誘導PC12細胞向神經(jīng)元分化的實驗研究
發(fā)布時間：2018-07-23 13:49
【摘要】： 本實驗研究了NGF、RA及BMSCs培養(yǎng)上清誘導PC12細胞向神經(jīng)元分化在形態(tài)上的變化。結果顯示未經(jīng)處理的PC12細胞呈圓形、短梭形或三角形,有的細胞兩極有短突起。添加不同濃度的NGF及RA組的PC12細胞在培養(yǎng)24h后即有突起伸出,有的較長增粗,其上還可見小的突起,類似神經(jīng)元軸突。除軸突樣突起外,細胞還伸出多條樹突狀突起,長短不一,突起數(shù)目不等。培養(yǎng)至72h,發(fā)現(xiàn)NGF和RA誘導組的細胞突起明顯增粗,且明顯比對照組伸長,以50ng/mlNGF組和1.0μg/ml RA組最為明顯,細胞之間相互連接交織成網(wǎng)。用10、20和50ng/mlNGF和0.1、0.3、0.5及1.0μg/mlRA處理PC12細胞后,隨著NGF和RA使用劑量的增加,細胞突起長度和突起數(shù)目均增大和增多,而將NGF濃度增加到80ng/ml及RA濃度增加到2.0μg/ml和5.0μg/ml時,其細胞突起長度和突起數(shù)目卻沒有50ng/mlNGF組和1.0μg/ml RA組明顯。隨著誘導時間的延長,2.0μg/ml濃度的RA即可導致細胞中黑色顆粒增多,胞體回縮,細胞老化,視野中多為細胞碎屑顆粒,有的細胞融合成片。BMSCs培養(yǎng)上清對PC12細胞的生長分化影響作用較顯著。120μl BMSCs培養(yǎng)上清誘導PC12細胞48h后就使細胞分化,突起增粗,且比對照組伸長,培養(yǎng)至72h,發(fā)現(xiàn)BMSCs培養(yǎng)上清誘導組的細胞突起明顯比對照組伸長,細胞之間同樣也相互連接交織成網(wǎng),而且隨著BMSCs培養(yǎng)上清使用量的增加,細胞突起長度和突起數(shù)目均增大和增多。將誘導72h的各組細胞用圖像分析系統(tǒng)測量分化細胞的細胞直徑和突起長度,結果發(fā)現(xiàn)10、20、50、80ng/mlNGF組和0.1、0.3、0.5、1.0、2.0及5.0μg/ml RA組均可促進分化細胞突起的生長,且以50ng/mlNGF組1.0μg/mlRA組為著;40、80、120及160μl BMSCs培養(yǎng)上清可促進分化細胞突起的生長。 本實驗在觀察了NGF、RA及BMSCs培養(yǎng)上清誘導PC12細胞向神經(jīng)元分化在形態(tài)上變化的基礎上,利用常規(guī)免疫細胞化學染色方法探討了神經(jīng)元的標志蛋白MAP2在用NGF、RA及BMSCs培養(yǎng)上清誘導PC12細胞72h后的陽性表達情況。結果發(fā)現(xiàn)10、20、50、80ng/mlNGF組和0.3、0.5、1.0、2.0μg/mL RA組MAP2陽性細胞數(shù)均明顯比對照組增加,且以50ng/mlNGF組和1.0μg/mLRA組最為明顯。40、80、120及160μl BMSCs培養(yǎng)上清組MAP2陽性細胞數(shù)也明顯比對照組增加,而且隨著加入培養(yǎng)上清量的增加,MAP2陽性細胞數(shù)也增加。此結果進一步證明了NGF、RA及BMSCs培養(yǎng)上清具有誘導PC12細胞向神經(jīng)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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329
本文編號：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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329
【引證文獻】
相關碩士學位論文 前1條
1 王維;刺參巖藻多糖對PC12細胞缺氧/復氧損傷的保護作用研究[D];山東大學;2012年
，本文編號：2139610
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2139610.html
最近更新
教材專著
