肌萎縮側(cè)索硬化G93A SOD1轉(zhuǎn)基因小鼠腦內(nèi)源性神經(jīng)前體細(xì)胞增殖，遷移，分化的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2019-01-05 07:37
【摘要】：目的:觀察分析成年正常野生型小鼠及不同發(fā)病階段G93A-SOD1 ALS轉(zhuǎn)基因小鼠腦相關(guān)解剖區(qū)NPCs的增殖、遷移、分化情況,為進(jìn)一步調(diào)控NPCs的增殖、定向遷移、分化提供理論依據(jù),為治療ALS提供可能途徑。方法:構(gòu)建G93A-SOD1 C57BL/6J轉(zhuǎn)基因小鼠動(dòng)物模型,用PCR技術(shù)檢測(cè)篩選轉(zhuǎn)基因小鼠動(dòng)物模型。將G93A-SOD1轉(zhuǎn)基因小鼠分為未發(fā)病階段(60-70天),發(fā)病階段(90-100天)和進(jìn)展階段(120-130天)。腹腔注射Brdu(5-溴脫氧尿核苷)標(biāo)記野生型和G93A-SOD1 C57BL/6J轉(zhuǎn)基因小鼠增殖的新生細(xì)胞。采用熒光免疫組織化學(xué)雙標(biāo)技術(shù),用NPCs生物學(xué)標(biāo)志物(Nestin,Vimentin)標(biāo)記NPCs,用Brdu標(biāo)記增殖的新生細(xì)胞,用NeuN標(biāo)記神經(jīng)元細(xì)胞,用GFAP標(biāo)記星形膠質(zhì)細(xì)胞,用Olig標(biāo)記少突膠質(zhì)細(xì)胞。在顯微鏡同一視野、同一放大倍數(shù)下,用圖像處理技術(shù)將不同染色的陽(yáng)性細(xì)胞兩兩疊加成像,觀察是否存在共免疫反應(yīng)。統(tǒng)計(jì)不同發(fā)病階段不同解剖區(qū)陽(yáng)性細(xì)胞數(shù)量,觀察陽(yáng)性細(xì)胞分布特征。分析野生型和G93A-SOD1轉(zhuǎn)基因小鼠腦相關(guān)解剖區(qū)NPCs增殖、定向遷徙、分化特征。結(jié)果:1、VCCs廣泛分布于成年野生型小鼠腦的多個(gè)部位,包括室管膜區(qū)、室管膜下區(qū)、海馬、大腦皮層、嗅球。2、G93A-SOD1 ALS轉(zhuǎn)基因小鼠腦干腹側(cè)多個(gè)核團(tuán)出現(xiàn)VCCs的表達(dá),隨著疾病的進(jìn)展,VCCs陽(yáng)性細(xì)胞數(shù)進(jìn)行性增多,存在進(jìn)展組起病組未發(fā)病組的規(guī)律。3、腦干增多的VCCs幾乎全與GFAP存在共免疫反應(yīng)性,提示VCCs幾乎全部分化成星形膠質(zhì)細(xì)胞。4、與正常對(duì)照相比,G93A-SOD1 ALS轉(zhuǎn)基因小鼠的嗅皮層、扣帶回、運(yùn)動(dòng)皮層的VCCs數(shù)量減少。5、增多的VCCs極少數(shù)BrdU陽(yáng)性,提示增多的VCCs可能有兩種來(lái)源:1)少數(shù)與BrdU存在共免疫反應(yīng)的VCCs為增殖的新生細(xì)胞;2)大多數(shù)與BrdU不存在共免疫反應(yīng)的VCCs可能是增殖分化晚期的NPCs,這部分VCCs也有可能是從其他部位遷移分化而來(lái)。6、在G93A-SOD1 ALS轉(zhuǎn)基因小鼠腦橋與延髓,所有nestin陽(yáng)性細(xì)胞均表達(dá)Vimentin,僅有一部分Vimentin陽(yáng)性細(xì)胞與nestin存在共免疫反應(yīng),說(shuō)明VCCs大部分是分化晚期的NPCs。結(jié)論:正常成年野生型小鼠腦內(nèi)多個(gè)部位廣泛存在大量NPCs。ALS的發(fā)病刺激NPCs在腦干腹側(cè)多個(gè)核團(tuán)大量增殖,小部分處于增殖早期,大部分處于增殖分化晚期,并大多分化為星形膠質(zhì)細(xì)胞。
[Abstract]:Objective: to observe and analyze the proliferation, migration and differentiation of NPCs in brain related anatomical regions of adult normal wild type mice and G93A-SOD1 ALS transgenic mice in different stages, so as to provide theoretical basis for further regulating the proliferation, directional migration and differentiation of NPCs. To provide a possible way for the treatment of ALS. Methods: G93A-SOD1 C57BL/6J transgenic mouse model was constructed, and the transgenic mouse model was screened by PCR assay. The G93A-SOD1 transgenic mice were divided into three stages: non-onset stage (60-70 days), pathogenetic stage (90-100 days) and progressive stage (120-130 days). Brdu (5-bromodeoxyuridine) labeled newborn cells of wild type and G93A-SOD1 C57BL/6J transgenic mice were injected intraperitoneally. NPCs biomarkers (Nestin,Vimentin) were used to label the proliferating neonate cells with Brdu, neuron cells with NeuN, astrocytes with GFAP and oligodendrocytes with Olig. In the same field of view and magnification of microscope, the image processing technique was used to image the positive cells with different staining, and to observe the existence of co-immune reaction. The number of positive cells in different anatomical regions and the distribution characteristics of positive cells were observed. The characteristics of NPCs proliferation, migration and differentiation in brain related anatomical regions of wild-type and G93A-SOD1 transgenic mice were analyzed. Results: 1VCCs were widely distributed in many parts of brain of adult wild-type mice, including ependymal area, subependymal area, hippocampus, cerebral cortex and olfactory bulb. 2 the expression of VCCs was found in ventral nuclei of brainstem of G93A-SOD1 ALS transgenic mice. With the progression of the disease, the number of VCCs positive cells increased gradually, and the regularity of no onset group in the progressive group. 3. The VCCs with increased brainstem almost all had co-immunoreactivity with GFAP, suggesting that almost all VCCs differentiated into astrocytes. 4. Compared with the normal control, the number of VCCs in olfactory cortex, cingulate cortex and motor cortex of G93A-SOD1 ALS transgenic mice was decreased by 0.5, and the number of VCCs positive in increased VCCs was very few. These results suggest that there may be two sources of increased VCCs: 1) a few VCCs with co-immunoreaction with BrdU are newly proliferated cells; 2) most of the VCCs which did not co-immunize with BrdU may be the NPCs, in the late stage of proliferation and differentiation, and this part of VCCs may also migrate and differentiate from other sites. 6, in the pons and medulla oblongata of G93A-SOD1 ALS transgenic mice. All nestin positive cells expressed Vimentin,. Only a part of Vimentin positive cells had co-immunoreaction with nestin, indicating that most of VCCs was NPCs. in late differentiation. Conclusion: a large number of NPCs.ALS stimulates the proliferation of NPCs in the ventral nucleus of brain stem in normal adult wild-type mice, a small number of them are in the early stage of proliferation, and most of them are in the late stage of proliferation and differentiation. And most of them differentiate into astrocytes.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R744.8
本文編號(hào)：2401480
[Abstract]:Objective: to observe and analyze the proliferation, migration and differentiation of NPCs in brain related anatomical regions of adult normal wild type mice and G93A-SOD1 ALS transgenic mice in different stages, so as to provide theoretical basis for further regulating the proliferation, directional migration and differentiation of NPCs. To provide a possible way for the treatment of ALS. Methods: G93A-SOD1 C57BL/6J transgenic mouse model was constructed, and the transgenic mouse model was screened by PCR assay. The G93A-SOD1 transgenic mice were divided into three stages: non-onset stage (60-70 days), pathogenetic stage (90-100 days) and progressive stage (120-130 days). Brdu (5-bromodeoxyuridine) labeled newborn cells of wild type and G93A-SOD1 C57BL/6J transgenic mice were injected intraperitoneally. NPCs biomarkers (Nestin,Vimentin) were used to label the proliferating neonate cells with Brdu, neuron cells with NeuN, astrocytes with GFAP and oligodendrocytes with Olig. In the same field of view and magnification of microscope, the image processing technique was used to image the positive cells with different staining, and to observe the existence of co-immune reaction. The number of positive cells in different anatomical regions and the distribution characteristics of positive cells were observed. The characteristics of NPCs proliferation, migration and differentiation in brain related anatomical regions of wild-type and G93A-SOD1 transgenic mice were analyzed. Results: 1VCCs were widely distributed in many parts of brain of adult wild-type mice, including ependymal area, subependymal area, hippocampus, cerebral cortex and olfactory bulb. 2 the expression of VCCs was found in ventral nuclei of brainstem of G93A-SOD1 ALS transgenic mice. With the progression of the disease, the number of VCCs positive cells increased gradually, and the regularity of no onset group in the progressive group. 3. The VCCs with increased brainstem almost all had co-immunoreactivity with GFAP, suggesting that almost all VCCs differentiated into astrocytes. 4. Compared with the normal control, the number of VCCs in olfactory cortex, cingulate cortex and motor cortex of G93A-SOD1 ALS transgenic mice was decreased by 0.5, and the number of VCCs positive in increased VCCs was very few. These results suggest that there may be two sources of increased VCCs: 1) a few VCCs with co-immunoreaction with BrdU are newly proliferated cells; 2) most of the VCCs which did not co-immunize with BrdU may be the NPCs, in the late stage of proliferation and differentiation, and this part of VCCs may also migrate and differentiate from other sites. 6, in the pons and medulla oblongata of G93A-SOD1 ALS transgenic mice. All nestin positive cells expressed Vimentin,. Only a part of Vimentin positive cells had co-immunoreaction with nestin, indicating that most of VCCs was NPCs. in late differentiation. Conclusion: a large number of NPCs.ALS stimulates the proliferation of NPCs in the ventral nucleus of brain stem in normal adult wild-type mice, a small number of them are in the early stage of proliferation, and most of them are in the late stage of proliferation and differentiation. And most of them differentiate into astrocytes.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R744.8
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 徐仁O5;;肌萎縮側(cè)索硬化的治療研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(老年醫(yī)學(xué)分冊(cè));2006年04期
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