fù)反應(yīng)

37、脊髓挫傷后嘴側(cè)軸突變性誘導(dǎo)：修復(fù)+細(xì)胞凋亡,Piaton G, et al. (2010),在發(fā)育髓鞘、脫髓鞘、修復(fù)過(guò)程中，軸突與少突膠質(zhì)細(xì)胞相互作用髓磷脂的生物合成和髓鞘修復(fù)：神經(jīng)元和少突膠質(zhì)細(xì)胞之間的相互溝通必不可少I(mǎi)n MS, CNS demyelination is often followed by spontaneous repair, mostly achieved by adult oligodendrocyte

38、precursor cells. Extent of this myelin repair differs, ranging from very low, limited to the plaque border, to extensive, with remyelination throughout the 'shadow plaques.' In addition to restoring neuronal co

39、nnectivity, new myelin is neuroprotective. It reduces axonal loss and thus disability progression.,Neural stem cell (NSC) 神經(jīng)干細(xì)胞,Hwang DH, et al. (2009),轉(zhuǎn)導(dǎo)OLIG2轉(zhuǎn)錄因子的人類神經(jīng)干細(xì)胞（NSCs）脊髓挫傷損傷后大鼠：提高運(yùn)動(dòng)功能的恢復(fù)增強(qiáng)脊髓白質(zhì)髓鞘修復(fù)再生,Sher F

40、, et al. (2009),生物發(fā)光成像（bioluminescence imaging）Olig2-NSCs增加在脫髓鞘小鼠模型的植入效果,Yang J, et al. (2010),比較骨髓和腦源性神經(jīng)干細(xì)胞在中樞神經(jīng)系統(tǒng)自身免疫性疾病的治療效果類似a similar ability to differentiate into neurons, astrocytes, and oligodendrocytes both

41、in vitro and in vivo both types of NSCs suppressed chronic experimental autoimmune encephalomyelitis therapeutic effects of NSCs include immunomodulation in the PNS and the CNS, neuron/oligodendrocyte repopulation by t

42、ransplanted cells, and enhanced endogenous remyelination and axonal recovery,Carbajal KS, et al. (2010),多發(fā)性硬化病毒模型神經(jīng)干細(xì)胞遷移通過(guò)CXCR4介導(dǎo)CXCL12的信號(hào),Adipose mesenchymal stem cell脂肪來(lái)源間充質(zhì)干細(xì)胞,Radtke C, et al. (2009),脂肪間充質(zhì)干細(xì)胞衍生的神經(jīng)球

43、可分化成周?chē)窠?jīng)膠質(zhì)樣細(xì)胞,Constantin G, et al. (2009),脂肪來(lái)源的間質(zhì)干細(xì)胞改善慢性實(shí)驗(yàn)性自身免疫性腦脊髓炎 (EAE)靜脈給藥：顯著降低免疫反應(yīng)所致EAE的嚴(yán)重性、減少脊髓炎和脫髓鞘、軸突損失ASC優(yōu)先歸巢到淋巴器官，遷移中樞神經(jīng)系統(tǒng)雙峰機(jī)制的治療潛力：在疾病的早期階段：抑制自身免疫反應(yīng)誘導(dǎo)內(nèi)源性祖細(xì)胞的神經(jīng)再生,,,,Age of the Donor Reduces the Ability o

44、f Human Adipose-Derived Stem Cells to Alleviate Symptoms in the Experimental Autoimmune Encephalomyelitis Mouse Model,ASCs from older donors failed to ameliorate the neurodegeneration associated with EAE, and mice treate

45、d with older donor cells had increased CNS inflammation, demyelination, splenocyte proliferation in vitro compared with the mice receiving cells from younger donors.,STEM CELLS TRANSLATIONALMEDICINE 2013;2:797,Ghasemi

46、 N. Transplantation of human adipose-derived stem cells enhances remyelination in lysolecithin-induced focal demyelination of rat spinal cord. Mol Biotechnol. 2014 .,transplanted human ADSCs (hADSCs) into a lysophosphati

47、dylcholine (lysolecithin) model of multiple sclerosis (MS) and determined the efficiency of these cells in remyelination process. Forty adult rats were randomly divided into control, lysolecithin, vehicle, and transplan

48、tation groups, and focal demyelination was induced by lysolecithin injection into spinal cord. To assess motor performance, all rats were examined weekly with a standard EAE scoring scale. Four weeks after cell transpla

49、ntation, to assess the extent of demyelination and remyelination, Luxol Fast Blue staining was used. In addition, immunohistochemistry technique was used for assessment of the presence of oligodendrocyte phenotype cells

50、in damaged spinal cord. hADSCs had ability to differentiate into oligodendrocyte phenotype cells and improved remyelination process. Moreover, the evaluation of rat motor functions showed that animals which were treated

51、 with hADSC compared to other groups had significant improvement. hADSCs transplantation for cell-based therapies may play a proper cell source in the treatment of neurodegenerative diseases such as MS.,Cord blood deriv

52、ed cell臍帶血細(xì)胞,,Skin derived precursor cell皮膚干細(xì)胞,Radtke C, et al. (2009),促進(jìn)周?chē)窠?jīng)再生a 12 mm gap created in the sciatic nerve of Lewis rats bridged by a freeze-thawed nerve graft SKP衍生的雪旺細(xì)胞（SKP- SCS）：nerve-derived SCsSKP

53、 – SCS：分泌具有生物活性的神經(jīng)營(yíng)養(yǎng)因子,,,2015,,,,,,,,,,Embryonic stem (ES) cell胚胎干細(xì)胞,Salehi M, et al. (2009),聯(lián)合移植胚胎干細(xì)胞（ES）細(xì)胞衍生的運(yùn)動(dòng)神經(jīng)元（ESMN）和嗅鞘細(xì)胞協(xié)同作用：促進(jìn)挫傷脊髓損傷神經(jīng)再生ESMN生存神經(jīng)功能恢復(fù),Sundberg M, et al. (2010),一種高效的生產(chǎn)和分離方法人類胚胎干細(xì)胞來(lái)源的NG2（+）少突

54、膠質(zhì)細(xì)胞前體無(wú)血清培養(yǎng)基,Bone marrow-derived MSC骨髓基質(zhì)細(xì)胞(BM-MSC),Goel RK, et al. (2009),BM-MNC靜脈移植：髓鞘修復(fù)大鼠脊髓大鼠坐骨神經(jīng)切斷模型：軸突再生髓鞘化修復(fù)厚髓鞘形成,,,Umbilical cord-derived mesenchymal stromal cell臍帶來(lái)源間充質(zhì)基質(zhì)細(xì)胞,Matsuse D, et al. (2010),人臍帶源性間

55、充質(zhì)基質(zhì)干細(xì)胞具有分化成雪旺細(xì)胞的能力維持周?chē)窠?jīng)再生,Liu AM, et al. (2010),肝細(xì)胞生長(zhǎng)因子（HGF）轉(zhuǎn)導(dǎo)的MSCs大鼠腦出血模型提高髓鞘再生、軸突再生和功能恢復(fù),iPS cell多能誘導(dǎo)干細(xì)胞,Tsuji O, et al. (2010),iPS源神經(jīng)球脊髓損傷模型體內(nèi)，體外：分化成所有3個(gè)神經(jīng)譜系（神經(jīng)元，星形膠質(zhì)細(xì)胞，少突膠質(zhì)細(xì)胞 ），沒(méi)有形成畸胎瘤髓鞘修復(fù)，誘導(dǎo)宿主5-HT（+） - 羥色胺

56、纖維軸突再生，促進(jìn)運(yùn)動(dòng)功能的恢復(fù),,,Schwann cell雪旺細(xì)胞,Côté MP, et al. (2010),神經(jīng)嫁接的方法促進(jìn)非嚙齒類動(dòng)物--大動(dòng)物模型脊髓損傷后的再生周?chē)窠?jīng)移植到脊髓損傷貓?jiān)偕妮S突與宿主神經(jīng)元形成功能突觸,,,Route of cell therapy,立體定向手術(shù)腦內(nèi)移植腦室穿刺（或Ommya囊）全麻下顯微鏡下脊髓內(nèi)移植局麻下CT引導(dǎo)脊髓內(nèi)移植局部蛛網(wǎng)膜下腔移植（小腦