鹿茸多肽促骨髓间充质干细胞向神经元样细胞转化的实验研究
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摘要
目的:本文通过生物实验技术,研究以鹿茸多肽(Pilose antler polypeptides,PAP)为促诱导剂诱导大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向神经元样细胞分化的作用。并通过观察不同浓度的PAP体外促诱导BMSCs向神经元样细胞分化不同时段的情况,以找出最优诱导方案。为PAP更好的应用于临床提供更多的实验基础,为脊髓损伤提供新的治疗途径。
方法:在无菌条件下,取健康雄性SD大鼠的骨髓组织,用密度梯度离心法获取有核细胞层,接种于细胞培养瓶,放置于细胞培养箱中培养。通过换液传代去除非BMSCs,得到纯化的BMSCs。经细胞周期测定及鉴定其是否具有多项分化能力的方法鉴定,该细胞确为BMSCs。用以bFGF+EGF为基础加入不同剂量PAP的的培养液为实验组,与bFGF+EGF的基础培养液组及标准培养液组作为对照研究。分别在诱导后1,5,7d在倒置显微镜下观察细胞形态学变化,并在第7天时用透射电镜观察细胞形态学变化,再以免疫染色法鉴定神经元特异性烯醇化酶(NSE)、神经元细胞特异性标志神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达。
结果:在诱导前,大鼠BMSCs以长梭形为主,有些呈圆形、多角形,少数有短小突起。(l)在诱导1d后,倒置显微镜下可见部分细胞胞体收缩,折光性增强,伸出突起,部分胞体收缩成三角形或成为简单的双极或多极细胞。5d后较长的突起末端形成生长锥样的膨大,呈现神经元样的形态。7d后神经元样细胞数量增多,细胞突起延长。第7天时用透射电镜观察呈现神经细胞形态,神经元样的细胞数量进一步增多,且几个细胞的突起之间互相连接成网状。诱导7d后细胞表达NF、NSE但不表达GFAP。(2)在以bFGF+EGF+PAP为诱导剂诱导后BMSCs逐渐向神经元样细胞分化,并呈现神经元样细胞形态,在诱导7d后免疫染色法鉴定NSE、NF为阳性表达,GFAP为阴性表达。
结论:
1. PAP可促诱导BMSCs向神经元样细胞分化,分化过程是: BMSCs-神经干样细胞-神经元样细胞。
2. bFGF+EGF+PAP上清液可诱导大鼠BMSCs分化为神经元样细胞,其中PAP用量为40ug的诱导效果最佳,明显优于单纯使用诱导剂组,且诱导效果在一定程度上受PAP剂量的影响。
Objective: In this paper, biological experimental techniques, research polypeptide (Pilose antler polypeptides, PAP) to promote inducer inducing bone marrow mesenchymal stem cells (bone mesenchymal stem cells, BMSCs) into neuron-like cell differentiation. Through the effects of different concentrations of PAP in vitro induced BMSCs promote neuron-like cells to different periods, so as to identify the optimal induction program. Clinical application for the PAP to provide more and better experimental basis for spinal cord injury to provide a new therapeutic approach.
Methods: In aseptic conditions, taking healthy bone marrow of male SD rats by density gradient centrifugation to obtain nuclear cell layer, was inoculated in cell culture flask, placed in the cell incubator. The medium was changed to go through unless passage BMSCs, are purified BMSCs. Determined by the cell cycle and identification of whether the method has identified a number of differentiation, the cells are indeed BMSCs. For bFGF + EGF-based with different doses of PAP of the culture medium for the experimental group, and bFGF + EGF group based medium and standard culture medium as a control study group. Used to observe the 7th day showed nerve cell morphology, the number of neuron-like cells further increased, and the number of cells connected into a network between processes. 1,5,7 d after induction, respectively, under inverted microscope to examine cell morphology, and in the first 7 days was used to observe cell morphology, then immunostaining in the identification of neuron-specific enolase (NSE), neuron specific markers neurofilament (NF), glial fibrillary acidic protein (GFAP) expression.
Results: Before induction, rat BMSCs to the main spindle, and some were round, polygonal, a small number of short processes. (L) in the induction of 1d, the inverted microscope visible part of the cell body contraction, refraction, extended processes, some contraction of cell body, forming a triangle or a simple bipolar or multipolar cells. 5d longer processes after the formation of growth cone-like end of the enlargement, showing neuron-like morphology. 7d after the increased number of neuron-like cells, cell processes extend. 7d induced expression after NF, NSE, but cells did not express GFAP. (2) to bFGF + EGF + PAP for the induction agent BMSCs gradually after the induction of neuron-like cells, and showed neuron-like cell morphology, the induction of immune staining after 7d identified NSE, NF is positive, GFAP negative expression.
Conclusion:
1. PAP can promote the induction of BMSCs into neuron-like cells, differentiation is: BMSCs-neural stem-like cells - neuron-like cells.
2. BFGF + EGF + PAP supernatant can induce BMSCs to differentiate into neuron-like cells, which the dosage of 40ug PAP induced the best, better than only using the inductor group, and to some extent, induction dose by PAP effects.
方法:在无菌条件下,取健康雄性SD大鼠的骨髓组织,用密度梯度离心法获取有核细胞层,接种于细胞培养瓶,放置于细胞培养箱中培养。通过换液传代去除非BMSCs,得到纯化的BMSCs。经细胞周期测定及鉴定其是否具有多项分化能力的方法鉴定,该细胞确为BMSCs。用以bFGF+EGF为基础加入不同剂量PAP的的培养液为实验组,与bFGF+EGF的基础培养液组及标准培养液组作为对照研究。分别在诱导后1,5,7d在倒置显微镜下观察细胞形态学变化,并在第7天时用透射电镜观察细胞形态学变化,再以免疫染色法鉴定神经元特异性烯醇化酶(NSE)、神经元细胞特异性标志神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达。
结果:在诱导前,大鼠BMSCs以长梭形为主,有些呈圆形、多角形,少数有短小突起。(l)在诱导1d后,倒置显微镜下可见部分细胞胞体收缩,折光性增强,伸出突起,部分胞体收缩成三角形或成为简单的双极或多极细胞。5d后较长的突起末端形成生长锥样的膨大,呈现神经元样的形态。7d后神经元样细胞数量增多,细胞突起延长。第7天时用透射电镜观察呈现神经细胞形态,神经元样的细胞数量进一步增多,且几个细胞的突起之间互相连接成网状。诱导7d后细胞表达NF、NSE但不表达GFAP。(2)在以bFGF+EGF+PAP为诱导剂诱导后BMSCs逐渐向神经元样细胞分化,并呈现神经元样细胞形态,在诱导7d后免疫染色法鉴定NSE、NF为阳性表达,GFAP为阴性表达。
结论:
1. PAP可促诱导BMSCs向神经元样细胞分化,分化过程是: BMSCs-神经干样细胞-神经元样细胞。
2. bFGF+EGF+PAP上清液可诱导大鼠BMSCs分化为神经元样细胞,其中PAP用量为40ug的诱导效果最佳,明显优于单纯使用诱导剂组,且诱导效果在一定程度上受PAP剂量的影响。
Objective: In this paper, biological experimental techniques, research polypeptide (Pilose antler polypeptides, PAP) to promote inducer inducing bone marrow mesenchymal stem cells (bone mesenchymal stem cells, BMSCs) into neuron-like cell differentiation. Through the effects of different concentrations of PAP in vitro induced BMSCs promote neuron-like cells to different periods, so as to identify the optimal induction program. Clinical application for the PAP to provide more and better experimental basis for spinal cord injury to provide a new therapeutic approach.
Methods: In aseptic conditions, taking healthy bone marrow of male SD rats by density gradient centrifugation to obtain nuclear cell layer, was inoculated in cell culture flask, placed in the cell incubator. The medium was changed to go through unless passage BMSCs, are purified BMSCs. Determined by the cell cycle and identification of whether the method has identified a number of differentiation, the cells are indeed BMSCs. For bFGF + EGF-based with different doses of PAP of the culture medium for the experimental group, and bFGF + EGF group based medium and standard culture medium as a control study group. Used to observe the 7th day showed nerve cell morphology, the number of neuron-like cells further increased, and the number of cells connected into a network between processes. 1,5,7 d after induction, respectively, under inverted microscope to examine cell morphology, and in the first 7 days was used to observe cell morphology, then immunostaining in the identification of neuron-specific enolase (NSE), neuron specific markers neurofilament (NF), glial fibrillary acidic protein (GFAP) expression.
Results: Before induction, rat BMSCs to the main spindle, and some were round, polygonal, a small number of short processes. (L) in the induction of 1d, the inverted microscope visible part of the cell body contraction, refraction, extended processes, some contraction of cell body, forming a triangle or a simple bipolar or multipolar cells. 5d longer processes after the formation of growth cone-like end of the enlargement, showing neuron-like morphology. 7d after the increased number of neuron-like cells, cell processes extend. 7d induced expression after NF, NSE, but cells did not express GFAP. (2) to bFGF + EGF + PAP for the induction agent BMSCs gradually after the induction of neuron-like cells, and showed neuron-like cell morphology, the induction of immune staining after 7d identified NSE, NF is positive, GFAP negative expression.
Conclusion:
1. PAP can promote the induction of BMSCs into neuron-like cells, differentiation is: BMSCs-neural stem-like cells - neuron-like cells.
2. BFGF + EGF + PAP supernatant can induce BMSCs to differentiate into neuron-like cells, which the dosage of 40ug PAP induced the best, better than only using the inductor group, and to some extent, induction dose by PAP effects.
引文
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