【摘要】：研究背景偏頭痛的發(fā)病機(jī)制尚未完全清楚,三叉神經(jīng)血管痛覺通路激活被認(rèn)為是最能全面揭示偏頭痛發(fā)病進(jìn)展機(jī)制的學(xué)說,這種學(xué)說認(rèn)為在偏頭痛大鼠模型中,三叉神經(jīng)節(jié)作為經(jīng)典上行疼痛傳導(dǎo)通路的起始部位,它的激活在偏頭痛的產(chǎn)生和維持過程中起關(guān)鍵作用。然而目前關(guān)于三叉神經(jīng)節(jié)細(xì)胞的激活研究尚不清楚。傳統(tǒng)的方法證實三叉神經(jīng)節(jié)細(xì)胞的激活可引起細(xì)胞內(nèi)鈣離子一次爆炸性的升高,且有高度的細(xì)胞特異性,但在偏頭痛發(fā)病進(jìn)展過程中,具體如何激活,激活順序等尚不清楚。CaMPARI熒光蛋白因為其對胞內(nèi)鈣離子高度敏感,且具有不可逆轉(zhuǎn)變?yōu)樽霞t色熒光的特性,故可用來給激活的三叉神經(jīng)元作標(biāo)記,即可實現(xiàn)將瞬間的神經(jīng)元激活轉(zhuǎn)變?yōu)橛谰玫臒晒獾鞍椎谋磉_(dá)。然而目前,CaMPARI是2015年新合成的一段基因序列,僅被證實可在斑馬魚、果蠅、小鼠模式生物組織中表達(dá),它是否可用通過腺相關(guān)病毒轉(zhuǎn)導(dǎo)至大鼠三叉神經(jīng)節(jié)細(xì)胞,從而為我們研究三叉神經(jīng)節(jié)細(xì)胞激活在偏頭痛大鼠模型發(fā)病進(jìn)展中的作用提供一個新的思路呢?這是本研究想要解決的問題。目的CaMPARI是否可通過腺相關(guān)病毒轉(zhuǎn)導(dǎo)辦法表達(dá)在大鼠三叉神經(jīng)節(jié)細(xì)胞。方法1.驗證空載AAV-9-hSyn-GFP可成功轉(zhuǎn)染至大鼠三叉神經(jīng)節(jié)細(xì)胞首先使用亞甲藍(lán)確定200-250gSD雄性大鼠三叉神經(jīng)節(jié)的立體定位注射坐標(biāo),將空載 AAV-hSyn-2-GFP、AAV-hSyn-8-GFP、AAV-hSyn-9-GFP 三個血清型分別以不同劑量2uL,5ul,8uL直接注射至大鼠三叉神經(jīng)節(jié)細(xì)胞。對照組大鼠給予等量生理鹽水注射。2-3周后,取材冰凍切片于熒光顯微鏡下觀察GFP綠色熒光蛋白表達(dá)情況,從而確定后續(xù)實驗選用的血清型和病毒劑量。2. AAV-hSyn-9-CaMPARI腺相關(guān)病毒的制備首先 Addgene 官網(wǎng)上訂購 pcDNA3-CaMPARI(Plasmid #60421),挑穿刺菌,單克隆培養(yǎng),質(zhì)粒提取,測質(zhì)粒濃度,測序鑒定比對,病毒包裝。3.驗證CaMPARI熒光蛋白在大鼠三叉神經(jīng)節(jié)細(xì)胞表達(dá)健康雄性SD大鼠,將8uLAAV-hSyn-9-CaMPARI立體定位注射入大鼠三叉神經(jīng)節(jié),2-6周后取材熒光顯微鏡下成像。生理鹽水作空白對照。過程中避光,保證三叉神經(jīng)節(jié)置于不含鈣的kerb氏溶液中。結(jié)果AAV-hSyn-9-CaMPARI在注射后2-3周可高效率表達(dá)在大鼠三叉神經(jīng)節(jié)細(xì)胞中,至少持續(xù)28天。結(jié)論CaMPARI可通過腺相關(guān)病毒轉(zhuǎn)導(dǎo)表達(dá)在大鼠三叉神經(jīng)節(jié)細(xì)胞,這為以后進(jìn)一步研究三叉神經(jīng)節(jié)細(xì)胞在偏頭痛大鼠模型的激活奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R747.2;R-332

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