CAJ | 학술논문

背景:骨髓基质细胞是贴壁黏附的非造血多潜能干细胞,其在一定条件体外培养、扩增可被诱导分化为神经元和胶质细胞.目的:观察家猫骨髓基质细胞体外培养及诱导分化成神经干细胞的生长情况.设计:随机取样.单位:南方医科大学附属珠江医院神经外科.材料:实验于2002-01/12在解放军第一军医大学珠江医院全军神经医学研究所中心实验室进行,实验动物是由第一军医大学动物中心提供健康家猫20只,年龄1.0～2.0岁,体质量2.5～4.0 kg,雌雄各半.干预:随机抽取家猫左右下肢的骨髓,从中分离得到骨髓基质细胞,提取的骨髓基质细胞悬液在体外给予神经干细胞培养基培养,增殖后用维甲酸为分化诱导因子进行诱导骨髓基质细胞向神经干细胞转化.CK2型倒置相差光学显微镜(OLYMPAS,JAPAN)追踪观察活细胞培养生长情况,同时观察4,12,24,48 h去除非贴壁细胞及不去除贴壁细胞骨髓基质细胞生长情况.采用免疫组织化学染色改良法对干细胞阶段的骨髓基质细胞进行免疫细胞化学检测,OLYMPAS光学显微镜观察.主要观察指标:对接受实验干预的骨髓基质细胞分别进行倒置相差显微镜下观察活细胞生长情况及光镜下观察免疫细胞化学染色情况.结果:20只家猫全部进入结果分析.猫骨髓基质细胞在体外培养中胞体增大,倒置相差显微镜下可见丰富的胞浆颗粒,继之出芽,贴壁生长,可形成克隆团,同时可传代培养.免疫组化染色分析结果显示能表达神经干细胞特异性抗原Nestin,而且能分化出胶质细胞样细胞和神经元样细胞.结论:家猫骨髓基质细胞具有干细胞特征,在合适条件下可扩增、形成克隆并诱导分化出神经胶质细胞和神经元特征细胞.
배경:골수기질세포시첩벽점부적비조혈다잠능간세포,기재일정조건체외배양、확증가피유도분화위신경원화효질세포.목적:관찰가묘골수기질세포체외배양급유도분화성신경간세포적생장정황.설계:수궤취양.단위:남방의과대학부속주강의원신경외과.재료:실험우2002-01/12재해방군제일군의대학주강의원전군신경의학연구소중심실험실진행,실험동물시유제일군의대학동물중심제공건강가묘20지,년령1.0～2.0세,체질량2.5～4.0 kg,자웅각반.간예:수궤추취가묘좌우하지적골수,종중분리득도골수기질세포,제취적골수기질세포현액재체외급여신경간세포배양기배양,증식후용유갑산위분화유도인자진행유도골수기질세포향신경간세포전화.CK2형도치상차광학현미경(OLYMPAS,JAPAN)추종관찰활세포배양생장정황,동시관찰4,12,24,48 h거제비첩벽세포급불거제첩벽세포골수기질세포생장정황.채용면역조직화학염색개량법대간세포계단적골수기질세포진행면역세포화학검측,OLYMPAS광학현미경관찰.주요관찰지표:대접수실험간예적골수기질세포분별진행도치상차현미경하관찰활세포생장정황급광경하관찰면역세포화학염색정황.결과:20지가묘전부진입결과분석.묘골수기질세포재체외배양중포체증대,도치상차현미경하가견봉부적포장과립,계지출아,첩벽생장,가형성극륭단,동시가전대배양.면역조화염색분석결과현시능표체신경간세포특이성항원Nestin,이차능분화출효질세포양세포화신경원양세포.결론:가묘골수기질세포구유간세포특정,재합괄조건하가확증、형성극륭병유도분화출신경효질세포화신경원특정세포.
BACKGROUND: Bone marrow mesenchymal cells, multiple-potential non-hematopoiefic stem cells adhering to the wall in vitro culture, can be induced to proliferate and differentiate towards neurons and glia cells.OBJECTIVE: To investigate the growth state of cat bone marrow mesenchymal cells in vitro culture, as well as the capability to differentiate towards neural stem cells.DESIGN: A randomized sampling study.SETTING: Department of Neurosurgery, Zhujiang Hospital, Southern Medical University.MATERIALS: This study was carried out at the Central Laboratory of the General Military Neurological Research Institute, Zhujiang Hospital, Southern Medical University between January and December 2002. Twenty healthy home-raised cats, aged 1.0 - 2.0 years and weighing 2. 5 - 4.0 kg, male and female in half, were provided by the Animal Center of the First Military Medical University of Chinese PLA.INTERVENTIONS: Bone marrows were randomly aspirated from the left or right hindlimbs in order to separate bone marrow mesenchymal cells, then the bone marrow mesenchymal cells single cell suspension was co-cultured with neural stem cell culture media in vitro so as to induce differentiation to neural stem cells with tretinoin. CK2 type inverted optical microscope(Olympus,Japan) was used to observe the growth of bone marrow mesenchymal cells in vitro culture, as well as 4, 12, 24, 48 hours of induction upon eliminating or not eliminating the wall-adhering cells. Bone marrow mesenchymal cells in stem cell stage were identified under Olympus optical microscope with modified immunohistochemical staining.MAIN OUTCOME MEASURES: The growth state and the immunocytochemical staining of living bone marrow mesenchymal cells exposed to experimental intervention were observed under the Olympus inverted optical microscope.RESULTS: Data from the 20 cats were analyzed without loss. Reversed microscopic observation revealed that cat bone marrow mesenchymal cells becrame larger when cultured in vitro, which were rich in plasmic granules with prominence projecting, adhering to the wall and forming cell clones. These cells were then successively cultured, and imnunohistochemical staining analysis suggested that the passaged bone marrow mesenchymal cells could express neural stem cells-specific antigen Nestin and differentiate towards glia-like cells and neuron-like cells.CONCLUSION: Cat bone marrow mesenchymal cells possess the characteristics of stem cells; they can be amplified into cell clones and induced to express the property of neural glia cells and neuron-like cells under proper condition.