井沆,肖雁,官志忠
JING Hang,XIAO Yan,GUAN Zhizhong

• 1:贵阳医学院分子生物学重点实验室
• 2:贵阳医学院
• 3:贵阳医学院病理学教研室

摘要(Abstract)：

目的:建立体外分离、培养大鼠骨髓间充质干细胞(BMSCs),体外诱导BMSCs向神经元样细胞方向分化、鉴定并移植至血管性痴呆大鼠模型的方法。方法:体外分离培养SD大鼠BMSCs,采用贴壁筛选法分离纯化BMSCs,显微镜下观察不同时期BMSCs的细胞形态,流式细胞术检测细胞表面标志物表达鉴定BMSCs,用含10μg/L bFGF的L-DMEM及含200μmol/L BHA、2%DMSO的无血清L-DMEM诱导BMSCs向神经元样细胞分化;免疫细胞化学检测分化细胞的巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)的表达,以确定其分化特性;用尿嘧啶脱氧核苷(BrdU)标记分化好的细胞,移植到改良Pulsinelli’s四血管复制的血管性痴呆(VaD)模型大鼠侧脑室内,免疫组织化学检测BrdU表达以反映移植BMSCs在大鼠脑内的生长情况,Morris水迷宫检测大鼠学习记忆能力。结果:大鼠BMSCs可通过贴壁筛选法成功分离并在体外大量扩增,流式细胞仪检测显示BMSCs第5代表面标志可达90%以上,细胞表型CD90、CD29、CD44表达阳性,CD45表达阴性;bFGF/BHA诱导BMSCs分化后有Nestin和NSE表达,分化细胞具有神经细胞的形态;与对照组相比,模型组大鼠逃避潜伏期明显延长,第1次穿越平台时间延长,穿越平台次数减少,差异有统计学意义(P<0.05);BrdU成功标记诱导后的BMSCs,并可在移植大鼠脑内检测到BrdU标记阳性细胞。结论:体外成功分离、培养大鼠BMSCs,生长稳定、可多次传代,bFGF/BHA可诱导BMSCs分化为神经元细胞,BMSCs成功移植到VaD大鼠,BMSCs有望为神经疾病细胞移植治疗提供丰富、易得的细胞来源。
Objective: To establish the method of isolation and culture of rat bone marrow mesenchymal stem cells( BMSCs) in vitro,and to induce them to differentiate into neuron like cell,identify them and transplant them into vascular dementia rats. Methods: BMSCs of SD rats were isolated and purified by adherent screening method. Morphology of BMSCs at different stages was observed under microscope. Cell membrane markers were detected by flow cytometry to identify BMSCs. BMSCs were induced to differentiate into neuron like cell by using L-DMEM including 10 μg / L bFGF and DMEM including 200 μmol / L BHA and 2 % DMSO. Immunocytochemical staining of neural markers such as NSE and Nestin was performed to determine the phenotype characteristics of the differentiated cells. After the cell was well differentiated and authenticated,Pyrimidine nucleoside( BrdU) was used to label well differentiated cells,which then were transplanted into lateral ventricle of vascular dementia( VaD) model rats established with improved Pulsinelli's four vessels. The expressions of BrdU and growth conditions of transplanted cells in rats were observed with immunohistochemistry staining. Abilities of learning and memory of rats were tested by using Morris water maze. Results: BMSCs was successfully isolated and amplified. 99% of the fifth generation of BMSCs expressed surface markers. CD90,CD28 and CD44 were positive,and CD45 was negative. The differentiated BMSCs induced by bFGF / BHA expressed NSE and Nestin and exhibited neuronal morphological characteristics. Compared with the control group,the rat escape latency period and the period for crossing the platform at the first time in model group significantly prolonged,the number of crossing the platform reduced( P < 0. 05). BrdU positive cells were detectable in rat brain. Conclusions: BMSCs are successfully isolated and cultured in vitro and grow stably and can passage time and again. BMSCs can be induced to differentiate into neuron-like cells by bFGF / BHA. BMSCs may provide cellular source for the treatment of neurologic diseases.

关键词(KeyWords)： 骨髓间充质干细胞;血管性痴呆;神经元;分化;诱导;移植
bone marrow mesenchymal stem cells;vascular dementia;neurons;differentiation;in-duction;transplantation

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(No.81160149);; 贵州省科技厅社会攻关计划项目[黔科合SY(2010)3076]

作者(Author): 井沆,肖雁,官志忠
JING Hang,XIAO Yan,GUAN Zhizhong

DOI: 10.19367/j.cnki.1000-2707.2014.02.003

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