SHH信號(hào)通路成分在成年大鼠脊髓損傷后的表達(dá)研究
發(fā)布時(shí)間：2019-06-22 12:47
【摘要】：目的：在大鼠脊髓組織受到急性損傷后,觀察脊髓內(nèi)源性神經(jīng)前體細(xì)胞的增殖與分化規(guī)律,觀察與神經(jīng)前體細(xì)胞生長(zhǎng)發(fā)育相關(guān)的Sonic Hedgehog (SHH)信號(hào)通路成分SHH和Gli-1分子的表達(dá)變化,探討成年大鼠脊髓損傷后內(nèi)源性神經(jīng)前體細(xì)胞分化調(diào)控的分子機(jī)制過(guò)程。方法：構(gòu)建大鼠脊髓急性損傷模型,運(yùn)用隨機(jī)數(shù)表法將健康成年大鼠分為正常組,假手術(shù)組與脊髓損傷各時(shí)間組,用5-溴-2-脫氧尿嘧啶核苷(BrdU)示蹤劑、膠質(zhì)原纖維酸性蛋白(glial fibrillary acidic protein、GFAP)、髓鞘堿性蛋白(myelin basic protein,MBP)分別標(biāo)記神經(jīng)前體細(xì)胞、星形膠質(zhì)細(xì)胞與少突膠質(zhì)細(xì)胞,通過(guò)免疫熒光技術(shù)觀察不同組BrdU標(biāo)記陽(yáng)性細(xì)胞數(shù)和BrdU/GFAP、 BrdU/MBP雙標(biāo)陽(yáng)性細(xì)胞數(shù)目的變化趨勢(shì),探討脊髓損傷后神經(jīng)前體細(xì)胞的增殖分化規(guī)律。此外,應(yīng)用實(shí)時(shí)熒光定量PCR技術(shù)和Western blotting技術(shù)檢測(cè)大鼠各組SHH信號(hào)通路成分SHH和Gli-1信號(hào)分子的變化趨勢(shì),探討SHH信號(hào)通路在大鼠脊髓損傷后的調(diào)控作用。結(jié)果：對(duì)于神經(jīng)前體細(xì)胞的研究發(fā)現(xiàn),正常組及假手術(shù)組大鼠脊髓均可觀察到少量BrdU陽(yáng)性細(xì)胞以及BrdU/GFAP、BrdU/MBP雙標(biāo)陽(yáng)性細(xì)胞,而脊髓損傷后BrdU陽(yáng)性細(xì)胞與雙標(biāo)陽(yáng)性細(xì)胞的表達(dá)呈現(xiàn)出顯著增高的趨勢(shì)(P0.05)。此外,對(duì)于SHH信號(hào)通路成分的研究發(fā)現(xiàn),正常大鼠脊髓低度表達(dá)SHH和Gli-1信號(hào)分子,脊髓損傷后SHH和Gli-1的表達(dá)量持續(xù)升高,并在損傷后第7天達(dá)到峰值,脊髓損傷后第21天仍然處于高水平表達(dá)(P0.05)。結(jié)論：大鼠脊髓損傷神經(jīng)前體細(xì)胞增殖明顯增多并向星形膠質(zhì)細(xì)胞與少突膠質(zhì)細(xì)胞大量分化。在健康正常大鼠可觀察到SHH信號(hào)轉(zhuǎn)導(dǎo)通路成分的表達(dá),脊髓損傷后,SHH和Gli-1蛋白的表達(dá)明顯增多,其變化規(guī)律與神經(jīng)前體細(xì)胞的增殖變化規(guī)律基本一致,提示SHH信號(hào)通路可能參與脊髓損傷后神經(jīng)細(xì)胞修復(fù)的調(diào)控作用。
[Abstract]:Aim: to observe the proliferation and differentiation of intrinsic neural progenitor cells in spinal cord after acute spinal cord injury, to observe the expression of SHH and Gli-1, which are related to the growth and development of neural progenitor cells, and to explore the molecular mechanism of differentiation regulation of endogenous neural progenitor cells after spinal cord injury in adult rats. Methods: the rat model of acute spinal cord injury was established. Healthy adult rats were divided into normal group, pseudo-operation group and spinal cord injury time group. 5-bromo-2-deoxyuridine (BrdU) tracer, glial fibrillar acidic protein (glial fibrillary acidic protein,GFAP), myelin basic protein (myelin basic protein,MBP) were used to label neural precursor cells, astrocytes and oligodendrocytes, respectively. The number of BrdU labeled positive cells and the number of BrdU/GFAP, BrdU/MBP double labeled positive cells in different groups were observed by immunofluorescence technique, and the proliferation and differentiation of neural progenitor cells after spinal cord injury were investigated. In addition, real-time fluorescence quantitative PCR and Western blotting techniques were used to detect the changing trend of SHH and Gli-1 signal molecules in SHH signaling pathway in rats, and to explore the regulatory effect of SHH signaling pathway after spinal cord injury in rats. Results: a small number of BrdU positive cells and BrdU/GFAP,BrdU/MBP double labeled positive cells were observed in the spinal cord of normal group and false operation group, while the expression of BrdU positive cells and double labeled positive cells increased significantly after spinal cord injury (P 0.05). In addition, the study of SHH signaling pathway components found that the low expression of SHH and Gli-1 signal molecules in the spinal cord of normal rats, the expression of SHH and Gli-1 continued to increase after spinal cord injury, and reached the peak on the 7th day after injury, and remained at a high level on the 21st day after spinal cord injury (P 0.05). Conclusion: the proliferation of neural progenitor cells in spinal cord injury in rats is significantly increased and differentiated into astrocytes and oligodendrocytes. The expression of SHH signal transduction pathway components could be observed in healthy rats. The expression of SHH and Gli-1 protein increased significantly after spinal cord injury, which was basically consistent with the proliferation of neural progenitor cells, suggesting that SHH signaling pathway may be involved in the regulation of nerve cell repair after spinal cord injury.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.2
本文編號(hào)：2504595
[Abstract]:Aim: to observe the proliferation and differentiation of intrinsic neural progenitor cells in spinal cord after acute spinal cord injury, to observe the expression of SHH and Gli-1, which are related to the growth and development of neural progenitor cells, and to explore the molecular mechanism of differentiation regulation of endogenous neural progenitor cells after spinal cord injury in adult rats. Methods: the rat model of acute spinal cord injury was established. Healthy adult rats were divided into normal group, pseudo-operation group and spinal cord injury time group. 5-bromo-2-deoxyuridine (BrdU) tracer, glial fibrillar acidic protein (glial fibrillary acidic protein,GFAP), myelin basic protein (myelin basic protein,MBP) were used to label neural precursor cells, astrocytes and oligodendrocytes, respectively. The number of BrdU labeled positive cells and the number of BrdU/GFAP, BrdU/MBP double labeled positive cells in different groups were observed by immunofluorescence technique, and the proliferation and differentiation of neural progenitor cells after spinal cord injury were investigated. In addition, real-time fluorescence quantitative PCR and Western blotting techniques were used to detect the changing trend of SHH and Gli-1 signal molecules in SHH signaling pathway in rats, and to explore the regulatory effect of SHH signaling pathway after spinal cord injury in rats. Results: a small number of BrdU positive cells and BrdU/GFAP,BrdU/MBP double labeled positive cells were observed in the spinal cord of normal group and false operation group, while the expression of BrdU positive cells and double labeled positive cells increased significantly after spinal cord injury (P 0.05). In addition, the study of SHH signaling pathway components found that the low expression of SHH and Gli-1 signal molecules in the spinal cord of normal rats, the expression of SHH and Gli-1 continued to increase after spinal cord injury, and reached the peak on the 7th day after injury, and remained at a high level on the 21st day after spinal cord injury (P 0.05). Conclusion: the proliferation of neural progenitor cells in spinal cord injury in rats is significantly increased and differentiated into astrocytes and oligodendrocytes. The expression of SHH signal transduction pathway components could be observed in healthy rats. The expression of SHH and Gli-1 protein increased significantly after spinal cord injury, which was basically consistent with the proliferation of neural progenitor cells, suggesting that SHH signaling pathway may be involved in the regulation of nerve cell repair after spinal cord injury.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.2
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