肌萎缩侧索硬化G93A SOD1转基因小鼠脑内源性神经前体细胞增殖，迁移，分化的实验研究
发布时间：2019-01-05 07:37
【摘要】：目的:观察分析成年正常野生型小鼠及不同发病阶段G93A-SOD1 ALS转基因小鼠脑相关解剖区NPCs的增殖、迁移、分化情况,为进一步调控NPCs的增殖、定向迁移、分化提供理论依据,为治疗ALS提供可能途径。方法:构建G93A-SOD1 C57BL/6J转基因小鼠动物模型,用PCR技术检测筛选转基因小鼠动物模型。将G93A-SOD1转基因小鼠分为未发病阶段(60-70天),发病阶段(90-100天)和进展阶段(120-130天)。腹腔注射Brdu(5-溴脱氧尿核苷)标记野生型和G93A-SOD1 C57BL/6J转基因小鼠增殖的新生细胞。采用荧光免疫组织化学双标技术,用NPCs生物学标志物(Nestin,Vimentin)标记NPCs,用Brdu标记增殖的新生细胞,用NeuN标记神经元细胞,用GFAP标记星形胶质细胞,用Olig标记少突胶质细胞。在显微镜同一视野、同一放大倍数下,用图像处理技术将不同染色的阳性细胞两两叠加成像,观察是否存在共免疫反应。统计不同发病阶段不同解剖区阳性细胞数量,观察阳性细胞分布特征。分析野生型和G93A-SOD1转基因小鼠脑相关解剖区NPCs增殖、定向迁徙、分化特征。结果:1、VCCs广泛分布于成年野生型小鼠脑的多个部位,包括室管膜区、室管膜下区、海马、大脑皮层、嗅球。2、G93A-SOD1 ALS转基因小鼠脑干腹侧多个核团出现VCCs的表达,随着疾病的进展,VCCs阳性细胞数进行性增多,存在进展组起病组未发病组的规律。3、脑干增多的VCCs几乎全与GFAP存在共免疫反应性,提示VCCs几乎全部分化成星形胶质细胞。4、与正常对照相比,G93A-SOD1 ALS转基因小鼠的嗅皮层、扣带回、运动皮层的VCCs数量减少。5、增多的VCCs极少数BrdU阳性,提示增多的VCCs可能有两种来源:1)少数与BrdU存在共免疫反应的VCCs为增殖的新生细胞;2)大多数与BrdU不存在共免疫反应的VCCs可能是增殖分化晚期的NPCs,这部分VCCs也有可能是从其他部位迁移分化而来。6、在G93A-SOD1 ALS转基因小鼠脑桥与延髓,所有nestin阳性细胞均表达Vimentin,仅有一部分Vimentin阳性细胞与nestin存在共免疫反应,说明VCCs大部分是分化晚期的NPCs。结论:正常成年野生型小鼠脑内多个部位广泛存在大量NPCs。ALS的发病刺激NPCs在脑干腹侧多个核团大量增殖,小部分处于增殖早期,大部分处于增殖分化晚期,并大多分化为星形胶质细胞。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.8
本文编号：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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.8
【参考文献】
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1 徐仁O5;;肌萎缩侧索硬化的治疗研究进展[J];国外医学(老年医学分册);2006年04期
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