摘要
背景:选择一种高效、细胞损伤低的诱导人脐血间充质干细胞分化为神经细胞的方法是其应用于临床的前提。目的:拟采用传统中药香丹注射液联合生长因子诱导脐血间充质干细胞向神经细胞方向分化,并且与单纯生长因子的诱导作用进行比较。设计、时间及地点:免疫细胞化学水平的细胞观察实验,于2006-09/2008-04在潍坊医学院组织胚胎学教研室完成。材料:人脐血标本取自潍坊市妇幼保健院,香丹注射液的有效成分为丹参素。方法:采用密度梯度离心法分离人脐血单个核细胞,贴壁法筛选出人脐血间充质干细胞,体外扩增,以免疫荧光方法检测表面抗原。分为2组进行诱导分化,香丹注射液联合生长因子诱导组采用30g/L香丹注射液联合表皮生长因子和碱性成纤维细胞生长因子诱导脐血间充质干细胞向神经细胞方向分化,并且与单纯生长因子诱导组进行比较。主要观察指标:采用免疫细胞化学方法和免疫荧光方法检测诱导前后神经元特异性标志(神经元核抗原、β-TubulinⅢ)和神经胶质纤维酸性蛋白的表达。结果:香丹注射液联合生长因子诱导法对脐血间充质干细胞损伤作用小,诱导效率高。脐血间充质干细胞经香丹注射液诱导后出现类似神经细胞的形态改变,胞体呈椭圆形,伸出长突起。免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能特异性表达神经元核抗原和β-TubulinⅢ,而神经胶质纤维酸性蛋白阳性细胞较少。香丹注射液联合生长因子诱导组的β-TubulinⅢ和神经元核抗原的阳性细胞率显著高于生长因子诱导组(P<0.05),神经胶质纤维酸性蛋白阳性细胞率明显低于生长因子诱导组(P<0.05)。结论:香丹注射液联合生长因子诱导对脐血间充质干细胞损伤作用小,诱导效率高,优于生长因子诱导法。且体外诱导后细胞主要分化为神经元样细胞,而非星形胶质细胞。
BACKGROUND： Selecting an effective, optimal condition of human umbilical cord blood mesenchymal stem cells （UCB-MSCs） differentiation into neural cells was the prerequisite for clinical applications. OBJECTIVE： To study the effect of Xiangdan injection and growth factors on directional differentiation of UCB-MSCs into neural ceils, and to compare with the effect of simple growth factor induction. DESIGN, TIME AND SETTING： The immunocytochemistry study was conducted at the Department of Histology and Embryology, Weifan Medical College between September 2006 and April 2008. MATERIALS： The umbilical cord blood was collected at Weifang Woman and Infants Nursing Center. The effective integrant of Xiangdan injection was tanshinol. METHODS： The umbilical cord blood mononuclear cells were isolated by density gradient centrifugation. Human UCB-MSCs was purified by the adherence screening, and amplified in vitro, the cell surface marker was detected by immunofluorescence method. 30 g/L Xiangdan injection with epidermal growth factor and basic fibroblast growth factor was used to induce differentiation of MSCs into neural cells in the Xiangdan injection ＋ growth factor group, and the results were compared with growth factor induction group. MAIN OUTCOME MEASURES： The expression of neuron-specific marker （NeuN, β -TubutinⅢ） and gliat fibrillary acidic protein was measured using immunocytochemical and immunofluorescence methods before and after induction. RESULTS： Xiangclan injection combined with growth factors had small injuries to UCB-MSCs, with high induction efficiency. UCB-MSCs displayed neuronal morphologies and there were long processes out from the cell body when the MSCs were induced by Xiangdan injection. Immunohistochemical and immunofluorescence methods showed that induced cells derived from MSCs expressed NeuN and β -TubulinⅢ, but few expressed glial fibrillary acidic protein. The positive cell rates of NeuN and β - TubulinⅢ were significantly higher in the Xiangdan injection ＋
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第14期2656-2660,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
山东省自然科学基金项目(Y2008C26)
山东省教育厅资助项目(J07YD01)~~
