诱导PC12细胞向神经元分化的实验研究
发布时间：2018-07-23 13:49
【摘要】： 本实验研究了NGF、RA及BMSCs培养上清诱导PC12细胞向神经元分化在形态上的变化。结果显示未经处理的PC12细胞呈圆形、短梭形或三角形,有的细胞两极有短突起。添加不同浓度的NGF及RA组的PC12细胞在培养24h后即有突起伸出,有的较长增粗,其上还可见小的突起,类似神经元轴突。除轴突样突起外,细胞还伸出多条树突状突起,长短不一,突起数目不等。培养至72h,发现NGF和RA诱导组的细胞突起明显增粗,且明显比对照组伸长,以50ng/mlNGF组和1.0μg/ml RA组最为明显,细胞之间相互连接交织成网。用10、20和50ng/mlNGF和0.1、0.3、0.5及1.0μg/mlRA处理PC12细胞后,随着NGF和RA使用剂量的增加,细胞突起长度和突起数目均增大和增多,而将NGF浓度增加到80ng/ml及RA浓度增加到2.0μg/ml和5.0μg/ml时,其细胞突起长度和突起数目却没有50ng/mlNGF组和1.0μg/ml RA组明显。随着诱导时间的延长,2.0μg/ml浓度的RA即可导致细胞中黑色颗粒增多,胞体回缩,细胞老化,视野中多为细胞碎屑颗粒,有的细胞融合成片。BMSCs培养上清对PC12细胞的生长分化影响作用较显著。120μl BMSCs培养上清诱导PC12细胞48h后就使细胞分化,突起增粗,且比对照组伸长,培养至72h,发现BMSCs培养上清诱导组的细胞突起明显比对照组伸长,细胞之间同样也相互连接交织成网,而且随着BMSCs培养上清使用量的增加,细胞突起长度和突起数目均增大和增多。将诱导72h的各组细胞用图像分析系统测量分化细胞的细胞直径和突起长度,结果发现10、20、50、80ng/mlNGF组和0.1、0.3、0.5、1.0、2.0及5.0μg/ml RA组均可促进分化细胞突起的生长,且以50ng/mlNGF组1.0μg/mlRA组为著;40、80、120及160μl BMSCs培养上清可促进分化细胞突起的生长。 本实验在观察了NGF、RA及BMSCs培养上清诱导PC12细胞向神经元分化在形态上变化的基础上,利用常规免疫细胞化学染色方法探讨了神经元的标志蛋白MAP2在用NGF、RA及BMSCs培养上清诱导PC12细胞72h后的阳性表达情况。结果发现10、20、50、80ng/mlNGF组和0.3、0.5、1.0、2.0μg/mL RA组MAP2阳性细胞数均明显比对照组增加,且以50ng/mlNGF组和1.0μg/mLRA组最为明显。40、80、120及160μl BMSCs培养上清组MAP2阳性细胞数也明显比对照组增加,而且随着加入培养上清量的增加,MAP2阳性细胞数也增加。此结果进一步证明了NGF、RA及BMSCs培养上清具有诱导PC12细胞向神经元分化的能力。
[Abstract]:The morphological changes of PC12 cells induced by NGF RA and BMSCs supernatants were studied in this study. The results showed that the untreated PC12 cells were round, fusiform or triangular, and some had short protuberances at the poles. After 24 hours of culture, PC12 cells with different concentrations of NGF and RA had protrusions, some of which were longer and thicker, and small processes, similar to neuronal axons, could be seen on them. In addition to axon-like protrusions, cells also extend multiple dendritic processes, varying in length and number. After 72 hours of culture, it was found that the cells in NGF and RA induced group were significantly thicker and longer than those in control group, especially in 50ng/mlNGF group and 1.0 渭 g/ml RA group, and the cells were connected and interwoven into a network. When PC12 cells were treated with 10 渭 g/mlRA, 50ng/mlNGF and 0.1 渭 g/mlRA, the length and number of processes increased with the increase of NGF and RA dosage, but when the NGF concentration increased to 2.0 渭 g/ml and 5.0 渭 g/ml, the concentration of 80ng/ml and RA increased to 2.0 渭 g/ml and 5.0 渭 g/ml. The length and number of protrusions were not obvious in 50ng/mlNGF group and 1.0 渭 g/ml RA group. With the prolongation of induction time, RA at the concentration of 2.0 渭 g/ml could lead to the increase of black particles, the retraction of cell bodies, the aging of cells, and the majority of cellular debris particles in the field of vision. The effect of supernatant of cell fusion. BMSCs culture supernatant on the growth and differentiation of PC12 cells was more significant. The supernatants of BMSCs culture induced PC12 cells to differentiate after 48 hours, and the processes were thicker and longer than those of the control group. After 72 hours of culture, it was found that the cell processes in the supernatant induced by BMSCs culture were significantly longer than those in the control group, and the cells were also intertwined with each other to form a network, and with the increase of the amount of supernatant used in the BMSCs culture, The length and number of processes increased. The cell diameter and process length of differentiated cells were measured by image analysis system at 72 h after induction. The results showed that the growth of differentiated cells could be promoted in 102050ng / ml NGF group, 0.3ng / ml NGF group and 0.1 渭 g/ml RA group. The supernatant cultured with 40 渭 l BMSCs and 160 渭 l BMSCs in 50ng/mlNGF group (1.0 渭 g/mlRA) could promote the growth of differentiated cell processes. In this study, we observed the morphological changes in the differentiation of PC12 cells into neurons induced by NGF RA and BMSCs supernatants. The positive expression of neuronal marker protein MAP2 in PC12 cells induced by NGFRA and BMSCs supernatants for 72 hours was investigated by routine immunocytochemical staining. The results showed that the number of MAP2 positive cells in NGF group and 0.1 渭 g/mL RA group was significantly higher than that in control group, and the number of MAP2 positive cells in 50ng/mlNGF group and 1.0 渭 g/mLRA group was significantly higher than that in control group, and the number of MAP2 positive cells in 50ng/mlNGF group and 1.0 渭 g/mLRA group was significantly higher than that in control group, and the number of MAP2 positive cells in 50ng/mlNGF group and 1.0 渭 g/mLRA group was significantly higher than that in control group. Moreover, the number of MAP2 positive cells increased with the addition of culture supernatant. The results further demonstrated that NGF RA and the supernatant of BMSCs could induce the differentiation of PC12 cells into neurons.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
本文编号：2139610
[Abstract]:The morphological changes of PC12 cells induced by NGF RA and BMSCs supernatants were studied in this study. The results showed that the untreated PC12 cells were round, fusiform or triangular, and some had short protuberances at the poles. After 24 hours of culture, PC12 cells with different concentrations of NGF and RA had protrusions, some of which were longer and thicker, and small processes, similar to neuronal axons, could be seen on them. In addition to axon-like protrusions, cells also extend multiple dendritic processes, varying in length and number. After 72 hours of culture, it was found that the cells in NGF and RA induced group were significantly thicker and longer than those in control group, especially in 50ng/mlNGF group and 1.0 渭 g/ml RA group, and the cells were connected and interwoven into a network. When PC12 cells were treated with 10 渭 g/mlRA, 50ng/mlNGF and 0.1 渭 g/mlRA, the length and number of processes increased with the increase of NGF and RA dosage, but when the NGF concentration increased to 2.0 渭 g/ml and 5.0 渭 g/ml, the concentration of 80ng/ml and RA increased to 2.0 渭 g/ml and 5.0 渭 g/ml. The length and number of protrusions were not obvious in 50ng/mlNGF group and 1.0 渭 g/ml RA group. With the prolongation of induction time, RA at the concentration of 2.0 渭 g/ml could lead to the increase of black particles, the retraction of cell bodies, the aging of cells, and the majority of cellular debris particles in the field of vision. The effect of supernatant of cell fusion. BMSCs culture supernatant on the growth and differentiation of PC12 cells was more significant. The supernatants of BMSCs culture induced PC12 cells to differentiate after 48 hours, and the processes were thicker and longer than those of the control group. After 72 hours of culture, it was found that the cell processes in the supernatant induced by BMSCs culture were significantly longer than those in the control group, and the cells were also intertwined with each other to form a network, and with the increase of the amount of supernatant used in the BMSCs culture, The length and number of processes increased. The cell diameter and process length of differentiated cells were measured by image analysis system at 72 h after induction. The results showed that the growth of differentiated cells could be promoted in 102050ng / ml NGF group, 0.3ng / ml NGF group and 0.1 渭 g/ml RA group. The supernatant cultured with 40 渭 l BMSCs and 160 渭 l BMSCs in 50ng/mlNGF group (1.0 渭 g/mlRA) could promote the growth of differentiated cell processes. In this study, we observed the morphological changes in the differentiation of PC12 cells into neurons induced by NGF RA and BMSCs supernatants. The positive expression of neuronal marker protein MAP2 in PC12 cells induced by NGFRA and BMSCs supernatants for 72 hours was investigated by routine immunocytochemical staining. The results showed that the number of MAP2 positive cells in NGF group and 0.1 渭 g/mL RA group was significantly higher than that in control group, and the number of MAP2 positive cells in 50ng/mlNGF group and 1.0 渭 g/mLRA group was significantly higher than that in control group, and the number of MAP2 positive cells in 50ng/mlNGF group and 1.0 渭 g/mLRA group was significantly higher than that in control group, and the number of MAP2 positive cells in 50ng/mlNGF group and 1.0 渭 g/mLRA group was significantly higher than that in control group. Moreover, the number of MAP2 positive cells increased with the addition of culture supernatant. The results further demonstrated that NGF RA and the supernatant of BMSCs could induce the differentiation of PC12 cells into neurons.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 王维;刺参岩藻多糖对PC12细胞缺氧/复氧损伤的保护作用研究[D];山东大学;2012年
，本文编号：2139610
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