血管内皮生长因子体外诱导大鼠骨髓基质细胞分化为神经元样细胞
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摘要
背景与目的
已有许多文献报道骨髓基质细胞(bone marrow stromal cells,BMSCs)在一定的体外条件下能分化为神经元样细胞。然而目前用以诱导BMSCs分化为神经元样细胞的诱导剂很少被应用于临床。研究者们对血管内皮生长因子(vascular endothelial growth factor,VEGF)体外诱导BMSCs分化为神经元样细胞的影响和作用机制还了解甚少。本实验旨在探索VEGF体外对BMSCs分化为神经元样细胞的影响作用,并确定实验中VEGF的最佳的诱导浓度。
方法
(1) BMSCs取自6～8周雄性和雌性Sprague-Dawley大鼠体内。细胞传代至第3代时,流式细胞分析鉴定BMSCs表面干细胞标志物CD90和造血干细胞标志物CD45。
(2) BMSCS经传代3次后经10ng/mL碱性成纤维细胞生长因子(basic fibroblast growth factor,b-FGF)预诱导24h。细胞被随机分为5,10,和20ng/mLVEGF处理组和未经VEGF处理的对照组。倒置相差显微镜(×200)下逐日观察细胞形态变化。
(3)诱导后的第3天和第10天采用免疫组织化学方法鉴定神经元标志物神经特异性烯醇化酶(neuron specific enolase,NSE),分别计数每20个随机视野下NSE阳性细胞总数。
结果
(1)培养第3天可见贴壁细胞,细胞形态各异,以圆形为主,少部分细胞呈三角形、梭形,有粗短突起。原代第7～10天,较多梭形细胞形成集落生长。传代培养2～3代后细胞形态变得较均一,细长梭形细胞较多,呈漩涡样生长,部分细胞呈扁平形。
(2)流式细胞分析结果示第3代BMSCs细胞表面干细胞标志物CD90(97.5%)阳性,而造血干细胞标志物CD45阴性。
(3)诱导第3天经5,10,和20ng/mLVEGF诱导的组别均可观察到细胞胞体回缩,呈锥形或圆形,胞体折光性增强,伸出较细长双或多极突起,并分出次级突起。而对照组则无此类似现象出现。
(4)诱导第10天,经10ng/mLVEGF处理的组别NSE阳性细胞数最多(P<0.05)。诱导第3天和第10天,对照组中NSE阳性细胞数均最少(P<0.05)。诱导第10天各组NSE阳性细胞数目均较第3天增多(P<0.05)。
结论
(1)在一定条件下,大鼠骨髓基质细胞体外可向神经元样细胞分化。
(2)本实验中VEGF能促进骨髓基质细胞体外向神经元样细胞分化;与5和20ng/mL相比,10ng/mL为最合适的浓度。
(3)VEGF可能具有应用于诱导BMSCs体外向神经元定向分化和修复中枢神经损伤和神经变性疾病的潜能。
Background and objective
Studies have demonstrated that bone marrow stromal cells (BMSCs) undergo neuronal differentiation under certain in vitro conditions.However, very few inducers of BMSCs neural differentiation have been used in clinical application.The effects and mechanism of vascular endothelial growth factor (VEGF) on neural differentiation of BMSCs in vitro remain poorly understood.In this experiment,we aimed at investigating the effect of VEGF on inducing BMSCs into neuron-like cells in vitro, and at determining the best VEGF concentration for experimental induction.
Methods
(1)BMSCs were harvested from 6～8-weeks-old male and female adult Sprague Dawley rats. The stem cell marker CD90 and the hematopoietic cell marker CD45 were detected by flow cytometry to identify the third passage of BMSCs.
(2)After 3 passages, the BMSCs were pre-induced with 10ng/mL basic fibroblast growth factor(b-FGF) for 24 hours,followed by randomly being assigned to 5,10,and 20ng/mL VEGF-treated groups,as well as the control groups,in which the BMSCs were not treated with VEGF.The morphological changes in BMSCs prior to and following VEGF induction was observed under a phase-contrast microscope (×200).
(3)Expression of NSE following induction was determined by immunocytochemistry after 3 and 10 days after induction.The total number of NSE-positive cells was quantified from 20 randomly selected visual fields (×200) per coverslip.
Results
(1)After 72 hours in culture, some adherent BMSCs were observed with varying appearances:primarily round cells accompanied by triangle and spindle-shaped cell bodies with short processes.At 7–10 days in primary culture,the majority of spindle-shaped cells formed colonies.After 2–3 passages,the BMSCs were relatively homogeneous in appearance,and the majority of cells were spindle-shaped and displayed a whirlpool pattern;some cells were large and flat.
(2)Flow cytometry demonstrated that BMSCs from the third passage were positive for the stem cell marker CD90 (97.5%) and negative for the hematopoietic cell marker CD45.
(3)Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20ng/mL VEGF. However, these changes were not observed in the control group.
(4)At 10 days after induction, the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group(P< 0.05).The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction(P<0.05).Moreover, the number of NSE-positive cells was greater at 10 days compared with 3 days after induction(P<0.05).
Conclusion
(1)rat BMSCs were capable of differentiating into neuronal-like cells.
(2)In this experiment,VEGF played an important role on inducing BMSCs into neuron-like cells in vitro and compared with 5 and 20ng/mL,10ng/mL was the most appropriate VEGF concentration.
(3)VEGF could be utilized to determine neuronal differentiation of BMSCs in vitro and repair central neural damage and neural degeneration.
已有许多文献报道骨髓基质细胞(bone marrow stromal cells,BMSCs)在一定的体外条件下能分化为神经元样细胞。然而目前用以诱导BMSCs分化为神经元样细胞的诱导剂很少被应用于临床。研究者们对血管内皮生长因子(vascular endothelial growth factor,VEGF)体外诱导BMSCs分化为神经元样细胞的影响和作用机制还了解甚少。本实验旨在探索VEGF体外对BMSCs分化为神经元样细胞的影响作用,并确定实验中VEGF的最佳的诱导浓度。
方法
(1) BMSCs取自6～8周雄性和雌性Sprague-Dawley大鼠体内。细胞传代至第3代时,流式细胞分析鉴定BMSCs表面干细胞标志物CD90和造血干细胞标志物CD45。
(2) BMSCS经传代3次后经10ng/mL碱性成纤维细胞生长因子(basic fibroblast growth factor,b-FGF)预诱导24h。细胞被随机分为5,10,和20ng/mLVEGF处理组和未经VEGF处理的对照组。倒置相差显微镜(×200)下逐日观察细胞形态变化。
(3)诱导后的第3天和第10天采用免疫组织化学方法鉴定神经元标志物神经特异性烯醇化酶(neuron specific enolase,NSE),分别计数每20个随机视野下NSE阳性细胞总数。
结果
(1)培养第3天可见贴壁细胞,细胞形态各异,以圆形为主,少部分细胞呈三角形、梭形,有粗短突起。原代第7～10天,较多梭形细胞形成集落生长。传代培养2～3代后细胞形态变得较均一,细长梭形细胞较多,呈漩涡样生长,部分细胞呈扁平形。
(2)流式细胞分析结果示第3代BMSCs细胞表面干细胞标志物CD90(97.5%)阳性,而造血干细胞标志物CD45阴性。
(3)诱导第3天经5,10,和20ng/mLVEGF诱导的组别均可观察到细胞胞体回缩,呈锥形或圆形,胞体折光性增强,伸出较细长双或多极突起,并分出次级突起。而对照组则无此类似现象出现。
(4)诱导第10天,经10ng/mLVEGF处理的组别NSE阳性细胞数最多(P<0.05)。诱导第3天和第10天,对照组中NSE阳性细胞数均最少(P<0.05)。诱导第10天各组NSE阳性细胞数目均较第3天增多(P<0.05)。
结论
(1)在一定条件下,大鼠骨髓基质细胞体外可向神经元样细胞分化。
(2)本实验中VEGF能促进骨髓基质细胞体外向神经元样细胞分化;与5和20ng/mL相比,10ng/mL为最合适的浓度。
(3)VEGF可能具有应用于诱导BMSCs体外向神经元定向分化和修复中枢神经损伤和神经变性疾病的潜能。
Background and objective
Studies have demonstrated that bone marrow stromal cells (BMSCs) undergo neuronal differentiation under certain in vitro conditions.However, very few inducers of BMSCs neural differentiation have been used in clinical application.The effects and mechanism of vascular endothelial growth factor (VEGF) on neural differentiation of BMSCs in vitro remain poorly understood.In this experiment,we aimed at investigating the effect of VEGF on inducing BMSCs into neuron-like cells in vitro, and at determining the best VEGF concentration for experimental induction.
Methods
(1)BMSCs were harvested from 6～8-weeks-old male and female adult Sprague Dawley rats. The stem cell marker CD90 and the hematopoietic cell marker CD45 were detected by flow cytometry to identify the third passage of BMSCs.
(2)After 3 passages, the BMSCs were pre-induced with 10ng/mL basic fibroblast growth factor(b-FGF) for 24 hours,followed by randomly being assigned to 5,10,and 20ng/mL VEGF-treated groups,as well as the control groups,in which the BMSCs were not treated with VEGF.The morphological changes in BMSCs prior to and following VEGF induction was observed under a phase-contrast microscope (×200).
(3)Expression of NSE following induction was determined by immunocytochemistry after 3 and 10 days after induction.The total number of NSE-positive cells was quantified from 20 randomly selected visual fields (×200) per coverslip.
Results
(1)After 72 hours in culture, some adherent BMSCs were observed with varying appearances:primarily round cells accompanied by triangle and spindle-shaped cell bodies with short processes.At 7–10 days in primary culture,the majority of spindle-shaped cells formed colonies.After 2–3 passages,the BMSCs were relatively homogeneous in appearance,and the majority of cells were spindle-shaped and displayed a whirlpool pattern;some cells were large and flat.
(2)Flow cytometry demonstrated that BMSCs from the third passage were positive for the stem cell marker CD90 (97.5%) and negative for the hematopoietic cell marker CD45.
(3)Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20ng/mL VEGF. However, these changes were not observed in the control group.
(4)At 10 days after induction, the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group(P< 0.05).The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction(P<0.05).Moreover, the number of NSE-positive cells was greater at 10 days compared with 3 days after induction(P<0.05).
Conclusion
(1)rat BMSCs were capable of differentiating into neuronal-like cells.
(2)In this experiment,VEGF played an important role on inducing BMSCs into neuron-like cells in vitro and compared with 5 and 20ng/mL,10ng/mL was the most appropriate VEGF concentration.
(3)VEGF could be utilized to determine neuronal differentiation of BMSCs in vitro and repair central neural damage and neural degeneration.
引文
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