發(fā)布時(shí)間：2018-01-22 07:26

  本文關(guān)鍵詞： 帕金森病 核酸疫苗 紋狀體 免疫治療　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的： 探討優(yōu)化核酸疫苗改善PD模型小鼠紋狀體DA能神經(jīng)末梢脫失及其可能的機(jī)制。方法： 提取足量高純度空質(zhì)粒（pVAX1）和優(yōu)化核酸疫苗（pVAX1-IL-4/SYN-B）。將45只正常C57BL/6小鼠（雄性，22-25克）隨機(jī)分為4組：正常對照組（PBS+PBS，10只）、PBS PD模型組（MPTP+PBS,10只）、空質(zhì)粒PD模型組(MPTP+pVAX1，10只)和優(yōu)化核酸疫苗PD模型組（MPTP+pVAX1-IL-4/SYN-B，15只）。后三組小鼠腹腔注射丙磺舒30min后，再向其腹腔注射MPTP建立慢性MPTP PD小鼠模型。而正常對照組小鼠腹腔注射等體積PBS。成功建立PD動物模型后，分別向正常對照組和3組PD組小鼠每側(cè)后肢脛前肌分別注射PBS、PBS、pVAX1、pVAX1-IL-4/SYN-B50μL共100μL，共進(jìn)行3次免疫。免疫結(jié)束后，5只優(yōu)化核酸疫苗PD模型組小鼠再立體定位于側(cè)腦室注射3μL磷酸氯喹，以同樣的方式向剩余各組小鼠注射等體積PBS。對比各組小鼠的行為學(xué)變化。通過western blot，免疫組化和免疫熒光檢測各組小鼠紋狀體區(qū)酪氨酸羥化酶（tyrosine hydroxylase，，TH）、突觸素（synaptophysin，SYP）、α-突觸核蛋白（alpha-synuclein，α-syn）、微管相關(guān)蛋白1輕鏈3（microtubule-associated protein1lightchain3，LC3）、溶酶體相關(guān)膜蛋白1（lysosomal-associated membraneprotein1，Lamp1）組織蛋白酶D（cathepsin D，CD）的表達(dá)水平。 結(jié)果： 1.行為學(xué)觀察在建立慢性MPTP PD小鼠模型的過程中，PD模型組小鼠均先后出現(xiàn)類PD癥狀：肢體抖動、尾巴上翹，活動緩慢、減少，雙后肢站立時(shí)后仰等姿勢不穩(wěn)。免疫之后，與正常對照組相比較，PD模型組小鼠運(yùn)動障礙顯著（P＜0.05）。與PBS PD模型組或空質(zhì)粒PD模型組比較，優(yōu)化核酸疫苗PD模型組小鼠運(yùn)動障礙顯著改善（P＜0.05）。 2.免疫組化改變免疫組化結(jié)果顯示：PD模型組小鼠紋狀體區(qū)TH和SYP表達(dá)量較正常對照組明顯減少（P＜0.05），優(yōu)化核酸疫苗PD模型組小鼠紋狀體區(qū)TH和SYP表達(dá)量減少較PBS PD模型組或空質(zhì)粒PD模型組明顯減輕（P＜0.05）。 3. Western blot對比Western blot結(jié)果顯示：PD模型組小鼠紋狀體區(qū)TH和SYP表達(dá)量較正常對照組明顯減少（P＜0.05），優(yōu)化核酸疫苗PD模型組小鼠紋狀體區(qū)TH和SYP表達(dá)量減少較PBS PD模型組或空質(zhì)粒PD模型組明顯減輕（P＜0.05）。與正常對照組相比較，PD模型組小鼠紋狀體區(qū)α-syn二聚體明顯增多（P＜0.05），CD明顯減少（P＜0.05），Lamp1明顯減少（P＜0.05）。與PBS PD模型組或空質(zhì)粒PD模型組相比較，優(yōu)化核酸疫苗PD模型組小鼠紋狀體區(qū)α-syn二聚體明顯減少（P＜0.05），CD明顯增多（P＜0.05），Lamp1明顯增多（P＜0.05）。 結(jié)論： 本研究結(jié)果表明：慢性MPTP暴露可以損害C57BL/6小鼠運(yùn)動功能和紋狀體DA能神經(jīng)末梢，分子病理機(jī)制可能是溶酶體消耗引起α-syn二聚體增加或二者互為因果。而優(yōu)化核酸疫苗免疫治療可以改善慢性MTPT PD模型小鼠運(yùn)動障礙和紋狀體DA能神經(jīng)末梢脫失，免疫治療的神經(jīng)保護(hù)機(jī)制可能是通過修復(fù)溶酶體，促進(jìn)α-syn二聚體通過自噬-溶酶體途徑降解。
[Abstract]:Objective: Objective: to investigate the mechanism of improving the loss of DA nerve endings in striatum of PD model mice by optimizing nucleic acid vaccine. The high purity empty plasmid pVAX1 was extracted and the nucleic acid vaccine pVAX1-IL-4 / SYN-B1 was optimized. 45 normal C57BL / 6 mice (male) were selected. 22-25 g) were randomly divided into 4 groups: 10 PBS PBSs in the normal control group and 10 MPTP PBSs in the PBS PD model group. The empty plasmid PD model group (10 mice) and the optimized nucleic acid vaccine PD model group (MPTP pVAX1-IL-4/SYN-B). 15 mice in the latter three groups were intraperitoneally injected with propanecid for 30 minutes. The model of chronic MPTP PD mice was established by intraperitoneal injection of MPTP, while the normal control group mice were injected intraperitoneally with the same volume of PBSs. After the establishment of PD animal model, the model of PD was successfully established. The normal control group and three groups of PD mice were injected into the anterior tibial muscle of each side of the hind limb, respectively. The PBSS-pVAX1-IL-4 / SYN-B50 渭 L was 100 渭 L. After immunization, 5 mice in PD model group of optimized nucleic acid vaccine were stereoscopically injected with 3 渭 L chloroquine phosphate in lateral ventricle. In the same way, the remaining mice were injected with iso-volume PBSs. The behavioral changes of each group were compared by western blot. The tyrosine hydroxylase (tyrosine hydroxylaseTHN) and synaptophysin (synaptophysin) in the striatum of each group were detected by immunohistochemistry and immunofluorescence. SYP, alpha-synuclein, 偽 -synn. Microtubule-associated protein 1lightchain3 (LC3). Lysosomal-associated membraneprotein1. The expression level of cathepsin DCD in Lamp1. Results: 1.Behavioral observation showed that PD like symptoms occurred successively in the model group of chronic MPTP PD mice: limb jitter, tail upwardness, slow activity and decrease. The postures were unstable when the hind limbs were standing. After immunization, they were compared with the normal control group. The motor dysfunction of PD model group was significantly lower than that of PBS PD model group or blank plasmid PD model group (P < 0.05). In PD model group, the dyskinesia was significantly improved (P < 0.05). 2.Immunohistochemical changes showed that the expression of th and SYP in the striatum of the mice in the weight PD group was significantly lower than that in the normal control group (P < 0.05). The expression of th and SYP in the striatum of PD mice with optimized nucleic acid vaccine was significantly lower than that of PBS PD model or blank plasmid PD model group (P < 0.05). 3. The results of Western blot and Western blot showed that the expression of th and SYP in the striatum of the mice in the group of 10% PD was significantly lower than that in the control group (2). P < 0.05). The expression of th and SYP in the striatum of mice in the PD model group of optimized nucleic acid vaccine was significantly lower than that in the PBS PD model group or the blank plasmid PD model group (P < 0.05), and was significantly lower than that in the normal control group. In PD model group, 偽 -syn dimer in striatum increased significantly (P < 0.05) and CD decreased significantly (P < 0.05). Compared with PBS PD model group or empty plasmid PD model group, Lamp1 decreased significantly (P < 0.05). In PD model group, 偽 -syn dimer in striatum decreased significantly (P < 0.05) and CD increased significantly (P < 0.05). Lamp1 increased significantly (P < 0.05). Conclusion: The results showed that chronic MPTP exposure could damage motor function and striatal DA nerve endings in C57BL / 6 mice. The mechanism of molecular pathology may be the increase of 偽 -syn dimer caused by lysosome consumption or the causality of the two. Optimizing the immunotherapy of nucleic acid vaccine can improve chronic MTPT. PD model mice showed motor disorders and striatal DA nerve endings loss. The neuroprotective mechanism of immunotherapy may be to promote the degradation of 偽 -syn dimer through autophage-lysosome pathway by repairing lysosomes.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R742.5

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相關(guān)期刊論文 前1條

1 王少君;王加才;彭國光;羅琴;涂文斌;江思德;徐芙蓉;;含CpG基元核酸疫苗免疫慢性MPTP帕金森病小鼠的治療作用[J];中國實(shí)驗(yàn)動物學(xué)報(bào);2008年03期

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