【摘要】： 本實(shí)驗(yàn)研究了NGF、RA及BMSCs培養(yǎng)上清誘導(dǎo)PC12細(xì)胞向神經(jīng)元分化在形態(tài)上的變化。結(jié)果顯示未經(jīng)處理的PC12細(xì)胞呈圓形、短梭形或三角形,有的細(xì)胞兩極有短突起。添加不同濃度的NGF及RA組的PC12細(xì)胞在培養(yǎng)24h后即有突起伸出,有的較長(zhǎng)增粗,其上還可見(jiàn)小的突起,類(lèi)似神經(jīng)元軸突。除軸突樣突起外,細(xì)胞還伸出多條樹(shù)突狀突起,長(zhǎng)短不一,突起數(shù)目不等。培養(yǎng)至72h,發(fā)現(xiàn)NGF和RA誘導(dǎo)組的細(xì)胞突起明顯增粗,且明顯比對(duì)照組伸長(zhǎng),以50ng/mlNGF組和1.0μg/ml RA組最為明顯,細(xì)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(xì)胞后,隨著NGF和RA使用劑量的增加,細(xì)胞突起長(zhǎng)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(shí),其細(xì)胞突起長(zhǎng)度和突起數(shù)目卻沒(méi)有50ng/mlNGF組和1.0μg/ml RA組明顯。隨著誘導(dǎo)時(shí)間的延長(zhǎng),2.0μg/ml濃度的RA即可導(dǎo)致細(xì)胞中黑色顆粒增多,胞體回縮,細(xì)胞老化,視野中多為細(xì)胞碎屑顆粒,有的細(xì)胞融合成片。BMSCs培養(yǎng)上清對(duì)PC12細(xì)胞的生長(zhǎng)分化影響作用較顯著。120μl BMSCs培養(yǎng)上清誘導(dǎo)PC12細(xì)胞48h后就使細(xì)胞分化,突起增粗,且比對(duì)照組伸長(zhǎng),培養(yǎng)至72h,發(fā)現(xiàn)BMSCs培養(yǎng)上清誘導(dǎo)組的細(xì)胞突起明顯比對(duì)照組伸長(zhǎng),細(xì)胞之間同樣也相互連接交織成網(wǎng),而且隨著B(niǎo)MSCs培養(yǎng)上清使用量的增加,細(xì)胞突起長(zhǎng)度和突起數(shù)目均增大和增多。將誘導(dǎo)72h的各組細(xì)胞用圖像分析系統(tǒng)測(cè)量分化細(xì)胞的細(xì)胞直徑和突起長(zhǎng)度,結(jié)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(jìn)分化細(xì)胞突起的生長(zhǎng),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(jìn)分化細(xì)胞突起的生長(zhǎng)。 本實(shí)驗(yàn)在觀察了NGF、RA及BMSCs培養(yǎng)上清誘導(dǎo)PC12細(xì)胞向神經(jīng)元分化在形態(tài)上變化的基礎(chǔ)上,利用常規(guī)免疫細(xì)胞化學(xué)染色方法探討了神經(jīng)元的標(biāo)志蛋白MAP2在用NGF、RA及BMSCs培養(yǎng)上清誘導(dǎo)PC12細(xì)胞72h后的陽(yáng)性表達(dá)情況。結(jié)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(yáng)性細(xì)胞數(shù)均明顯比對(duì)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(yáng)性細(xì)胞數(shù)也明顯比對(duì)照組增加,而且隨著加入培養(yǎng)上清量的增加,MAP2陽(yáng)性細(xì)胞數(shù)也增加。此結(jié)果進(jìn)一步證明了NGF、RA及BMSCs培養(yǎng)上清具有誘導(dǎo)PC12細(xì)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王維;刺參巖藻多糖對(duì)PC12細(xì)胞缺氧/復(fù)氧損傷的保護(hù)作用研究[D];山東大學(xué);2012年

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本文編號(hào)：2139610