甘露醇对骨髓间充质干细胞治疗血管性痴呆模型大鼠行为学及Nogo蛋白表达影响

发布时间：2018-06-12 12:47

本文选题：血管性痴呆 + 动物模型　；参考：《广西医科大学》2013年硕士论文

【摘要】：第一章血管性痴呆大鼠模型的制备 [目的]探讨制备理想VD动物模型的方法,为进行BMSCs移植制备模型。 [方法]使用Morris水迷宫对实验大鼠进行初筛,以剔除在95%参考值范围以外的SD大鼠,将筛选出的实验鼠根据随机原则分为假手术组(10只)和模型组(77只)。模型组采用改良二血管结扎法(间隔三天分次结扎双侧颈总动脉)制备VD大鼠模型,假手术组实施同样的手术操作,但是不结扎双侧颈总动脉,同等条件笼养4周后,再次行水迷宫实验,以测定两组大鼠的空间学习记忆能力变化,筛选出造模成功的VD大鼠。 [结果]90只大鼠经水迷宫实验初筛后剔除3只,纳入实验87只大鼠随机分成假手术组(10只)和模型组(77只)。造模前,两组的逃避潜伏期和平台象限滞留时间差异没有统计学意义(P0.05),造模后,两组的逃避潜伏期和平台象限滞留时间差异有统计学意义(P0.05)。假手术组由于饲养不当死亡1只,余9只；模型组大鼠因麻醉意外死亡4只,感染死亡2只,肠梗阻死亡5只,出血过多死亡2只,肺淤血死亡5只,1只出现眼球病变,3只未达痴呆标准,共制备VD大鼠模型55只,成活率为76.62%,成模率93.22%。 [结论]改良二血管结扎法制备VD模型,不仅操作简易,可重复性好,而且有较高的存活率,成模率高,是较理想的制备VD模型的方法,适用于实验研究。第二章SD大鼠骨髓间充质干细胞的分离、培养及鉴定 [目的]应用全骨髓贴壁改良法分离培养SD大鼠BMSCs,形成稳定分离培养和扩增BMSCs的培养方法,以获得纯度高、活性好、形态均一的大鼠BMSCs,为移植治疗VD提供充足的细胞来源,保证实验顺利进行。 [方法]断头处死七日龄SD乳鼠,用培养基反复冲洗股骨和胫骨,收集其骨髓,采用改良的全骨髓贴壁培养法分离、培养、纯化BMSCs,观察BMSCs的形态、生长情况并绘制细胞生长曲线,了解BMSCs的形态特征及生长特性。采用流式细胞仪测定细胞表面标志物和进行成骨诱导对BMSCs进行鉴定。 [结果]光学显微镜下观察分离培养的原代BMSCs呈梭形贴壁生长。传代后细胞生长迅速,5-6天达到融合即可再次传代,细胞形态仍为梭形,鱼群样排列,集落生长呈漩涡状,第7天细胞达到饱和出现接触性抑制而生长缓慢。流式细胞仪鉴定第三代BMSCs均一表达CD29, CD90,阳性率分别为99%,99.6%；几乎不表达CD34, CD45,阳性率分别为0.169%,1.19%。对BMSCs进行成骨诱导后行茜素红染色,钙结节呈深红色。 [结论]SD大鼠的BMSCs能在体外分离、培养和纯化。全骨髓贴壁改良培养法能稳定分离培养和扩增BMSCs,获得纯度高、活性好、形态均一的大鼠BMSCs,可提供充足的实验细胞来源。第三章甘露醇对骨髓间充质干细胞移植治疗血管性痴呆大鼠效果观察 [目的]比较直接移植BMSCs与甘露醇预处理后再进行BMSCs移植的VD大鼠模型的行为学改变,海马CA1区Nogo蛋白及其受体表达量的变化,探讨甘露醇对静脉移植BMSCs治疗VD大鼠模型疗效的影响及机制。 [方法]造模成功的VD大鼠随机分为五组,①模型对照组8只(不进行任何处理),②甘露醇组10只(尾静脉注射37℃、20%甘露醇溶液,1.5g/kg),③培养基组10只(尾静脉注射1m工无血清低糖培养基),④BMSCs组13只(尾静脉注射细胞浓度为1×106个/ml的第三代BMSCs)⑤甘露醇预处理BMSCs组14只(尾静脉注射37℃、20%甘露醇溶液,1.5g/kg,10-20min后,注射细胞浓度为1×108个/m1的第三代BMSCs),此外设立假手术组9只。同等条件下笼养4周后,行水迷宫实验检测各组大鼠的空间学习记忆能力,使用免疫组化检测各组大鼠海马CA1区的Nogo A蛋白及其受体表达水平的变化。 [结果]BMSCs组与模型对照组、甘露醇组、培养基组比较,逃避潜伏期缩短,平台象限滞留时间延长,差异具有统计学意义(P0.05)；甘露醇预处理BMSCs组的逃避潜伏期比BMSCs组明显缩短,其平台象限滞留时间也比BMSCs组显著延长,差异均具有统计学意义(P0.05),尚未达到假手术组的水平。但是,甘露醇预处理BMSCs组与假手术组第3-5天的逃避潜伏期比较,差异没有统计学意义(P0.05),两组的平台象限滞留时间比较,差异也没有统计学意义(P0.05)。 BMSCs组海马CA1区Nogo A蛋白及其受体免疫组化阳性产物比模型对照组、甘露醇组、培养基组表达量少,染色浅；甘露醇预处理BMSCs组的Nogo A蛋白及其受体染色不但比模型对照组、甘露醇组、培养基组浅,而且也比BMSCs组浅,阳性产物也较少,但染色的深度及面积超过假手术组。 [结论]尾静脉注射移植第三代BMSCs能够改善VD大鼠的空间学习记忆能力。经过甘露醇预处理后再行尾静脉注射移植BMSCs使VD大鼠的空间学习记忆能力改善效果优于单独注射BMSCs的VD大鼠。其机制可能是甘露醇提高血脑屏障对BMSCs及各种脑营养因子的通透率,进而增加VD大鼠脑内各种脑营养因子的表达水平,减低Nogo蛋白信号系统对突触生长的抑制作用,从而促进神经功能恢复。
[Abstract]:The preparation of vascular dementia rat model in the first chapter
[Objective] to explore the method of preparing ideal VD animal model, and prepare a model for BMSCs transplantation.
[Methods] the experimental rats were screened by Morris water maze to remove the SD rats outside the reference range of 95%. The selected experimental rats were divided into sham operation group (10 rats) and model group (77 rats) according to the random principle. The model group was prepared by modified two vascular ligation (Jian Gesan ligation of bilateral common carotid artery) to prepare the VD rat model. In the operation group, the same operation was performed, but the bilateral common carotid artery was not ligated. After 4 weeks of the same condition, the water maze test was carried out again to determine the spatial learning and memory ability of the two groups of rats and to screen out the successful VD rats.
[results in]90 rats, only 3 rats were removed after the water maze test, and 87 rats were randomly divided into the sham operation group (10) and the model group (77). There was no significant difference between the two groups of escape latency and platform quadrant time (P0.05). The difference between the escape latency and the platform quadrant time of the two groups was different. Statistical significance (P0.05). In the sham operation group, 1 died of improper feeding and the remaining 9 were killed, 4 rats died of anaesthesia, 2 died of infection, 5 died of intestinal obstruction, 2 died of excessive bleeding, 5 died of pulmonary congestion, 1 had eyeball lesions, 3 had not reached dementia standard, and 55 VD rat models were prepared, the survival rate was 76.62%, molding rate 9. 3.22%.
[Conclusion] the modified two vascular ligation method for the preparation of VD model is not only easy to operate, good reproducibility, but also has high survival rate and high mold forming rate. It is an ideal method for preparing VD model. It is suitable for experimental research.
The second chapter is the isolation, culture and identification of SD rat bone marrow mesenchymal stem cells.
[Objective] to isolate and culture SD rat BMSCs by full bone marrow adherent method, and to form a stable isolation culture and amplification of BMSCs, so as to obtain the BMSCs of rat with high purity, good activity and homogeneous form, which provides sufficient cell source for the transplantation of VD to ensure the smooth progress of the experiment.
[Methods] the seven day old SD rats were killed and the femur and tibia were rinsed repeatedly with the medium. The bone marrow was collected and the bone marrow was collected. The modified full bone marrow adherent culture method was used to separate, cultivate and purify BMSCs. The morphology of BMSCs, the growth condition and the cell growth curve were observed to understand the shape characteristics and growth characteristics of BMSCs. Flow cytometry was used to determine the cell surface. BMSCs was identified by surface markers and osteogenic induction.
[results] the primary BMSCs was observed under the optical microscope. The cell growth was rapid and the cell growth was rapid. The cell morphology was still fusiform after 5-6 days of fusion. The cell morphology was still spindle shaped, the fish group was arranged, the colony growth was whirlpool, and the cells reached saturation in the seventh day and grew slowly. The three generation BMSCs expressed CD29, CD90, and the positive rate was 99%, 99.6%, respectively, and almost did not express CD34, CD45, and the positive rate was 0.169% respectively. 1.19%. was stained with alizarin red after osteogenesis of BMSCs, and the calcium nodule was dark red.
[conclusion the BMSCs of]SD rats can be isolated, cultured and purified in vitro. The whole bone marrow adherent culture method can be isolated and cultured and amplified BMSCs, and the rat BMSCs with high purity, good activity and homogeneous form can provide sufficient experimental cell source.
The third chapter
Effect of mannitol on transplantation of bone marrow mesenchymal stem cells in rats with vascular dementia
[Objective] to compare the behavior changes of the VD rat model of the direct transplantation of BMSCs and mannitol pretreated with BMSCs transplantation, the changes in the expression of Nogo protein and its receptor in the hippocampus CA1 region, and to explore the effect and mechanism of mannitol on the therapeutic effect of BMSCs on the treatment of VD rat model with intravenous transplantation.
[Methods] the VD rats were divided into five groups, including 8 rats in the model control group (without any treatment) and 10 in the mannitol group (37 centigrade and 20% mannitol solution, 1.5g/kg) in the mannitol group, and 10 in the medium group (the tail vein was injected with 1m workers without serum low sugar medium), and (4) the BMSCs group 13 (the tail vein injection cell concentration was 1 * 106 /ml. Three generation of BMSCs) BMSCs group pretreated with mannitol (37 degrees centigrade, 20% mannitol solution, 1.5g/kg, 10-20min, 1 x 108 /m1 third generation BMSCs), and 9 rats in the sham operation group. After the same condition for 4 weeks, the water maze test was used to detect the spatial learning and memory ability of each group and use immunohistochemistry. The changes of Nogo A protein and its receptor expression in hippocampus CA1 region of each group were detected.
[results group]BMSCs and model control group, mannitol group, culture group comparison, escape latency shortened, platform quadrant retention time extended, the difference was statistically significant (P0.05); the escape latency of mannitol pretreated group BMSCs was significantly shorter than that of the BMSCs group, and its platform image limited retention time was also significantly longer than that of the BMSCs group. The study significance (P0.05) had not reached the level of the sham operation group. However, there was no significant difference between the BMSCs group and the 3-5 day escape latency of the mannitol preconditioning group (P0.05), and there was no significant difference between the two groups of platform quadrant retention time (P0.05).
The positive products of Nogo A protein and its receptor in the hippocampal CA1 region of BMSCs group were compared with that of the model control group, and the expression in the mannitol group was less and the staining was shallow, and the Nogo A protein and its receptor staining of the mannitol pretreated group BMSCs were not only lighter than the model control group, the mannitol group and the culture group, but also lighter than the BMSCs group, but the positive products were also less, but also less, but the positive products were less than those in the BMSCs group. The depth and area of the stain were more than that of the sham operation group.
[Conclusion] the third generation of BMSCs from the tail vein can improve the spatial learning and memory ability of VD rats. The effect of BMSCs on the spatial learning and memory ability of VD rats after mannitol preconditioning is better than that of VD rats injected with BMSCs alone. The mechanism may be that mannitol improves the blood brain barrier to BMSCs and various kinds of blood brain barrier. The penetration rate of brain nutrient factors increases the expression level of various brain nutrition factors in the brain of VD rats, and reduces the inhibitory effect of the Nogo protein signal system on the growth of synapses, thus promoting the recovery of nerve function.
【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;R965

【参考文献】