赵永波,沈楠

• 1:上海交通大学附属第一人民医院神经科

摘要(Abstract)：

目的通过应用骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)移植治疗帕金森病(Parkinson's disease,PD)大鼠,检测大鼠外周血中CD4+CD25highFoxP3+调节性T细胞(regulatory T cell,Treg)比例的变化及相关细胞因子的漂移,观察脑内免疫排斥反应的发生状况。以期探讨干细胞移植治疗PD后,外周血中Treg的改变与脑内免疫排斥反应间的相关性及其在干细胞移植后免疫耐受中的作用。方法 SD大鼠随机分为正常对照组、PD组、(PD+PBS)组、(PD+BMSCs)组。正常对照组大鼠不进行任何处理,为完全空白对照;余下大鼠均通过脑内立体定向注射6-羟基多巴胺(6-hydmxydopamine,6-OHDA)建立PD大鼠模型。成功建模后第3周,(PD+PBS)组大鼠脑内定向注射PBS缓冲液;(PD+BMSCs)组大鼠脑内定向注射应用全骨髓贴壁法培养的大鼠BMSCs悬液;PD组大鼠不做任何处理。PBS缓冲液和BMSCs悬液注射2周后,各组大鼠均应用流式细胞术(flow cytometry,FCM)检测其外周血中CD4+CD25highFoxP3+Treg占T淋巴细胞比例的变化,并运用酶联免疫吸附测定(ELISA)检测外周血中白介素(IL)-4、IL-10和干扰素-γ(INF-γ)等相关细胞因子的改变。另外,通过免疫组织化学染色法观察大鼠脑内酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性神经元、离子钙接头蛋白分子-1(ionized calcium bindingadaptor molecule-1,iba-1)阳性细胞、主要组织相容性复合体-Ⅱ(major histocompatibility complex-Ⅱ,MHC-Ⅱ)分子的变化。同时,(PD+BMSCs)组大鼠采用EdU染色法监测移植后BMSCs的存活情况。结果脑内立体定向注射6-OHDA 3周后,部分大鼠腹腔注射阿扑吗啡可诱导出旋转行为,表现为向健侧旋转,呈首尾相接状,以210圈/30分钟为标准,建模成功率达65.45%,并且免疫组织化学染色显示,建模成功的PD大鼠偏侧TH阳性神经元毁损率可达90%以上。体外培养的大鼠BMSCs至P4时,细胞形态良好且均一,经流式细胞术鉴定,其纯度达95%以上。应用P4的BMSCs移植治疗PD大鼠2周后,BMSCs可在移植注射部位少量存活,但无法自我分化为多巴胺(dopamine,DA)能神经元,且(PD+BMSCs)组大鼠阿扑吗啡诱导旋转行为较PD组、(PD+PBS)组未见好转。另外,FCM示(PD+BMSCs)组大鼠外周血中CD4+CD25highFoxP3+Treg占T淋巴比例为(0.45±0.10)%,较正常对照组、PD组及(PD+PBS)组均显著减少(P<0.01)。ELISA示(PD+BMSCs)组大鼠外周血中IL-4、IL-10和INF-γ水平也较其他对照组显著降低(P<0.01)。同时,(PD+BMSCs)组大鼠脑内iba-1阳性细胞在移植处增生激活,MHC-Ⅱ类抗原分子表达上调,且二者与正常对照组、PD组及(PD+PBS)组相比均显著增加(P<0.01)。另外,进一步分析发现大鼠外周血中Treg比例与iba-1阳性细胞增加率、MHC-Ⅱ类抗原分子表达量均呈负相关(P<0.01)。结论本研究通过立体定向注射6-OHDA建立PD的大鼠模型,并给予脑内注射体外培养至P4的BMSCs移植治疗。此为基础,笔者验证了体外未经诱导分化的BMSCs移植治疗PD大鼠后无法自我分化为DA能神经元,甚至还可引起脑内免疫排斥反应,导致最终的治疗失败。另外,BMSCs移植治疗PD大鼠后外周血中CD4+CD25highFoxP3+Treg比例显著降低、相关细胞因子水平也明显下降,并且这些改变与移植后脑内免疫排斥反应的发生相关。

关键词(KeyWords)： 帕金森病;骨髓间充质干细胞;CD4+CD25highFoxP3+调节性T细胞;免疫排斥反应

Abstract:

Keywords:

基金项目(Foundation): 上海市科委基础研究重点项目(11JC1411402)

作者(Author): 赵永波,沈楠

参考文献(References)：

• [1]Dawson TM,Dawson VL.Molecular pathways of neurodegeneration in Parkinson's disease[J].Science,2003,302(5646):819-822.
• [2]Schapira AH.Neurobiology and treatment of Parkinson's disease[J].Trends Pharmacol Sci,2009,30(1):41-47.
• [3]Benabid AL,Chabardes S,Mitrofanis J,et al.Deep brain stimulation of the subthalamic nucleus for the treatment of Parkinson's disease[J].Lancet Neurol,2009,8(1):67-81.
• [4]Christine CW,Starr PA,Larson PS,et al.Safety and tolerability of putaminal AADC gene therapy for Parkinson disease[J].Neurology,2009,73(20):1662-1669.
• [5]Kim SU,de Vellis J.Stem cell-based cell therapy in neurological diseases:a review[J].J Neurosci Res,2009,87(10):2183-2200.
• [6]Fahn S,Oakes D,Shoulson I,et al.Levodopa and the progression of Parkinson's disease[J].N Engl J Med,2004,351(24):2498-2508.
• [7]Krack P,Batir A,Van Blercom N,et al.Five-year follow-up of bilateral stimulation of the subthalamic nucleus in advanced Parkinson's disease[J].N Engl J Med,2003,349(20):1925-1934.
• [8]Gervais-Bernard H,Xie-Brustolin J,Mertens P,et al.Bilateral subthalamic nucleus stimulation in advanced Parkinson's disease:five year follow-up[J].J Neurol,2009,256(2):225-233.
• [9]Fasano A,Romito LM,Daniele A,et al.Motor and cognitive outcome in patients with Parkinson's disease 8 years after subthalamic implants[J].Brain,2010,133(9):2664-2676.
• [10]Gogel S,Gubernator M,Minger SL.Progress and prospects:stem cells and neurological diseases[J].Gene Ther,2011,18(1):1-6.
• [11]Gibson SA,Gao GD,Mcdonagh K,et al.Progress on stem cell research towards the treatment of Parkinson's disease[J].Stem Cell Res Ther,2012,3(2):11.
• [12]Allan LE,Petit GH,Brundin P.Cell transplantation in Parkinson's disease:problems and perspectives[J].Curr Opin Neurol,2010,23(4):426-432.
• [13]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.
• [14]Bae KS,Park JB,Kim HS,et al.Neuron-like differentiation of bone marrow-derived mesenchymal stem cells[J].Yonsei Med J,2011,52(3):401-412.
• [15]Si YL,Zhao YL,Hao HJ,et al.MSCs:Biological characteristics,clinical applications and their outstanding concerns[J].Ageing Res Rev,2011,10(1):93-103.
• [16]Le Blanc K.Immunomodulatory effects of fetal and adult mesenchymal stem cells[J].Cytotherapy,2003,5(6):485-489.
• [17]Le Blanc K,Ringden O.Immunomodulation by mesenchymal stem cells and clinical experience[J].J Intern Med,2007,262(5):509-525.
• [18]Uccelli A,Moretta L,Pistoia V.Immunoregulatory function of mesenchymal stem cells[J].Eur J Immunol,2006,36(10):2566-2573.
• [19]Bartholomew A,Sturgeon C,Siatskas M,et al.Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo[J].Exp Hematol,2002,30(1):42-48.
• [20]Le Blanc K,Rasmusson I,Sundberg B,et al.Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells[J].Lancet,2004,363(9419):1439-1441.
• [21]Tse WT,Pendleton JD,Beyer WM,et al.Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation[J].Transplantation,2003,75(3):389-397.
• [22]Aggarwal S,Pittenger MF.Human mesenchymal stem cells modulate allogeneic immune cell responses[J].Blood,2005,105(4):1815-1822.
• [23]Camp DM,Loeffler DA,Farrah DM,et al.Cellular immune response to intrastriatally implanted allogeneic bone marrow stromal cells in a rat model of Parkinson's disease[J].J Neuroinflammation,2009,6:17.
• [24]Carson MJ,Doose JM,Melchior B,et al.CNS immune privilege:hiding in plain sight[J].Immunol Rev,2006,213:48-65.
• [25]Galea I,Bechmann I,Perry VH.What is immune privilege(not)?[J].Trends Immunol,2007,28(1):12-18.
• [26]Muldoon LL,Alvarez JI,Begley DJ,et al.Immunologic privilege in the central nervous system and the blood-brain barrier[J].J Cereb Blood Flow Metab,2013,33(1):13-21.
• [27]Sakaguchi S,Sakaguchi N.Regulatory T cells in immunologic self-tolerance and autoimmune disease[J].Int Rev Immunol,2005,24(3-4):211-226.
• [28]Sakaguchi S,Sakaguchi N,Asano M,et al.Immunologic selftolerance maintained by activated T cells expressing IL-2receptor alpha-chains(CD25).Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases[J].J Immunol,1995,155(3):1151-1164.
• [29]Fontenot JD,Gavin MA,Rudensky AY.Foxp3 programs the development and function of CD4+CD25+regulatory T cells[J].Nat Immunol,2003,4(4):330-336.
• [30]Hori S,Nomura T,Sakaguchi S.Control of regulatory T cell development by the transcription factor Foxp3[J].Science,2003,299(5609):1057-1061.
• [31]Khattri R,Cox T,Yasayko SA,et al.An essential role for Scurfin in CD4+CD25+T regulatory cells[J].Nat Immunol,2003,4(4):337-342.
• [32]Brunkow ME,Jeffery EW,Hjerrild KA,et al.Disruption of a new forkhead/winged-helix protein,scurfin,results in the fatal lymphoproliferative disorder of the scurfy mouse[J].Nat Genet,2001,27(1):68-73.
• [33]Chen W,Jin W,Hardegen N,et al.Conversion of peripheral CD4+CD25-naive T cells to CD4+CD25+regulatory T cells by TGF-beta induction of transcription factor Foxp3[J].J Exp Med,2003,198(12):1875-1886.
• [34]Lan RY,Selmi C,Gershwin ME.The regulatory,inflammatory,and T cell programming roles of interleukin-2(IL-2)[J].J Autoimmun,2008,31(1):7-12.
• [35]Yates J,Rovis F,Mitchell P,et al.The maintenance of human CD4+CD25+regulatory T cell function:IL-2,IL-4,IL-7 and IL-15 preserve optimal suppressive potency in vitro[J].Int Immunol,2007,19(6):785-799.
• [36]Burrell BE,Nakayama Y,Xu J,et al.Regulatory T cell induction,migration,and function in transplantation[J].J Immunol,2012,189(10):4705-4711.
• [37]Maloy KJ,Salaun L,Cahill R,et al.CD4+CD25+T(R)cells suppress innate immune pathology through cytokine-dependent mechanisms[J].J Exp Med,2003,197(1):111-119.
• [38]Blum D,Torch S,Lambeng N,et al.Molecular pathways involved in the neurotoxicity of 6-OHDA,dopamine and MPTP:contribution to the apoptotic theory in Parkinson's disease[J].Prog Neurobiol,2001,65(2):135-172.
• [39]Deumens R,Blokland A,Prickaerts J.Modeling Parkinson's disease in rats:an evaluation of 6-OHDA lesions of the nigrostriatal pathway[J].Exp Neurol,2002,175(2):303-317.
• [40]Hudson JL,van Horne CG,Stromberg I,et al.Correlation of apomorphine-and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats[J].Brain Res,1993,626(1-2):167-174.
• [41]Friedenstein AJ,Gorskaja JF,Kulagina NN.Fibroblast precursors in normal and irradiated mouse hematopoietic organs[J].Exp Hematol,1976,4(5):267-274.
• [42]吴军,张燕燕.骨髓间充质干细胞生物学特性及临床应用进展[J].中国组织工程研究与临床康复,2007,11(50):10134-10137.
• [43]Soleimani M,Nadri S.A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow[J].Nat Protoc,2009,4(1):102-106.
• [44]Zhu H,Guo ZK,Jiang XX,et al.A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone[J].Nat Protoc,2010,5(3):550-560.
• [45]Baddoo M,Hill K,Wilkinson R,et al.Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection[J].J Cell Biochem,2003,89(6):1235-1249.
• [46]Yoshimura H,Muneta T,Nimura A,et al.Comparison of rat mesenchymal stem cells derived from bone marrow,synovium,periosteum,adipose tissue,and muscle[J].Cell Tissue Res,2007,327(3):449-462.
• [47]Karp JM,Leng TG.Mesenchymal stem cell homing:the devil is in the details[J].Cell Stem Cell,2009,4(3):206-216.
• [48]Mosna F,Sensebe L,Krampera M.Human bone marrow and adipose tissue mesenchymal stem cells:a user's guide[J].Stem Cells Dev,2010,19(10):1449-1470.
• [49]You M,Peng G,Li J,et al.Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate(PHA)scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide[J].Biomaterials,2011,32(9):2305-2313.
• [50]冯善伟,姚晓黎,李中,等.BrdU体外标记大鼠骨髓间充质干细胞的研究[J].第一军医大学学报,2005,25(2):184-186.
• [51]Hattan N,Kawaguchi H,Ando K,et al.Purified cardiomyocytes from bone marrow mesenchymal stem cells produce stable intracardiac grafts in mice[J].Cardiovasc Res,2005,65(2):334-344.
• [52]王小蕊,侯相麟,席亚明,等.成人骨髓间充质干细胞的鉴定与体外BrdU标记[J].中国组织工程研究与临床康复,2008,12(21):4131-4135.
• [53]Lu L,Zhao C,Liu Y,et al.Therapeutic benefit of THengineered mesenchymal stem cells for Parkinson's disease[J].Brain Res Brain Res Protoc,2005,15(1):46-51.
• [54]徐浩文,朱蔚文,叶钦勇,等.过表达Nurr1基因的骨髓间充质干细胞的诱导及移植治疗帕金森病的研究[J].中国临床解剖学杂志,2005,23(3):259-263.
• [55]Sadan O,Bahat-Stromza M,Barhum Y,et al.Protective effects of neurotrophic factor-secreting cells in a 6-OHDA rat model of Parkinson disease[J].Stem Cells Dev,2009,18(8):1179-1190.
• [56]张晓玲,戴尅戎,汤亭亭,等.未分化骨髓间充质干细胞的免疫学特性研究[J].中华实验外科杂志,2006,23(2):135-137.
• [57]董光龙,王为忠,李开宗.调节性T细胞及相关细胞因子与同种小肠移植耐受[J].免疫学杂志,2003,19(1):78-80.
• [58]Meloni F,Vitulo P,Bianco A M,et al.Regulatory CD4+CD25+T cells in the peripheral blood of lung transplant recipients:correlation with transplant outcome[J].Transplantation,2004,77(5):762-766.
• [59]贾维胜,张银甫,黄赤兵,等.Foxp3体外诱导产生的调节性T细胞对小鼠心脏移植物存活的影响[J].重庆医学,2006,35(4):203-205.