CaMPARI在三叉神经血管源性偏头痛大鼠模型中的应用研究
发布时间：2019-04-15 21:41
【摘要】：研究背景偏头痛的发病机制尚未完全清楚,三叉神经血管痛觉通路激活被认为是最能全面揭示偏头痛发病进展机制的学说,这种学说认为在偏头痛大鼠模型中,三叉神经节作为经典上行疼痛传导通路的起始部位,它的激活在偏头痛的产生和维持过程中起关键作用。然而目前关于三叉神经节细胞的激活研究尚不清楚。传统的方法证实三叉神经节细胞的激活可引起细胞内钙离子一次爆炸性的升高,且有高度的细胞特异性,但在偏头痛发病进展过程中,具体如何激活,激活顺序等尚不清楚。CaMPARI荧光蛋白因为其对胞内钙离子高度敏感,且具有不可逆转变为紫红色荧光的特性,故可用来给激活的三叉神经元作标记,即可实现将瞬间的神经元激活转变为永久的荧光蛋白的表达。然而目前,CaMPARI是2015年新合成的一段基因序列,仅被证实可在斑马鱼、果蝇、小鼠模式生物组织中表达,它是否可用通过腺相关病毒转导至大鼠三叉神经节细胞,从而为我们研究三叉神经节细胞激活在偏头痛大鼠模型发病进展中的作用提供一个新的思路呢?这是本研究想要解决的问题。目的CaMPARI是否可通过腺相关病毒转导办法表达在大鼠三叉神经节细胞。方法1.验证空载AAV-9-hSyn-GFP可成功转染至大鼠三叉神经节细胞首先使用亚甲蓝确定200-250gSD雄性大鼠三叉神经节的立体定位注射坐标,将空载 AAV-hSyn-2-GFP、AAV-hSyn-8-GFP、AAV-hSyn-9-GFP 三个血清型分别以不同剂量2uL,5ul,8uL直接注射至大鼠三叉神经节细胞。对照组大鼠给予等量生理盐水注射。2-3周后,取材冰冻切片于荧光显微镜下观察GFP绿色荧光蛋白表达情况,从而确定后续实验选用的血清型和病毒剂量。2. AAV-hSyn-9-CaMPARI腺相关病毒的制备首先 Addgene 官网上订购 pcDNA3-CaMPARI(Plasmid #60421),挑穿刺菌,单克隆培养,质粒提取,测质粒浓度,测序鉴定比对,病毒包装。3.验证CaMPARI荧光蛋白在大鼠三叉神经节细胞表达健康雄性SD大鼠,将8uLAAV-hSyn-9-CaMPARI立体定位注射入大鼠三叉神经节,2-6周后取材荧光显微镜下成像。生理盐水作空白对照。过程中避光,保证三叉神经节置于不含钙的kerb氏溶液中。结果AAV-hSyn-9-CaMPARI在注射后2-3周可高效率表达在大鼠三叉神经节细胞中,至少持续28天。结论CaMPARI可通过腺相关病毒转导表达在大鼠三叉神经节细胞,这为以后进一步研究三叉神经节细胞在偏头痛大鼠模型的激活奠定了基础。
[Abstract]:Background the pathogenesis of migraine is not fully understood, and the activation of the trigeminal vascular pain pathway is considered to be the most comprehensive theory to reveal the pathogenesis of migraine, which suggests that in migraine rat models, Trigeminal ganglion, as the starting point of classical ascending pain conduction pathway, plays a key role in the production and maintenance of migraine. However, the activation of trigeminal ganglion cells is still unclear. Traditional methods confirm that the activation of trigeminal ganglion cells can cause an explosive increase of intracellular calcium ion and high cell specificity, but during the pathogenesis of migraine, how to activate it? The activation sequence is not clear. CaMPARI fluorescent protein can be used to label activated trigeminal neurons because it is highly sensitive to intracellular calcium and has the characteristics of irreversible transition to purple-red fluorescence. The transient activation of neurons can be transformed into a permanent expression of fluorescent protein. At present, however, CaMPARI, a newly synthesized gene sequence in 2015, has only been demonstrated to be expressed in zebra fish, Drosophila melanogaster and mouse model biological tissues, and whether it can be transferred to rat trigeminal ganglion cells by adeno-associated viruses. This provides a new idea for us to study the role of trigeminal ganglion cell activation in the pathogenesis of migraine rat model. This is the problem that this research wants to solve. Objective to investigate whether CaMPARI can be expressed in rat trigeminal ganglion cells by adeno-associated virus transduction. Method 1. To verify that unloaded AAV-9-hSyn-GFP could be successfully transfected into rat trigeminal ganglion cells, the stereotaxic injection coordinates of trigeminal ganglia of 200-250gSD male rats were determined by methylene blue, and the unloaded AAV-hSyn-2-GFP,AAV-hSyn-8-GFP, was transferred to the trigeminal ganglion cells of rats. The three serotypes of AAV-hSyn-9-GFP were injected directly into the rat trigeminal ganglion cells at different doses of 2ul, 5ul and 8ul respectively. The rats in the control group were injected with the same amount of normal saline for 3 weeks. After 3 weeks, the frozen sections were taken to observe the expression of GFP green fluorescent protein under fluorescence microscope, so as to determine the serotype and virus dosage selected in the follow-up experiment. 2. Preparation of AAV-hSyn-9- CaMPARI adeno-associated virus first order pcDNA3-CaMPARI (Plasmid # 60421) on Addgene official website, select puncture bacteria, clone culture, plasmid extraction, measure plasmid concentration, sequence identification, virus packaging. To verify the expression of CaMPARI fluorescent protein in trigeminal ganglion cells of healthy male SD rats, 8uLAAV-hSyn-9-CaMPARI was stereotaxically injected into the trigeminal ganglia of rats. After 2 weeks and 6 weeks, the samples were obtained for imaging under fluorescence microscope. Normal saline was used as blank control. During the process, the trigeminal ganglion was kept in calcium-free kerb's solution. Results AAV-hSyn-9-CaMPARI was efficiently expressed in rat trigeminal ganglion cells 2 weeks after injection for at least 28 days. Conclusion CaMPARI can be expressed in rat trigeminal ganglion cells through adeno-associated virus transduction, which lays a foundation for further study on activation of trigeminal ganglion cells in migraine rat model.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R747.2;R-332
[Abstract]:Background the pathogenesis of migraine is not fully understood, and the activation of the trigeminal vascular pain pathway is considered to be the most comprehensive theory to reveal the pathogenesis of migraine, which suggests that in migraine rat models, Trigeminal ganglion, as the starting point of classical ascending pain conduction pathway, plays a key role in the production and maintenance of migraine. However, the activation of trigeminal ganglion cells is still unclear. Traditional methods confirm that the activation of trigeminal ganglion cells can cause an explosive increase of intracellular calcium ion and high cell specificity, but during the pathogenesis of migraine, how to activate it? The activation sequence is not clear. CaMPARI fluorescent protein can be used to label activated trigeminal neurons because it is highly sensitive to intracellular calcium and has the characteristics of irreversible transition to purple-red fluorescence. The transient activation of neurons can be transformed into a permanent expression of fluorescent protein. At present, however, CaMPARI, a newly synthesized gene sequence in 2015, has only been demonstrated to be expressed in zebra fish, Drosophila melanogaster and mouse model biological tissues, and whether it can be transferred to rat trigeminal ganglion cells by adeno-associated viruses. This provides a new idea for us to study the role of trigeminal ganglion cell activation in the pathogenesis of migraine rat model. This is the problem that this research wants to solve. Objective to investigate whether CaMPARI can be expressed in rat trigeminal ganglion cells by adeno-associated virus transduction. Method 1. To verify that unloaded AAV-9-hSyn-GFP could be successfully transfected into rat trigeminal ganglion cells, the stereotaxic injection coordinates of trigeminal ganglia of 200-250gSD male rats were determined by methylene blue, and the unloaded AAV-hSyn-2-GFP,AAV-hSyn-8-GFP, was transferred to the trigeminal ganglion cells of rats. The three serotypes of AAV-hSyn-9-GFP were injected directly into the rat trigeminal ganglion cells at different doses of 2ul, 5ul and 8ul respectively. The rats in the control group were injected with the same amount of normal saline for 3 weeks. After 3 weeks, the frozen sections were taken to observe the expression of GFP green fluorescent protein under fluorescence microscope, so as to determine the serotype and virus dosage selected in the follow-up experiment. 2. Preparation of AAV-hSyn-9- CaMPARI adeno-associated virus first order pcDNA3-CaMPARI (Plasmid # 60421) on Addgene official website, select puncture bacteria, clone culture, plasmid extraction, measure plasmid concentration, sequence identification, virus packaging. To verify the expression of CaMPARI fluorescent protein in trigeminal ganglion cells of healthy male SD rats, 8uLAAV-hSyn-9-CaMPARI was stereotaxically injected into the trigeminal ganglia of rats. After 2 weeks and 6 weeks, the samples were obtained for imaging under fluorescence microscope. Normal saline was used as blank control. During the process, the trigeminal ganglion was kept in calcium-free kerb's solution. Results AAV-hSyn-9-CaMPARI was efficiently expressed in rat trigeminal ganglion cells 2 weeks after injection for at least 28 days. Conclusion CaMPARI can be expressed in rat trigeminal ganglion cells through adeno-associated virus transduction, which lays a foundation for further study on activation of trigeminal ganglion cells in migraine rat model.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R747.2;R-332
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