本文選題：血管性癡呆 + 動物模型��； 參考：《廣西醫(yī)科大學》2013年碩士論文

【摘要】：第一章血管性癡呆大鼠模型的制備 [目的]探討制備理想VD動物模型的方法,為進行BMSCs移植制備模型。 [方法]使用Morris水迷宮對實驗大鼠進行初篩,以剔除在95%參考值范圍以外的SD大鼠,將篩選出的實驗鼠根據(jù)隨機原則分為假手術組(10只)和模型組(77只)。模型組采用改良二血管結扎法(間隔三天分次結扎雙側頸總動脈)制備VD大鼠模型,假手術組實施同樣的手術操作,但是不結扎雙側頸總動脈,同等條件籠養(yǎng)4周后,再次行水迷宮實驗,以測定兩組大鼠的空間學習記憶能力變化,篩選出造模成功的VD大鼠。 [結果]90只大鼠經(jīng)水迷宮實驗初篩后剔除3只,納入實驗87只大鼠隨機分成假手術組(10只)和模型組(77只)。造模前,兩組的逃避潛伏期和平臺象限滯留時間差異沒有統(tǒng)計學意義(P0.05),造模后,兩組的逃避潛伏期和平臺象限滯留時間差異有統(tǒng)計學意義(P0.05)。假手術組由于飼養(yǎng)不當死亡1只,余9只；模型組大鼠因麻醉意外死亡4只,感染死亡2只,腸梗阻死亡5只,出血過多死亡2只,肺淤血死亡5只,1只出現(xiàn)眼球病變,3只未達癡呆標準,共制備VD大鼠模型55只,成活率為76.62%,成模率93.22%。 [結論]改良二血管結扎法制備VD模型,不僅操作簡易,可重復性好,而且有較高的存活率,成模率高,是較理想的制備VD模型的方法,適用于實驗研究。 第二章SD大鼠骨髓間充質干細胞的分離、培養(yǎng)及鑒定 [目的]應用全骨髓貼壁改良法分離培養(yǎng)SD大鼠BMSCs,形成穩(wěn)定分離培養(yǎng)和擴增BMSCs的培養(yǎng)方法,以獲得純度高、活性好、形態(tài)均一的大鼠BMSCs,為移植治療VD提供充足的細胞來源,保證實驗順利進行。 [方法]斷頭處死七日齡SD乳鼠,用培養(yǎng)基反復沖洗股骨和脛骨,收集其骨髓,采用改良的全骨髓貼壁培養(yǎng)法分離、培養(yǎng)、純化BMSCs,觀察BMSCs的形態(tài)、生長情況并繪制細胞生長曲線,了解BMSCs的形態(tài)特征及生長特性。采用流式細胞儀測定細胞表面標志物和進行成骨誘導對BMSCs進行鑒定。 [結果]光學顯微鏡下觀察分離培養(yǎng)的原代BMSCs呈梭形貼壁生長。傳代后細胞生長迅速,5-6天達到融合即可再次傳代,細胞形態(tài)仍為梭形,魚群樣排列,集落生長呈漩渦狀,第7天細胞達到飽和出現(xiàn)接觸性抑制而生長緩慢。流式細胞儀鑒定第三代BMSCs均一表達CD29, CD90,陽性率分別為99%,99.6%；幾乎不表達CD34, CD45,陽性率分別為0.169%,1.19%。對BMSCs進行成骨誘導后行茜素紅染色,鈣結節(jié)呈深紅色。 [結論]SD大鼠的BMSCs能在體外分離、培養(yǎng)和純化。全骨髓貼壁改良培養(yǎng)法能穩(wěn)定分離培養(yǎng)和擴增BMSCs,獲得純度高、活性好、形態(tài)均一的大鼠BMSCs,可提供充足的實驗細胞來源。 第三章 甘露醇對骨髓間充質干細胞移植治療血管性癡呆大鼠效果觀察 [目的]比較直接移植BMSCs與甘露醇預處理后再進行BMSCs移植的VD大鼠模型的行為學改變,海馬CA1區(qū)Nogo蛋白及其受體表達量的變化,探討甘露醇對靜脈移植BMSCs治療VD大鼠模型療效的影響及機制。 [方法]造模成功的VD大鼠隨機分為五組,①模型對照組8只(不進行任何處理),②甘露醇組10只(尾靜脈注射37℃、20%甘露醇溶液,1.5g/kg),③培養(yǎng)基組10只(尾靜脈注射1m工無血清低糖培養(yǎng)基),④BMSCs組13只(尾靜脈注射細胞濃度為1×106個/ml的第三代BMSCs)⑤甘露醇預處理BMSCs組14只(尾靜脈注射37℃、20%甘露醇溶液,1.5g/kg,10-20min后,注射細胞濃度為1×108個/m1的第三代BMSCs),此外設立假手術組9只。同等條件下籠養(yǎng)4周后,行水迷宮實驗檢測各組大鼠的空間學習記憶能力,使用免疫組化檢測各組大鼠海馬CA1區(qū)的Nogo A蛋白及其受體表達水平的變化。 [結果]BMSCs組與模型對照組、甘露醇組、培養(yǎng)基組比較,逃避潛伏期縮短,平臺象限滯留時間延長,差異具有統(tǒng)計學意義(P0.05)；甘露醇預處理BMSCs組的逃避潛伏期比BMSCs組明顯縮短,其平臺象限滯留時間也比BMSCs組顯著延長,差異均具有統(tǒng)計學意義(P0.05),尚未達到假手術組的水平。但是,甘露醇預處理BMSCs組與假手術組第3-5天的逃避潛伏期比較,差異沒有統(tǒng)計學意義(P0.05),兩組的平臺象限滯留時間比較,差異也沒有統(tǒng)計學意義(P0.05)。 BMSCs組海馬CA1區(qū)Nogo A蛋白及其受體免疫組化陽性產(chǎn)物比模型對照組、甘露醇組、培養(yǎng)基組表達量少,染色淺；甘露醇預處理BMSCs組的Nogo A蛋白及其受體染色不但比模型對照組、甘露醇組、培養(yǎng)基組淺,而且也比BMSCs組淺,陽性產(chǎn)物也較少,但染色的深度及面積超過假手術組。 [結論]尾靜脈注射移植第三代BMSCs能夠改善VD大鼠的空間學習記憶能力。經(jīng)過甘露醇預處理后再行尾靜脈注射移植BMSCs使VD大鼠的空間學習記憶能力改善效果優(yōu)于單獨注射BMSCs的VD大鼠。其機制可能是甘露醇提高血腦屏障對BMSCs及各種腦營養(yǎng)因子的通透率,進而增加VD大鼠腦內各種腦營養(yǎng)因子的表達水平,減低Nogo蛋白信號系統(tǒng)對突觸生長的抑制作用,從而促進神經(jīng)功能恢復。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R749.13;R965

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