ERK1修饰的骨髓间充质干细胞移植治疗帕金森病认知障碍的实验研究

发布时间：2018-06-05 07:13

本文选题：帕金森病 + 认知障碍　；参考：《扬州大学》2017年硕士论文

【摘要】：目前对于帕金森病(PD)的关注逐渐从运动障碍到非运动障碍,非运动型障碍中的认知障碍已经成为一个研究热点。细胞外调节蛋白激酶1(ERK1)属于MAPK家族,其通过参与调节多条与学习记忆相关的信号通路,对长时程突触增强(LTP)介导的学习记忆的形成具有重要的调节控制作用。骨髓间充质干细胞(BMSCs)已被证实其对移植治疗PD模型大鼠有较好的治疗效果。本实验研究帕金森病伴随的认知障碍的发生发展与ERK1表达变化的关系,并以转染ERK1基因的BMSCs(ERK1-BMSCs)移植治疗PD模型大鼠,探讨ERK1-BMSCs移植对PD认知障碍的治疗效果及其机制。1.帕金森病模型大鼠脑内ERK1的水平的研究采用立体定位、单侧脑纹状体内注射6-OHDA方法制备PD大鼠模型,以行为学实验(避暗箱实验、穿梭箱实验、Morris水迷宫实验)检测造模后2周、5周、8周成功模型大鼠的学习记忆能力,以免疫组织化学、Western blot(WB)及Q-PCR法检测ERK1在帕金森病实验大鼠脑内黑质纹状体中表达变化的规律。结果显示,造模2周开始旋转圈数逐渐增加,8周时增加至9.92±2.43 r/min;PD大鼠的明箱观察时间百分比显著低于正常对照组(94.96±2.62%v 99.22±1.40%;p0.05,n=15);PD大鼠主动穿梭次数和平台象限时间均极显著低于正常对照组(p0.01,n=15),且呈下降趋势。模型大鼠的脑黑质区ERK1阳性棕染细胞数均明显下降;模型大鼠脑纹状体内ERK1蛋白表达量均极显著低于对照组(p0.01,n=5),且呈下降趋势;造模后2周大鼠脑纹状体内ERK1 mRNA相对表达量显著低于正常对照组(0.79±0.08 vs 1±0.07;p0.05,n=5),造模5周和8周后相对表达量极显著低于正常对照组(p0.01,n=5)。本实验提示,随病程的发展,PD模型大鼠的学习记忆能力明显下降,认知障碍的严重程度与PD病程加深呈正相关;认知障碍的发展与脑纹状体内ERK1的表达减少有密切关系。2.ERK1基因修饰BMSCs的构建利用Lipo 2000将构建的pCDNA3.1(+)-ERK1真核表达载体转染至BMSCs,获得ERK1-BMSCs。实验组分为ERK1-BMSCs组和BMSCs组。通过WB、Q-PCR、免疫细胞化学法检测两组的ERK1表达情况;在体外,利用β-巯基乙醇将ERK1-BMSCs定向诱导分化成神经元样细胞,探讨ERK1基因修饰后BMSCs的分化潜能及其分化后表达ERK1蛋白的能力。对重组载体的酶切电泳结果显示,酶切结果条带大小与理论值(1104 bp)一致。ERK1-BMSCs 中的 ERK1 蛋白表达量极显著高于 BMSCs(p0.01,n=5);ERK1-BMSCs 中ERK1 mRNA 相对表达量极显著高于 BMSCs 组(5.40±0.13 vs 1±0.07;p0.01,n=5)。ERK1-BMSCs经诱导分化后,神经元标志物生长蛋白Nestin和NSE蛋白表达呈阳性。结果提示转染ERK1基因不影响BMSCs的分化潜能,且ERK1-BMSCs诱导分化后仍能高表达ERK1蛋白。3.ERK1基因修饰的BMSCs移植治疗PD大鼠的研究将PD模型大鼠分为PBS对照组、BMSCs移植治疗组和ERK1-BMSCs移植治疗组3组,在3组动物的脑纹状体内分别定位注射PBS(10μL)、BMSCs(10μL,1×105个/μL细胞悬液)、ERK1-BMSCs(10μL,1×105个/μL细胞悬液)。各组大鼠在移植治疗后2周、5周、8周腹腔分别诱导转圈,并利用行为学实验检测各组治疗大鼠学习记忆能力的改变;并于上述时间点,取出大鼠纹状体组织,采用免疫组织化学法、WB技术以及Q-PCR方法检测各组治疗大鼠脑纹状体内ERK1的表达变化,分析对比各治疗组的治疗效果并探讨其作用机制。治疗2周后,ERK1-BMSCs组的转圈圈数极显著低于BMSCs组(5.08± 0.86 r/min vs 7.75士1.83 r/min,p0.01,n=10);学习记忆检测结果显示,BMSCs和ERK1-BMSCs移植治疗后2周大鼠明箱观察时间、主动逃避次数均极显著多于PBS对照组(p0.01,n=10)ERK1-BMSCs治疗8周主动逃避次数极显著多于BMSCs治疗组(p0.01,n=10);BMSCs治疗2周后,平台象限时间显著多于PBS对照组(p0.05,n=10),ERK1-BMSCs治疗2周平台象限时间显著多于 BMSCs 治疗组(p0.05,n=10)。治疗后 2 周,ERK1-BMSCs 和 BMSCs 治疗组ERK1mRNA相对表达量分别为2.76±0.06、1.21±0.15,均极显著高于PBS对照组(p0.01,n=5),ERK1-BMSCs组极显著高于BMSCs组(p0.01,n=5);ERK1-BMSCs组和BMSCs组动物脑纹状体内ERK1阳性细胞数均随治疗时间逐渐增多(n=5);移植治疗2周、5周、8周后,ERK1-BMSCs组大鼠的ERK1蛋白表达量均极显著高于BMSCs组(p0.01,n=5);与ERK1 mRNA表达结果相一致。实验结果提示,ERK1-BMSCs和BMSCs移植治疗PD模型大鼠后,大鼠的行为及学习记忆能力提高,PD及其认知障碍得到了改善,且ERK1-BMSCs移植治疗效果明显优于单独BMSCs移植治疗。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5

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