本文選題：帕金森病 + 認(rèn)知障礙�。� 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：目前對于帕金森病(PD)的關(guān)注逐漸從運(yùn)動(dòng)障礙到非運(yùn)動(dòng)障礙,非運(yùn)動(dòng)型障礙中的認(rèn)知障礙已經(jīng)成為一個(gè)研究熱點(diǎn)。細(xì)胞外調(diào)節(jié)蛋白激酶1(ERK1)屬于MAPK家族,其通過參與調(diào)節(jié)多條與學(xué)習(xí)記憶相關(guān)的信號(hào)通路,對長時(shí)程突觸增強(qiáng)(LTP)介導(dǎo)的學(xué)習(xí)記憶的形成具有重要的調(diào)節(jié)控制作用。骨髓間充質(zhì)干細(xì)胞(BMSCs)已被證實(shí)其對移植治療PD模型大鼠有較好的治療效果。本實(shí)驗(yàn)研究帕金森病伴隨的認(rèn)知障礙的發(fā)生發(fā)展與ERK1表達(dá)變化的關(guān)系,并以轉(zhuǎn)染ERK1基因的BMSCs(ERK1-BMSCs)移植治療PD模型大鼠,探討ERK1-BMSCs移植對PD認(rèn)知障礙的治療效果及其機(jī)制。1.帕金森病模型大鼠腦內(nèi)ERK1的水平的研究采用立體定位、單側(cè)腦紋狀體內(nèi)注射6-OHDA方法制備PD大鼠模型,以行為學(xué)實(shí)驗(yàn)(避暗箱實(shí)驗(yàn)、穿梭箱實(shí)驗(yàn)、Morris水迷宮實(shí)驗(yàn))檢測造模后2周、5周、8周成功模型大鼠的學(xué)習(xí)記憶能力,以免疫組織化學(xué)、Western blot(WB)及Q-PCR法檢測ERK1在帕金森病實(shí)驗(yàn)大鼠腦內(nèi)黑質(zhì)紋狀體中表達(dá)變化的規(guī)律。結(jié)果顯示,造模2周開始旋轉(zhuǎn)圈數(shù)逐漸增加,8周時(shí)增加至9.92±2.43 r/min;PD大鼠的明箱觀察時(shí)間百分比顯著低于正常對照組(94.96±2.62%v 99.22±1.40%;p0.05,n=15);PD大鼠主動(dòng)穿梭次數(shù)和平臺(tái)象限時(shí)間均極顯著低于正常對照組(p0.01,n=15),且呈下降趨勢。模型大鼠的腦黑質(zhì)區(qū)ERK1陽性棕染細(xì)胞數(shù)均明顯下降;模型大鼠腦紋狀體內(nèi)ERK1蛋白表達(dá)量均極顯著低于對照組(p0.01,n=5),且呈下降趨勢;造模后2周大鼠腦紋狀體內(nèi)ERK1 mRNA相對表達(dá)量顯著低于正常對照組(0.79±0.08 vs 1±0.07;p0.05,n=5),造模5周和8周后相對表達(dá)量極顯著低于正常對照組(p0.01,n=5)。本實(shí)驗(yàn)提示,隨病程的發(fā)展,PD模型大鼠的學(xué)習(xí)記憶能力明顯下降,認(rèn)知障礙的嚴(yán)重程度與PD病程加深呈正相關(guān);認(rèn)知障礙的發(fā)展與腦紋狀體內(nèi)ERK1的表達(dá)減少有密切關(guān)系。2.ERK1基因修飾BMSCs的構(gòu)建利用Lipo 2000將構(gòu)建的pCDNA3.1(+)-ERK1真核表達(dá)載體轉(zhuǎn)染至BMSCs,獲得ERK1-BMSCs。實(shí)驗(yàn)組分為ERK1-BMSCs組和BMSCs組。通過WB、Q-PCR、免疫細(xì)胞化學(xué)法檢測兩組的ERK1表達(dá)情況;在體外,利用β-巰基乙醇將ERK1-BMSCs定向誘導(dǎo)分化成神經(jīng)元樣細(xì)胞,探討ERK1基因修飾后BMSCs的分化潛能及其分化后表達(dá)ERK1蛋白的能力。對重組載體的酶切電泳結(jié)果顯示,酶切結(jié)果條帶大小與理論值(1104 bp)一致。ERK1-BMSCs 中的 ERK1 蛋白表達(dá)量極顯著高于 BMSCs(p0.01,n=5);ERK1-BMSCs 中ERK1 mRNA 相對表達(dá)量極顯著高于 BMSCs 組(5.40±0.13 vs 1±0.07;p0.01,n=5)。ERK1-BMSCs經(jīng)誘導(dǎo)分化后,神經(jīng)元標(biāo)志物生長蛋白Nestin和NSE蛋白表達(dá)呈陽性。結(jié)果提示轉(zhuǎn)染ERK1基因不影響B(tài)MSCs的分化潛能,且ERK1-BMSCs誘導(dǎo)分化后仍能高表達(dá)ERK1蛋白。3.ERK1基因修飾的BMSCs移植治療PD大鼠的研究將PD模型大鼠分為PBS對照組、BMSCs移植治療組和ERK1-BMSCs移植治療組3組,在3組動(dòng)物的腦紋狀體內(nèi)分別定位注射PBS(10μL)、BMSCs(10μL,1×105個(gè)/μL細(xì)胞懸液)、ERK1-BMSCs(10μL,1×105個(gè)/μL細(xì)胞懸液)。各組大鼠在移植治療后2周、5周、8周腹腔分別誘導(dǎo)轉(zhuǎn)圈,并利用行為學(xué)實(shí)驗(yàn)檢測各組治療大鼠學(xué)習(xí)記憶能力的改變;并于上述時(shí)間點(diǎn),取出大鼠紋狀體組織,采用免疫組織化學(xué)法、WB技術(shù)以及Q-PCR方法檢測各組治療大鼠腦紋狀體內(nèi)ERK1的表達(dá)變化,分析對比各治療組的治療效果并探討其作用機(jī)制。治療2周后,ERK1-BMSCs組的轉(zhuǎn)圈圈數(shù)極顯著低于BMSCs組(5.08± 0.86 r/min vs 7.75士1.83 r/min,p0.01,n=10);學(xué)習(xí)記憶檢測結(jié)果顯示,BMSCs和ERK1-BMSCs移植治療后2周大鼠明箱觀察時(shí)間、主動(dòng)逃避次數(shù)均極顯著多于PBS對照組(p0.01,n=10)ERK1-BMSCs治療8周主動(dòng)逃避次數(shù)極顯著多于BMSCs治療組(p0.01,n=10);BMSCs治療2周后,平臺(tái)象限時(shí)間顯著多于PBS對照組(p0.05,n=10),ERK1-BMSCs治療2周平臺(tái)象限時(shí)間顯著多于 BMSCs 治療組(p0.05,n=10)。治療后 2 周,ERK1-BMSCs 和 BMSCs 治療組ERK1mRNA相對表達(dá)量分別為2.76±0.06、1.21±0.15,均極顯著高于PBS對照組(p0.01,n=5),ERK1-BMSCs組極顯著高于BMSCs組(p0.01,n=5);ERK1-BMSCs組和BMSCs組動(dòng)物腦紋狀體內(nèi)ERK1陽性細(xì)胞數(shù)均隨治療時(shí)間逐漸增多(n=5);移植治療2周、5周、8周后,ERK1-BMSCs組大鼠的ERK1蛋白表達(dá)量均極顯著高于BMSCs組(p0.01,n=5);與ERK1 mRNA表達(dá)結(jié)果相一致。實(shí)驗(yàn)結(jié)果提示,ERK1-BMSCs和BMSCs移植治療PD模型大鼠后,大鼠的行為及學(xué)習(xí)記憶能力提高,PD及其認(rèn)知障礙得到了改善,且ERK1-BMSCs移植治療效果明顯優(yōu)于單獨(dú)BMSCs移植治療。
[Abstract]:The current attention to Parkinson's disease (PD) is gradually from dyskinesia to non motor disorders, and cognitive impairment in non motor disorders has become a focus of research. Extracellular regulated protein kinase 1 (ERK1) belongs to the MAPK family, which mediates long term synaptic enhancement (LTP) by modulating a number of signaling pathways associated with learning and memory. The formation of learning and memory has an important regulatory effect. Bone marrow mesenchymal stem cells (BMSCs) have been proved to have better therapeutic effects on the transplantation of PD model rats. This experiment studies the relationship between the development of cognitive impairment associated with Parkinson's disease and the changes in the expression of ERK1, and the transplantation of BMSCs (ERK1-BMSCs) to the ERK1 gene. Treatment of PD model rats, the effect of ERK1-BMSCs transplantation on PD cognitive impairment and the mechanism of the mechanism of ERK1 in the brain of.1. Parkinson's disease model rats were studied by stereotactic localization. The unilateral cerebral striatum was injected with 6-OHDA to prepare the PD rat model, and the behavior test (dark box experiment, shuttle box experiment, Morris water maze test) was tested. The learning and memory ability of the model rats after 2 weeks, 5 weeks and 8 weeks was successfully established. The changes of the expression of ERK1 in the substantia nigra of the brain of Parkinson's disease rats were detected by immunohistochemistry, Western blot (WB) and Q-PCR method. The results showed that the number of revolving circles gradually increased at the beginning of the model for 2 weeks, and increased to 9.92 + 2.43 r/min at 8 weeks; the clear box of PD rats The percentage of observation time was significantly lower than that of the normal control group (94.96 + 2.62%v 99.22 + 1.40%; P0.05, n=15). The active shuttle times and the platform quadrant time of the PD rats were significantly lower than those of the normal control group (P0.01, n=15), and showed a decline trend. The number of ERK1 positive Brown stained cells in the brain black mass of the model rats decreased obviously; the model rats' brain striatum ERK1 The expression of protein was significantly lower than that of the control group (P0.01, n=5) and showed a downward trend. The relative expression of ERK1 mRNA in the brain striatum was significantly lower than that of the normal control group (0.79 + 0.08 vs 1 + 0.07; P0.05, n=5). The relative expression of the model was significantly lower than that of the normal control group (P0.01, n=5) after 5 and 8 weeks (P0.01, n=5). Development, the learning and memory ability of PD model rats decreased significantly, the severity of cognitive impairment was positively correlated with the deepening of PD, and the development of cognitive impairment was closely related to the decrease of ERK1 expression in the brain striatum. The construction of the.2.ERK1 gene modified BMSCs was transfected to BMSCs by the pCDNA3.1 (+) -ERK1 eukaryotic expression vector constructed by Lipo 2000. The ERK1-BMSCs. experimental group was divided into group ERK1-BMSCs and group BMSCs. The expression of ERK1 in two groups was detected by WB, Q-PCR and immunocytochemical method. In vitro, ERK1-BMSCs was induced to differentiate into neuron like cells by beta mercapto ethanol, and the differentiation potential of BMSCs after ERK1 gene modification and the ability to express ERK1 protein after differentiation were investigated. The results of enzyme cut electrophoresis of the group carrier showed that the ERK1 protein expression in.ERK1-BMSCs was significantly higher than that of BMSCs (P0.01, n=5), and the relative expression of ERK1 mRNA in ERK1-BMSCs was significantly higher than that of BMSCs group (5.40 + 0.13 vs 1 + 0.07; P0.01, BP) in ERK1-BMSCs. The expression of Nestin and NSE protein was positive. The results suggested that the transfection of ERK1 gene did not affect the differentiation potential of BMSCs, and the ERK1 protein.3.ERK1 gene modified BMSCs transplantation for the treatment of PD rats after ERK1-BMSCs induced differentiation, the PD model rats were divided into PBS control group, the BMSCs transplantation treatment group and the transplant treatment. In the 3 groups of the treatment group, the injection of PBS (10 mu L), BMSCs (10 L, 1 x 105 / mu L cell suspension), ERK1-BMSCs (10 mu L, 1 x 105 / mu L cell suspension) in the brain striatum of the group of animals, respectively, were induced in each group after transplantation for 2 weeks, 5 weeks and 8 weeks respectively, and the learning memory ability of each group was detected by behavior test. At the above time point, the rat striatum tissue was removed, the expression of ERK1 in the brain striatum was detected by immunohistochemistry, WB and Q-PCR, and the therapeutic effect of each group was compared and discussed. After 2 weeks, the number of circling circles in group ERK1-BMSCs was significantly lower than that of group BMSCs (5.08 +. 0.86 r/min vs 7.75, 1.83 r/min, P0.01, n=10). The results of learning and memory test showed that the time of observation for 2 weeks after BMSCs and ERK1-BMSCs transplantation, the number of active evasion was significantly more than that of the PBS control group (P0.01, n=10) and ERK1-BMSCs treatment for 8 weeks was significantly more than that of the BMSCs treatment group. After 2 weeks, the treatment was flat. The stage quadrant time was significantly more than that of the PBS control group (P0.05, n=10), and the 2 week platform quadrant time of the ERK1-BMSCs treatment was significantly more than that of the BMSCs treatment group (P0.05, n=10). The relative expression of ERK1mRNA in the ERK1-BMSCs and BMSCs treatment groups was 2.76 + 0.15, respectively, at the end of the treatment, respectively, which were significantly higher than those in the PBS control group. In group BMSCs (P0.01, n=5), the number of ERK1 positive cells in group ERK1-BMSCs and BMSCs groups increased gradually with the time of treatment (n=5). The expression of ERK1 protein in ERK1-BMSCs group rats was significantly higher than that of BMSCs group (P0.01, n=5) after 2 weeks, 5 weeks and 8 weeks. After MSCs transplantation in PD model rats, the behavior and learning and memory ability of the rats were improved, and the PD and their cognitive impairment were improved, and the effect of ERK1-BMSCs transplantation was obviously better than that of the single BMSCs transplantation.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.5

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