甘露醇对骨髓间充质干细胞治疗血管性痴呆大鼠行为学及海马CA3区突触素表达水平的影响

发布时间：2018-12-18 10:43

【摘要】：第一章大鼠BMSCs体外分离、培养、鉴定和标记 [目的]探索SD大鼠骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells, BMSCs)体外分离、培养、鉴定及标记的方法。为下一步的移植研究提供充足的BMSCs。 [方法]应用全骨髓贴壁改良法分离培养SD大鼠BMSCs,显微镜观察细胞形态,绘制细胞生长曲线。使用流式细胞仪检测细胞表面标记物及茜素红成骨诱导染色对第三代细胞进行鉴定。并对第3代细胞进行BrdU标记。 [结果]体外培养的原代BMSCs生长旺盛,以梭形为主,细胞集落呈螺旋状。第3代BMSCs几乎均一表达CD29,CD90,而CD34,CD45几乎不表达。BMSCs经诱导能分化为成骨细胞,茜素红染色出现红色钙化结节。BrdU免疫组化结果显示,BMSCs标记率高达85.29%。 [结论]改良的全骨髓贴壁法体外分离、培养及传代的SD大鼠BMSCs纯度高,活力强,性状稳定。BrdU标记法安全、高效。第二章血管性痴呆大鼠模型的制备 [目的]探讨VD大鼠模型理想的制备方法,为下一步BMSCs移植研究提供VD大鼠模型。 [方法]采用间隔3天永久性结扎双侧颈总动脉法制备VD大鼠模型。假手术组不结扎双侧颈总动脉,其余手术操作步骤相同。术后4周,采用Morris水迷宫试验测定模型大鼠的行为学表现。 [结果]假手术组术后因饲养不当死亡1只,余9只。模型组大鼠存活59只,死亡18只,1只出现眼球变白、萎缩,3只未达VD标准。本实验成功制备55只VD大鼠。模型组大鼠造模后水迷宫的逃避潜伏期较假手术组明显延长,平台象限滞留时间比假手术组明显缩短(p0.05)。模型组大鼠的存活率为76.62%,成模率为93.22%。 [结论]改良双血管法具有死亡率低,成模率高,简单易行等优点,是制备VD大鼠模型的理想方法。第三章甘露醇对骨髓间充质干细胞静脉移植治疗血管性痴呆大鼠模型的疗效观察 [目的]探讨甘露醇预处理对骨髓间充质干细胞移植治疗VD大鼠模型疗效及海马CA3区突触素表达水平的影响。 [方法]造模4周后将VD大鼠随机分为空白对照组、培养基组、甘露醇组、BMSCs组、甘露醇预处理BMSCs组,分别给予相应的实验干预措施。观察干预4周后实验大鼠的行为学表现及应用免疫组化法检测海马CA3区突触素的表达水平。 [结果]甘露醇预处理BMSCs组行为学较BMSCs组、培养基组、甘露醇组、空白对照组有明显改善(P0.05),其逃避潜伏期缩短(P0.05),平台象限滞留时间延长(40.75±6.29s,P0.05),其海马CA3区突触素表达水平(39624±7797)亦比BMSCs组、培养基组、甘露醇组、空白对照组明显提高(27060±4667,19955±4243、18293±6445,18946±4953,P0.05)。 [结论]甘露醇预处理后静脉移植BMSCs显著改善VD大鼠行为学表现,并使其海马CA3区突触素表达显著增加。甘露醇预处理明显提高静脉移植BMSCs治疗VD大鼠的效果。
[Abstract]:Chapter 1: isolation, culture, identification and labeling of rat BMSCs in vitro [objective] to explore the methods of isolation, culture, identification and labeling of rat bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Stem Cells, BMSCs) from SD rats in vitro. Provide sufficient BMSCs. for the next transplant research [methods] the morphology of SD rats was observed by BMSCs, microscope, and the cell growth curve was drawn. The third generation cells were identified by flow cytometry and alizarin red osteoblast induction staining. The third passage cells were labeled with BrdU. [results] the primary BMSCs cultured in vitro grew vigorously, mainly fusiform, and the cell colony was spiral. The third generation of BMSCs expressed almost homogeneous CD29,CD90, and almost no CD34,CD45. BMSCs could differentiate into osteoblasts and alizarin red staining showed red calcified nodules. BrdU immunohistochemical results showed that BMSCs labeling rate was as high as 85.29%. [conclusion] the modified whole bone marrow adherent method was used to isolate, culture and subculture BMSCs from SD rats with high purity, strong activity and stable character. BrdU labeling method was safe and efficient. Chapter 2 the establishment of vascular dementia rat model [objective] to explore the ideal method for the preparation of VD rat model, and to provide VD rat model for the next step of BMSCs transplantation. [methods] VD rat model was established by permanent ligation of bilateral common carotid artery at intervals of 3 days. In the sham operation group, bilateral common carotid artery was not ligated, and the other procedures were the same. At 4 weeks after operation, Morris water maze test was used to determine the behavior of the model rats. [results] in the sham operation group, 1 died because of improper feeding, the remaining 9 died. In the model group, 59 rats survived, 18 rats died, 1 rats showed bleached eyes and atrophy, and 3 rats did not meet the VD standard. In this experiment, 55 VD rats were successfully prepared. The escape latency of water maze in the model group was significantly longer than that in the sham operation group, and the stay time in the platform quadrant was significantly shorter than that in the sham operation group (p0.05). The survival rate of the model group was 76.62 and the rate of model formation was 93.22. [conclusion] the modified double vessel method has the advantages of low mortality, high model rate, simple and easy to carry out, and is an ideal method for the preparation of VD rat model. The effect of mannitol on the treatment of vascular dementia rat model by vein transplantation of bone marrow mesenchymal stem cells [objective] to investigate the effect of mannitol preconditioning on the treatment of VD rat model by bone marrow mesenchymal stem cell transplantation And the effect of synaptophysin expression in hippocampal CA3. [methods] VD rats were randomly divided into control group, culture medium group, mannitol group, BMSCs group and BMSCs group pretreated with mannitol for 4 weeks. The behavioral manifestations and synaptophysin expression in hippocampal CA3 region were detected by immunohistochemical method after 4 weeks of intervention. [results] compared with BMSCs group, culture medium group, mannitol group and blank control group, the behavior of mannitol pretreated BMSCs group was significantly improved (P0.05), the escape latency was shortened (P0.05), and the retention time of platform quadrant was prolonged (40.75 卤6.29 s). The level of synaptophysin expression in hippocampal CA3 region (39624 卤7797) was significantly higher than that in BMSCs group, medium group, mannitol group and blank control group (27060 卤4667). [conclusion] after pretreatment with mannitol, BMSCs significantly improved the behavior of VD rats and increased synaptophysin expression in CA3 area of hippocampus. Mannitol pretreatment significantly improved the efficacy of intravenous BMSCs in the treatment of VD rats.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.13

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