bFGF对骨髓间充质干细胞向视网膜神经样细胞增殖分化的影响
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摘要
骨髓间充质干细胞(BMSCs)成为视网膜细胞移植的理想材料,通过对bFGF刺激骨髓间充质干细胞增殖分化后神经干细胞和神经细胞特异性标志物表达的检测,探讨bFGF在骨髓间充质干细胞向视网膜神经样细胞诱导分化过程中的作用。
体外分离提取骨髓细胞进行培养,传至第3代时经流式细胞仪检测细胞表面标志物CD90、CD71、CD29表达阳性,CD45表达阴性,免疫细胞化学方法鉴定CD71(+),CD90(+)。将BMSCs细胞分为两组,实验组分别加入浓度为10ng/ml,20ng/ml,30ng/ml的bFGF,对照组不加因子。培养9天后,免疫细胞化学染色检测实验组巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)表达阳性,对照组表达阴性。各组经RT-PCR检测均表达βⅢ-tubulin、nestin、NSE mRNA,实验组高于对照组,实验组各浓度组未见明显差异。两组细胞更换无血清的DMEM/F12培养基诱导2h后部分细胞成神经样形态改变。免疫细胞化学染色检测两组均表nestin、NSE;用RT-PCR方法均可检测到βⅢ-tubulin、nestin、NSE mRNA的表达,实验组表达高于对照组,实验组各浓度组未见明显差异。
对大鼠BMSCs进行有效的分离、培养可得到较均一的BMSCs。应用无血清培养基诱导的方法可将BMSCs诱导分化为神经样细胞。bFGF对BMSCs具有促进增殖的作用,并在向视网膜神经样细胞定向分化过程中起到促进作用。
Recently, the stem cell transplantation has attracted increasing attention in the retinal diseases regenerative therapy study. Therefore, seeking a new safe, effective and stable autologous cell source becomes more eager. The bone marrow mesenchymal stem cells (BMSCs) has strong point there in after: rich resource, easiness in obtaining materials, easy separation and purification culture, weak immunological rejection, high proliferative and differentiating plasticity, so it has been a hot study point as a kind of ideal material for cell transplantation. The contents of BMSCs are very few in bone marrow (part per 104~105), so we must find an effective way to proliferate cells in vitro for studying and application. Now, the dedifferentiation conditions of bone marrow mesenchymal stem cells to neuron-like cell remain uncertain, and the differentiation mechanism is unclear. We studied the effect of bFGF on the proliferation and differention of bone marrow mesenchymal stem cells to neuron-like cell by detecting the neural markers expression of differentiated cells. The results will provide theoretical and experimental basis for studying the mechanism of BMSCs committed differe- ntiation.
Methods proximal femurs of 10 days Wistar rats were isolated, bone marrow cells (BMSCs) were extracted from them and parified by attachment isolation. BMSCs had been cultured for 3rd generation, then cell surface markers were identified by flow cytometry, and markers CD71, CD90 of BMSCs were detected by immunocytochemical method. The third generation of BMSCs was divided into experimental group and control group. Experimental groups were added with three different concentration of bFGF (10ng/ml, 20ng/ml, 30ng/ml) and normal control group were cultured without bFGF. All groups were cultured in DMEM/12 medium with 10% fetal bovine serum for 9d.
The condition of BMSC’s growth and the cells growth activity in virto were observed under microscope in phase contrast method once daily. Nestin, NSE, were detected by immunocytochemical method and the relative expression of mRNA in Nestin, NSE andβIIItubulin were semi-quantitative detected by RT-PCR after being cultured for 9d. Part of the cultured cells was induced differentiation in serum-free DMEM/12 medium for 2h. The cell morphological changes after being induced were traced with inverted, Nestin, NSE, were detected by immunocytochemical and the relative expression of mRNA in Nestin, NSE andβIIItubulin were semi-quantitative detected by RT-PCR.
3rd generation adherent cells were detected by flow cytometry, the result showed that CD90, CD71 and CD29 were positive. Meanwhile, CD71 and CD90 were observed by immunocytochemistry. The cells proliferated fast after adding bFGF and were passaged every 2-3d, the cell bodies were slender and had high refraction. The neural markers expression of experimental groups and normal control group were detected by immunocytochemical, results showed that Nestin and NSE were positive in experimental groups, while they were negative in normal control group. Furthermore, the positive rate of experimental groups did not change significantly. mRNA was extracted to detect the expression of Nestin, NSE andβIIItubulin by RT-PCR. The result showed that the expressions of Nestin, NSE andβIIItubulin were observed in each group, and the concentration of markers in experimental groups was higher than normal control group, while it did not change significantly in each experimental group. Projection formed, cell body contracted and refraction enhanced could be observed after being induced 1h. Cells gradually changed to dendritic, bipolar or multipolar. Most cells had become to neuron-like shape in 2h. Compared with experimental groups, the change in normal control group appeared later, the number of neuron-like cells was also smaller, but the shape is similar. The neural markers expression of neuron-like cells cultured in serum-free medium were detected by immunocytochemical, results showed that Nestin and NSE were observed in each group. mRNA of cells cultured in serum-free medium was extracted to detect the expression of Nestin, NSE andβIIItubulin by RT-PCR. The result showed that the expressions of Nestin, NSE andβIIItubulin were observed in each group, and the concentration of markers in experimental groups was higher than normal control group, while it did not change significantly in each experimental group.
In this experiment, rat BMSCs were extracted and cultured effectively by attachment culture, early first time for the medium and digestion control method. 3rd generation adherent cells were detected by flow cytometry, the result showed that CD90, CD71 and CD29 were positive, while CD45 was negative, it suggested that the cells obtained were homogeneous BMSCs. BMSCs had been induced differentiation into cells in serum-free medium and cells obtained had representative neuron characters. Cells, which had been induced differentiation, were detected by immunocytochemical and RT-PCR, the result showed that the expression of neurons specific markers Nestin, NSE andβIIItubulin were observed. This suggested that these cells were neuron-like cells and had neuron characters. The neural markers expression of before and after being induced differentiation were detected by immunocytochemical and RT-PCR, the result showed that bFGF promoted proliferation and differentiation in the growth of BMSCs, it suggested that bFGF played an important role in the process of neural induction of the embryonic stem cells. All these results will play a fundamental role in the study of differentiation mechanism of the differention of BMSCs to neuron-like cell.
体外分离提取骨髓细胞进行培养,传至第3代时经流式细胞仪检测细胞表面标志物CD90、CD71、CD29表达阳性,CD45表达阴性,免疫细胞化学方法鉴定CD71(+),CD90(+)。将BMSCs细胞分为两组,实验组分别加入浓度为10ng/ml,20ng/ml,30ng/ml的bFGF,对照组不加因子。培养9天后,免疫细胞化学染色检测实验组巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)表达阳性,对照组表达阴性。各组经RT-PCR检测均表达βⅢ-tubulin、nestin、NSE mRNA,实验组高于对照组,实验组各浓度组未见明显差异。两组细胞更换无血清的DMEM/F12培养基诱导2h后部分细胞成神经样形态改变。免疫细胞化学染色检测两组均表nestin、NSE;用RT-PCR方法均可检测到βⅢ-tubulin、nestin、NSE mRNA的表达,实验组表达高于对照组,实验组各浓度组未见明显差异。
对大鼠BMSCs进行有效的分离、培养可得到较均一的BMSCs。应用无血清培养基诱导的方法可将BMSCs诱导分化为神经样细胞。bFGF对BMSCs具有促进增殖的作用,并在向视网膜神经样细胞定向分化过程中起到促进作用。
Recently, the stem cell transplantation has attracted increasing attention in the retinal diseases regenerative therapy study. Therefore, seeking a new safe, effective and stable autologous cell source becomes more eager. The bone marrow mesenchymal stem cells (BMSCs) has strong point there in after: rich resource, easiness in obtaining materials, easy separation and purification culture, weak immunological rejection, high proliferative and differentiating plasticity, so it has been a hot study point as a kind of ideal material for cell transplantation. The contents of BMSCs are very few in bone marrow (part per 104~105), so we must find an effective way to proliferate cells in vitro for studying and application. Now, the dedifferentiation conditions of bone marrow mesenchymal stem cells to neuron-like cell remain uncertain, and the differentiation mechanism is unclear. We studied the effect of bFGF on the proliferation and differention of bone marrow mesenchymal stem cells to neuron-like cell by detecting the neural markers expression of differentiated cells. The results will provide theoretical and experimental basis for studying the mechanism of BMSCs committed differe- ntiation.
Methods proximal femurs of 10 days Wistar rats were isolated, bone marrow cells (BMSCs) were extracted from them and parified by attachment isolation. BMSCs had been cultured for 3rd generation, then cell surface markers were identified by flow cytometry, and markers CD71, CD90 of BMSCs were detected by immunocytochemical method. The third generation of BMSCs was divided into experimental group and control group. Experimental groups were added with three different concentration of bFGF (10ng/ml, 20ng/ml, 30ng/ml) and normal control group were cultured without bFGF. All groups were cultured in DMEM/12 medium with 10% fetal bovine serum for 9d.
The condition of BMSC’s growth and the cells growth activity in virto were observed under microscope in phase contrast method once daily. Nestin, NSE, were detected by immunocytochemical method and the relative expression of mRNA in Nestin, NSE andβIIItubulin were semi-quantitative detected by RT-PCR after being cultured for 9d. Part of the cultured cells was induced differentiation in serum-free DMEM/12 medium for 2h. The cell morphological changes after being induced were traced with inverted, Nestin, NSE, were detected by immunocytochemical and the relative expression of mRNA in Nestin, NSE andβIIItubulin were semi-quantitative detected by RT-PCR.
3rd generation adherent cells were detected by flow cytometry, the result showed that CD90, CD71 and CD29 were positive. Meanwhile, CD71 and CD90 were observed by immunocytochemistry. The cells proliferated fast after adding bFGF and were passaged every 2-3d, the cell bodies were slender and had high refraction. The neural markers expression of experimental groups and normal control group were detected by immunocytochemical, results showed that Nestin and NSE were positive in experimental groups, while they were negative in normal control group. Furthermore, the positive rate of experimental groups did not change significantly. mRNA was extracted to detect the expression of Nestin, NSE andβIIItubulin by RT-PCR. The result showed that the expressions of Nestin, NSE andβIIItubulin were observed in each group, and the concentration of markers in experimental groups was higher than normal control group, while it did not change significantly in each experimental group. Projection formed, cell body contracted and refraction enhanced could be observed after being induced 1h. Cells gradually changed to dendritic, bipolar or multipolar. Most cells had become to neuron-like shape in 2h. Compared with experimental groups, the change in normal control group appeared later, the number of neuron-like cells was also smaller, but the shape is similar. The neural markers expression of neuron-like cells cultured in serum-free medium were detected by immunocytochemical, results showed that Nestin and NSE were observed in each group. mRNA of cells cultured in serum-free medium was extracted to detect the expression of Nestin, NSE andβIIItubulin by RT-PCR. The result showed that the expressions of Nestin, NSE andβIIItubulin were observed in each group, and the concentration of markers in experimental groups was higher than normal control group, while it did not change significantly in each experimental group.
In this experiment, rat BMSCs were extracted and cultured effectively by attachment culture, early first time for the medium and digestion control method. 3rd generation adherent cells were detected by flow cytometry, the result showed that CD90, CD71 and CD29 were positive, while CD45 was negative, it suggested that the cells obtained were homogeneous BMSCs. BMSCs had been induced differentiation into cells in serum-free medium and cells obtained had representative neuron characters. Cells, which had been induced differentiation, were detected by immunocytochemical and RT-PCR, the result showed that the expression of neurons specific markers Nestin, NSE andβIIItubulin were observed. This suggested that these cells were neuron-like cells and had neuron characters. The neural markers expression of before and after being induced differentiation were detected by immunocytochemical and RT-PCR, the result showed that bFGF promoted proliferation and differentiation in the growth of BMSCs, it suggested that bFGF played an important role in the process of neural induction of the embryonic stem cells. All these results will play a fundamental role in the study of differentiation mechanism of the differention of BMSCs to neuron-like cell.
引文
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