rhMG53联合hUC-MSCs移植对阿尔茨海默鼠的治疗作用

发布时间：2018-01-04 03:31

本文关键词：rhMG53联合hUC-MSCs移植对阿尔茨海默鼠的治疗作用　出处：《郑州大学》2016年硕士论文　论文类型：学位论文

【摘要】：阿尔茨海默病(Alzheimer disease,AD)是与年龄密切相关的神经退行性疾病,随着我国社会的老龄化,AD患者呈现逐年上升趋势。当前,AD的发病机制的阐明和治疗手段的改进仍然是医学界的难题,干细胞移植治疗作为新的疾病治疗方案,为AD的治疗带来了希望。提高移植治疗的干细胞的生存能力,促进更多的干细胞迁移到脑组织发挥作用,对于提高干细胞移植治疗效果具有重要作用。MG53蛋白(Mitsugumin53 protein)是一种新发现的TRIM家族肌特异性蛋白,在细胞膜损伤修复和多种急性器官损伤保护中发挥关键作用。但是重组人MG53蛋白(recombinant human Mitsugumin53 protein,rhMG53)对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)氧化损伤和AD慢性病理状态下的保护作用未见报道。目的体外实验建立hUC-MSCs的H2O2氧化损伤模型研究rhMG53对hUC-MSCs氧化损伤的保护作用。体内实验研究rhMG53联合hUC-MSCs移植对APP+转基因模型鼠的干预和治疗作用,为进一步了解MG53蛋白的功能并探讨对hUC-MSCs保护和对AD的治疗作用提供科学依据。方法1、体外实验研究rhMG53对hUC-MSCs氧化损伤的保护作用使用组织块培养法从健康人脐带分离培养hUC-MSCs,P3代细胞建立H2O2损伤模型;CCK-8法检测rhMG53蛋白对细胞增殖的影响,筛选合适处理条件,确定实验分组为NC组,rhMG53组,H2O2组,预处理rhMG53组(Pre组);Transwell小室观察rhMG53蛋白对细胞迁移的影响;流式细胞术检测rhMG53蛋白对细胞周期和细胞凋亡的影响;β-半乳糖苷酶染色检测rhMG53蛋白对细胞衰老的影响;分光光度法检测rhmg53蛋白对细胞sod活性和mda含量的影响。2、体内实验研究rhmg53联合huc-mscs移植对阿尔茨海默鼠的治疗作用使用4月龄app+转基因阿尔茨海默病模型鼠,分为app+组,huc-mscs组,rhmg53组,rhmg53联合huc-mscs组(联合组);一周后,免疫组化检测小鼠脑内huc-mscs移植的迁移率;两周后,用跑步机检测小鼠的运动能力;四周后,morris水迷宫检测各组小鼠的学习和记忆能力;分光光度法检测各组小鼠血清sod的活性和mda的含量;qrt-pcr和westernblot检测各组小鼠脑组织中衰老相关基因(sirt1、sirt2、pcna、p16、、p21、p53)的表达变化;免疫组化法检测各组小鼠脑组织神经标志物(dcx、nestin、nse)表达。结果1、体外实验发现,p3代huc-mscs贴壁生长,呈梭型,旋涡状,表面光滑,轮廓清晰;h2o2处理后,细胞贴壁能力减低,细胞贴壁宽度变窄,表面有泡状突起。h2o2对huc-mscs具有浓度和时间依赖性的损伤作用,考虑细胞的增殖能力和死亡率,选用200μmh2o2处理16h作为氧化损伤模型的最适条件。rhmg53处理对细胞的增殖能力具有浓度依赖的增强作用,选用30μg/mlrhmg53作为实验使用浓度。与nc组相比,rhmg53组细胞增殖和迁移能力显著提高,s期比例变化差异不大,细胞衰老和凋亡比例显著降低,细胞sod活力显著增加,mda含量显著降低;h2o2组细胞增殖、迁移能力和s期细胞比例显著降低,细胞衰老和凋亡比例显著增加,细胞sod活力显著降低,mda含量显著增加。与h2o2组相比,pre组细胞增殖和迁移能力显著提高,s期比例变化差异不显著,细胞衰老和凋亡比例显著降低,细胞sod活力显著增加,mda含量显著降低。2、体内实验鉴定获得app+转基因模型鼠40只。移植一周后,与huc-mscs组相比,联合组脑组织迁移的干细胞数目显著增加。移植两周后,与app+组和huc-mscs组相比,mg53组和联合组小鼠的运动能力均有显著提高。移植四周后,与app+组相比,各处理组小鼠逃避潜伏期时间缩短、穿越平台次数增多、平台所在象限停留时间增加,sod活性增加,mda含量下降,脑组织中sirt1、Sirt2、PCNA基因的mRNA和蛋白表达增加,而P16、P21、P53基因的mRNA和蛋白表达降低,差异显著;hUC-MSCs组和联合组小鼠脑组织DCX和Nestin的表达增多,NSE表达变化差异不显著。与hUC-MSCs组相比,联合组小鼠逃避潜伏期时间显著缩短,血清中SOD活性显著增加而MDA含量显著下降,脑组织中Sirt1、Sirt2、PCNA基因的mRNA和蛋白表达增加,而P16、P21、P53基因的mRNA和蛋白表达降低。结论1、rhMG53蛋白能够提高hUC-MSCs的存活和迁移,rhMG53预处理能够提高hUC-MSCs的抗氧化能力,减低H2O2对细胞造成的氧化损伤。2、外源rhMG53蛋白可以增加小鼠体内hUC-MSCs的存活和迁移数目,改善APP+鼠的学习和认知能力,促进神经细胞的增殖,对hUC-MSCs移植治疗阿尔茨海默鼠有促进作用。
[Abstract]:Alzheimer's disease (Alzheimer disease AD) is a neurodegenerative disease closely related with age, with the aging of our society, AD patients increased year by year. At present, the improvement of the pathogenesis of AD and elucidate the treatment is still a difficult problem in medicine, stem cell transplantation in the treatment of as new therapies that brings hope for the treatment of AD. To improve the transplantation of stem cell viability, promote more stem cells migrate to the brain play a role, plays an important role in the.MG53 protein to improve the therapeutic effects of stem cell transplantation (Mitsugumin53 protein) is a newly discovered TRIM family of muscle specific protein, plays a key role in cell membrane damage repair and a variety of acute organ injury. But the protection of recombinant human MG53 (recombinant human Mitsugumin53 protein, rhMG53) of human umbilical cord mesenchymal stem cells (Human Umbilical cord mesenchymal stem cells, hUC-MSCs) reported protective effect of chronic pathological oxidative damage and AD. The protective effect of in vitro objective to establish the hUC-MSCs model of rhMG53 H2O2 oxidative damage on hUC-MSCs oxidative damage. In vivo experimental study of rhMG53 combined with hUC-MSCs transplantation on APP+ transgenic mice of intervention and treatment, in order to further understand the MG53 the function of hUC-MSCs protein and explore the protective and therapeutic effect on AD to provide a scientific basis. 1 methods, the protective effect of in vitro rhMG53 on oxidative hUC-MSCs damage using tissue culture method for isolation of hUC-MSCs from healthy human umbilical cord P3 cells to establish H2O2 damage model; influence of CCK-8 method for detection of rhMG53 protein on cell proliferation. Select the appropriate processing conditions, determine the experimental group as NC group, rhMG53 group, H2O2 group, rhMG53 pretreatment group (Pre group); Transwell Room to observe the effect of rhMG53 protein on cell migration; the effect of rhMG53 protein by flow cytometry on cell cycle and apoptosis; beta galactosidase staining of rhMG53 protein was detected on cell senescence; rhmg53 protein was detected by spectrophotometry effect on cell SOD activity and MDA content of.2, therapeutic effect in vivo rhmg53 combined with hUC-MSCs transplantation on Alzheimer's mice using April old app+ transgenic rat model of Alzheimer's disease, divided into app+ group, hUC-MSCs group, rhmg53 group, rhmg53 combined with hUC-MSCs group (combined group); a week later, immunohistochemical detection of migration in the brain of mice hUC-MSCs transplantation rate; after two weeks, with the treadmill exercise capacity test mice; after four weeks, the ability of learning and memory in Morris water maze test in mice; Spectrophotometry Determination of serum SOD activity of mice and MDA; qRT-PCR and Westernblot to detect the The aging of brain tissue in mice related genes (SIRT1, sirt2, PCNA, p16, p21, p53) expression; immunohistochemistry in brain tissue of mice neural markers (DCX, nestin, NSE). Results the expression of P3 1, in vitro experiments found that the generation of hUC-MSCs adherent, spindle type, spiral shape, smooth surface, clear outline; after H2O2 treatment, cell adhesion ability decreased, cell wall width, surface damage blistery projections with.H2o2 concentration and time dependence on the hUC-MSCs, considering the cell proliferation ability and mortality, with 200 mh2o2 16h as oxidative damage model of the suitable conditions of.Rhmg53 on the proliferation of cells with a concentration dependent enhancement, using 30 g/mlrhmg53 as the experimental concentration. Compared with the NC group, the proliferation and migration of cells in rhmg53 group increased significantly, the proportion of s had no difference, cell senescence and apoptosis ratio Patients decreased significantly, the activity of SOD cells was significantly increased, MDA content decreased significantly; the proliferation of H2O2 cells, and the migration ability of cells in S phase decreased, the proportion of cell senescence and apoptosis cells significantly increased the activity of SOD decreased, MDA content increased significantly. Compared with the H2O2 group, the proliferation and migration of cells in pre group increased significantly. Changes in the proportion of S phase difference is not significant, the proportion of cell senescence and apoptosis of cells was significantly decreased, SOD activity increased significantly, MDA decreased the content of.2 in vivo identification of app+ transgenic mice was 40. One week after transplantation, compared with hUC-MSCs group, the number of stem cells in brain tissue of group migration increased significantly. After two weeks when compared with app+ group and hUC-MSCs group, mg53 group and combined group mice exercise capacity were significantly increased. Four weeks after transplantation, compared with app+ group, each group of mice escape latency time, cross platform The number increased, the platform quadrant dwell time increased, SOD activity increased, MDA decreased, SIRT1, Sirt2 in brain tissue, the expression of mRNA and protein of PCNA gene increased, while P16, P21, mRNA and protein expression of P53 gene decreased significantly; the increased expression of hUC-MSCs group and combined group mice brain tissue DCX and Nestin, NSE expression was not significantly different. Compared with the hUC-MSCs group, combined group mice escape latency was significantly shortened, the activity of serum SOD was significantly increased and MDA content decreased, Sirt1, Sirt2 in brain tissue, increase the expression of PCNA gene and mRNA protein and P16, P21, mRNA and protein expression of P53 gene reduced. Conclusion 1, rhMG53 protein can improve the survival and migration of hUC-MSCs, rhMG53 pretreatment can improve the oxidation resistance of hUC-MSCs,.2 decreased H2O2 on cell oxidative damage caused by the exogenous rhMG53 protein can increase the hUC-MSCs in mice The number of survival and migration, improve the learning and cognitive ability of APP+ rats, promote the proliferation of nerve cells, and promote the treatment of Alzheimer's rats by hUC-MSCs transplantation.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.16

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