中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2008年
8期
1577-1580
,共4页
张志英%任丛莉%张传森%李亮%党瑞山%孔征东
張誌英%任叢莉%張傳森%李亮%黨瑞山%孔徵東
장지영%임총리%장전삼%리량%당서산%공정동
骨髓%神经组织定向干细胞%细胞培养%鉴定%细胞移植
骨髓%神經組織定嚮榦細胞%細胞培養%鑒定%細胞移植
골수%신경조직정향간세포%세포배양%감정%세포이식
背景:骨髓间充质干细胞和造血干细胞具有分化为神经类细胞的潜能已得到共识.另有研究者认为骨髓中除骨髓间充质干细胞和造血干细胞以外,还寄居着CXCR4阳性的组织定向干细胞,包括骨骼肌、心、肝及神经组织定向干细胞.目的:实验拟进一步证实神经组织定向干细胞寄居于骨髓,并寻求确立神经组织定向干细胞分离、培养的新方法.设计:开放性实验.单位:解放军第二军医大学解剖学教研室.材料:所用成年清洁级SD大鼠由解放军第二军医大学动物中心提供.DMEM/F12,B27,N2均购自Invitrogen公司;bFGF源于CytoLab Ltd;EGF源于Invitrogen公司;Nestin、CXCR4,β-Tublin Ⅲ,神经胶质纤维酸性蛋白等一抗为Santa Cruz公司兔抗鼠多克隆抗体;MAP2ab(Clone11-5B),CNPase(Clone AP20)为NeoMarkers公司小鼠抗大鼠单克隆抗体;荧光(FITC,Cy3)标记试剂盒购自武汉博士德生物工程有限公司;免疫组化试剂盒购自Vector Labora-tories公司.NycoPrepTM分离液(1.077A)购自Axis-Shield 公司.方法:实验于2004-01/2006-12在解放军第二军医大学组织工程研究所完成.取SD大鼠双侧股骨及胫骨,冲洗骨髓腔,经NycoPrepTM分离液分离单核细胞层,DMEM/F12(1∶1) 无血清神经干细胞培养液(内含2%B27,1%N2,20 μg/L碱性成纤维细胞生长因子, 20 μg/L 表皮生长因子,1×105 U/L青霉素, 100 mg/L链霉素),通过先贴壁、后悬浮的方法从骨髓中分离、培养单克隆生长的神经组织定向干细胞.主要观察指标:通过免疫细胞化学法检测神经组织定向干细胞细胞球CXCR4和nestin蛋白,以及细胞球自然分化后神经元以及神经胶质细胞的标志蛋白.结果:①神经组织定向干细胞细胞球表达神经干细胞标志蛋白nestin和组织定向干细胞标志蛋白 CXCR4.②神经组织定向干细胞细胞球在含15%胎牛血清的DMEM培养液中可自然分化,分化细胞呈梭形或多角形,有2～5个细胞突起,免疫荧光检测显示神经元标志蛋白β-tublinIII及MAP2ab,以及神经胶质标志蛋白CNPase和神经胶质纤维酸性蛋白阳性.结论:骨髓中存在神经组织定向干细胞,可自然分化为神经元样细胞及神经胶质样细胞.
揹景:骨髓間充質榦細胞和造血榦細胞具有分化為神經類細胞的潛能已得到共識.另有研究者認為骨髓中除骨髓間充質榦細胞和造血榦細胞以外,還寄居著CXCR4暘性的組織定嚮榦細胞,包括骨骼肌、心、肝及神經組織定嚮榦細胞.目的:實驗擬進一步證實神經組織定嚮榦細胞寄居于骨髓,併尋求確立神經組織定嚮榦細胞分離、培養的新方法.設計:開放性實驗.單位:解放軍第二軍醫大學解剖學教研室.材料:所用成年清潔級SD大鼠由解放軍第二軍醫大學動物中心提供.DMEM/F12,B27,N2均購自Invitrogen公司;bFGF源于CytoLab Ltd;EGF源于Invitrogen公司;Nestin、CXCR4,β-Tublin Ⅲ,神經膠質纖維痠性蛋白等一抗為Santa Cruz公司兔抗鼠多剋隆抗體;MAP2ab(Clone11-5B),CNPase(Clone AP20)為NeoMarkers公司小鼠抗大鼠單剋隆抗體;熒光(FITC,Cy3)標記試劑盒購自武漢博士德生物工程有限公司;免疫組化試劑盒購自Vector Labora-tories公司.NycoPrepTM分離液(1.077A)購自Axis-Shield 公司.方法:實驗于2004-01/2006-12在解放軍第二軍醫大學組織工程研究所完成.取SD大鼠雙側股骨及脛骨,遲洗骨髓腔,經NycoPrepTM分離液分離單覈細胞層,DMEM/F12(1∶1) 無血清神經榦細胞培養液(內含2%B27,1%N2,20 μg/L堿性成纖維細胞生長因子, 20 μg/L 錶皮生長因子,1×105 U/L青黴素, 100 mg/L鏈黴素),通過先貼壁、後懸浮的方法從骨髓中分離、培養單剋隆生長的神經組織定嚮榦細胞.主要觀察指標:通過免疫細胞化學法檢測神經組織定嚮榦細胞細胞毬CXCR4和nestin蛋白,以及細胞毬自然分化後神經元以及神經膠質細胞的標誌蛋白.結果:①神經組織定嚮榦細胞細胞毬錶達神經榦細胞標誌蛋白nestin和組織定嚮榦細胞標誌蛋白 CXCR4.②神經組織定嚮榦細胞細胞毬在含15%胎牛血清的DMEM培養液中可自然分化,分化細胞呈梭形或多角形,有2～5箇細胞突起,免疫熒光檢測顯示神經元標誌蛋白β-tublinIII及MAP2ab,以及神經膠質標誌蛋白CNPase和神經膠質纖維痠性蛋白暘性.結論:骨髓中存在神經組織定嚮榦細胞,可自然分化為神經元樣細胞及神經膠質樣細胞.
배경:골수간충질간세포화조혈간세포구유분화위신경류세포적잠능이득도공식.령유연구자인위골수중제골수간충질간세포화조혈간세포이외,환기거착CXCR4양성적조직정향간세포,포괄골격기、심、간급신경조직정향간세포.목적:실험의진일보증실신경조직정향간세포기거우골수,병심구학립신경조직정향간세포분리、배양적신방법.설계:개방성실험.단위:해방군제이군의대학해부학교연실.재료:소용성년청길급SD대서유해방군제이군의대학동물중심제공.DMEM/F12,B27,N2균구자Invitrogen공사;bFGF원우CytoLab Ltd;EGF원우Invitrogen공사;Nestin、CXCR4,β-Tublin Ⅲ,신경효질섬유산성단백등일항위Santa Cruz공사토항서다극륭항체;MAP2ab(Clone11-5B),CNPase(Clone AP20)위NeoMarkers공사소서항대서단극륭항체;형광(FITC,Cy3)표기시제합구자무한박사덕생물공정유한공사;면역조화시제합구자Vector Labora-tories공사.NycoPrepTM분리액(1.077A)구자Axis-Shield 공사.방법:실험우2004-01/2006-12재해방군제이군의대학조직공정연구소완성.취SD대서쌍측고골급경골,충세골수강,경NycoPrepTM분리액분리단핵세포층,DMEM/F12(1∶1) 무혈청신경간세포배양액(내함2%B27,1%N2,20 μg/L감성성섬유세포생장인자, 20 μg/L 표피생장인자,1×105 U/L청매소, 100 mg/L련매소),통과선첩벽、후현부적방법종골수중분리、배양단극륭생장적신경조직정향간세포.주요관찰지표:통과면역세포화학법검측신경조직정향간세포세포구CXCR4화nestin단백,이급세포구자연분화후신경원이급신경효질세포적표지단백.결과:①신경조직정향간세포세포구표체신경간세포표지단백nestin화조직정향간세포표지단백 CXCR4.②신경조직정향간세포세포구재함15%태우혈청적DMEM배양액중가자연분화,분화세포정사형혹다각형,유2～5개세포돌기,면역형광검측현시신경원표지단백β-tublinIII급MAP2ab,이급신경효질표지단백CNPase화신경효질섬유산성단백양성.결론:골수중존재신경조직정향간세포,가자연분화위신경원양세포급신경효질양세포.
BACKGROUND: It has been widely accepted that both bone marrow-derived mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have the capacity to differentiate into neural lineages. Some scholars believe that in addition to HSCs and MSCs, bone marrow (BM) also harbors a highly mobile population of CXCR4+ tissue committed stem cells (TCSCs), including skeletal muscles, heart, liver, and neural tissue. OBJECTIVE: To make sure that neural tissue-committed stem cells (NTCSCs) reside in the bone marrow, and to establish a purification and culture method for bone marrow-derived NTCSCs.DESIGN: Opening animal study.SETTING: Department of Anatomy, the Second Military Medical University of Chinese PLA. MATERIALS: Adult Sprague-Dawley (SD) rats (pathogen-free) were provided by the Animal Center of the Second Military Medical University of Chinese PLA. Dulbecco's modified eagle's medium (DMEM)/F12, B27, N2 and epidermal growth factor (EGF, Invitrogen Company), basic fibroblast growth factor (bFGF, CytoLab Ltd), rabbit anti-rat Nestin,CXCR4, β-Tublin Ⅲ, glial fibrillary acidic protein (GFAP, Santa Cruz Company), mouse anti-rat microtubule associated protein 2ab (MAP2ab) (Clone11-5B), cyclic nucleotide 3'phosphohydrolase (CNPase, Clone AP20, NeoMarkers Company), fluorescent(fluorescein isothiocyanate, Cy3) marker reagents (Wuhan Boster Bioengineering Co., Ltd), nuclear fluorescent dyes 4,6-diamidino-2-phenylindole(DAPI)(Sigma), immunohistochemistry reagents (Vector Laboratories Company) , and NycoPrepTM separation liquid (1.077A, Axis-Shield Company) were used in this study.METHODS: This study was performed in the Department of Anatomy, the Second Military Medical University of Chinese PLA from January 2004 to December 2006. Bone marrow was harvested from bilateral femurs and tibias of 2-3 weeks SD rats. Mononuclear cell layer was isolated by NycoPrepTM separation liquid and suspended in DMEM/F12(1:1)serum-free medium supplemented with 2% B27,1% N2, 20 μg/L bFGF, 20μg/L EGF, 1×105 U/L penicillin and 100 mg/L streptomycin. NTCSCs were isolated and propagated by suspensive growing from adherent cells in bone marrow in DMEM/F12 free-serum medium. MAIN OUTCOME MEASURES: NTCSCs were identified by immunocytochemistry for CXCR4, a marker of TCSCs and nestin, a marker of neural stem cells, and neural lineages marker protein after differentiation of cellular spheres. RESULTS: The NTCSCs spheres expressed nestin, a neural stem cell marker as well as CXCR4, a marker of TCSCs. The NTCSCs' spheres were naturally differentiated in DMEM medium with 15% fetal bovine serum. The differentiated cells expressed β-Tublin Ⅲ, MAP2ab, CNPase and GFAP, markers of neural lineages. CONCLUSION: NTCSCs reside in bone marrow and naturally differentiate into neural lineages in vitro.
