成髓鞘細胞研究和應用進展

1、成髓鞘細胞研究和應用進展,陳 琳 清華大學第二附屬醫(yī)院神經(jīng)外科中心chenlin_china@163.com,第一屆全國兒童神經(jīng)修復學術會議暨第四屆全國兒童神經(jīng)康復論壇. 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：勇于擔當，發(fā)揮引領作用以苦為樂，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. 中英脫髓鞘倫敦會議,要點,一些基

2、本概念與影像學檢查兒童脫髓鞘疾病臨床分類成髓鞘細胞分類神經(jīng)修復的路徑各種細胞近期研究一覽,Year 1977~ 2016 40年 demyelination remyelination Pubmed文獻量趨勢圖,髓鞘構成成分,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)髓鞘相關糖蛋白(MAG) 髓鞘少突膠質(zhì)

3、細胞糖蛋白(MOG)Wolfram 蛋白脂DM 準大小蛋白其他微量蛋白,髓鞘功能,提供軸突與周圍組織電絕緣，避免干擾相鄰軸突之間軸突與其他結構之間加快動作電位傳遞：“跳躍式傳導”機制引導受損軸突：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)纖維與周圍(PNS)相似結構有髓鞘和郎飛結髓鞘外無基膜（神經(jīng)膜）相鄰神經(jīng)纖維有時融合（箭頭）,兒童發(fā)育,髓鞘化過程主要集中在：出生后18個月內(nèi)（1.5年）1歲半接近成人oligodendrocyte少突膠質(zhì)細胞：大多數(shù)細胞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、)髓磷脂體積分數(shù),myelin water fraction (MWF)髓磷脂水分,脫髓鞘疾病分類（一）,兒童脫髓鞘?。褐車?和 中樞性周圍性脫髓鞘病：多以雙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，但已無生理功能損傷部位遠端的軸突出現(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,細胞移植髓鞘

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

22、長因子體外：能促進軸突生長體內(nèi)：可形成髓鞘，促進髓鞘再生刺激軸突再生發(fā)芽優(yōu)于雪旺細胞：與星形膠質(zhì)細胞接觸,（1）嗅鞘細胞髓鞘化修復類于雪旺細胞,Babiarz J, et al. (2011)分離幼年和成年大鼠的嗅球，分析表達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。嗅鞘細胞與軸突組裝成束需要1周，如果形成可以檢測到的軸突髓鞘，需要1周以上SC：大鼠雪旺細胞不捆束軸突，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）染色陽性，雪旺細胞為陰性幾乎所有的OEC和雪旺細胞P75NTR和GFAP陽性兩種細胞之間只有細微的免疫標記差異,（1）嗅鞘細胞髓鞘化修復類于雪旺細胞,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）嗅鞘細胞修復腦缺血卒中,Shi X, et al. (2010)修復大腦中動脈閉塞（MCAO）大鼠腦白質(zhì) MCAO后56天結果：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，Western blot：OEC移植大

25、鼠髓鞘和軸突再生,（3）嗅鞘細胞修復周圍神經(jīng)損傷,Radtke C, et al. (2010) 嗅鞘細胞移植作為輔助治療周圍神經(jīng)損傷嗅鞘細胞移植促進再生過程：介導趨化因子，神經(jīng)營養(yǎng)和神經(jīng)保護作用髓鞘形成：橋接，建立一個允許軸突再生的環(huán)境,（4）嗅鞘細胞在體內(nèi)、體外與其他細胞的相互作用,Chuah MI, et al. (2010) 與其他類型的細胞：在體外和嗅鞘細胞移植后的膠質(zhì)疤痕和炎癥環(huán)境下的相互作用嗅鞘細胞和星形膠質(zhì)細胞

26、 克服膠質(zhì)瘢痕的有害影響不同脊髓損傷的實驗模型：OEC移植相關的膠質(zhì)瘢痕的形態(tài)學改變在體外：嗅鞘細胞和膠質(zhì)瘢痕的細胞類型組成之間的相互作用嗅鞘細胞：免疫細胞特性，移植到中樞神經(jīng)系統(tǒng)損傷部位時，調(diào)制神經(jīng)炎癥,（5）嗅鞘細胞移植聯(lián)合瘢痕切除修復脊髓挫傷,Zhang SX, et al. (2011)疤痕消融+ LP/ OEC移植促進大鼠脊髓慢性挫傷解剖恢復和P0（髓磷脂糖蛋白,P-zero）陽性髓鞘孟加拉玫瑰紅光毒性方法 單獨

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）嗅鞘細胞移植修復肌萎縮側索硬化,Li Y, et al. (2011) OEC移植到脊髓：延長SOD1（G93A）ALS大鼠生存期神經(jīng)保護作用和髓鞘化移植的嗅鞘細胞存活超過4周，在脊髓內(nèi)遷移4.2毫米,（7）嗅鞘細胞移植修復各種脫髓鞘模型,Sasaki M, et al. (2011)不同的脫髓鞘環(huán)境對OEC髓鞘化修復的影響OECs的遷移和髓鞘形成：炎癥處于活

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

29、屬特異性,Wewetzer K, et al. (2011) 細胞增殖控制：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/成年嗅鞘細胞移植修復效果存在差異,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)細胞,Li H, et al. (2009),兩層少突膠質(zhì)細胞分化的轉(zhuǎn)錄調(diào)控 “雙管齊下”的方式：創(chuàng)建一個基因控制的故障安全系統(tǒng)確保在發(fā)育過程、脫髓鞘病變的修復過程中：髓

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

37、脊髓挫傷后嘴側軸突變性誘導：修復+細胞凋亡,Piaton G, et al. (2010),在發(fā)育髓鞘、脫髓鞘、修復過程中，軸突與少突膠質(zhì)細胞相互作用髓磷脂的生物合成和髓鞘修復：神經(jīng)元和少突膠質(zhì)細胞之間的相互溝通必不可少In 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)干細胞,Hwang DH, et al. (2009),轉(zhuǎn)導OLIG2轉(zhuǎn)錄因子的人類神經(jīng)干細胞（NSCs）脊髓挫傷損傷后大鼠：提高運動功能的恢復增強脊髓白質(zhì)髓鞘修復再生,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)干細胞遷移通過CXCR4介導CXCL12的信號,Adipose mesenchymal stem cell脂肪來源間充質(zhì)干細胞,Radtke C, et al. (2009),脂肪間充質(zhì)干細胞衍生的神經(jīng)球

43、可分化成周圍神經(jīng)膠質(zhì)樣細胞,Constantin G, et al. (2009),脂肪來源的間質(zhì)干細胞改善慢性實驗性自身免疫性腦脊髓炎 (EAE)靜脈給藥：顯著降低免疫反應所致EAE的嚴重性、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)雙峰機制的治療潛力：在疾病的早期階段：抑制自身免疫反應誘導內(nèi)源性祖細胞的神經(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臍帶血細胞,,Skin derived precursor cell皮膚干細胞,Radtke C, et al. (2009),促進周圍神經(jīng)再生a 12 mm gap created in the sciatic nerve of Lewis rats bridged by a freeze-thawed nerve graft SKP衍生的雪旺細胞（SKP- SCS）：nerve-derived SCsSKP

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

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

55、充質(zhì)基質(zhì)干細胞具有分化成雪旺細胞的能力維持周圍神經(jīng)再生,Liu AM, et al. (2010),肝細胞生長因子（HGF）轉(zhuǎn)導的MSCs大鼠腦出血模型提高髓鞘再生、軸突再生和功能恢復,iPS cell多能誘導干細胞,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個神經(jīng)譜系（神經(jīng)元，星形膠質(zhì)細胞，少突膠質(zhì)細胞 ），沒有形成畸胎瘤髓鞘修復，誘導宿主5-HT（+） - 羥色胺

56、纖維軸突再生，促進運動功能的恢復,,,Schwann cell雪旺細胞,Côté MP, et al. (2010),神經(jīng)嫁接的方法促進非嚙齒類動物--大動物模型脊髓損傷后的再生周圍神經(jīng)移植到脊髓損傷貓再生的軸突與宿主神經(jīng)元形成功能突觸,,,Route of cell therapy,立體定向手術腦內(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引導脊髓內(nèi)移植局部蛛網(wǎng)膜下腔移植（小腦