發(fā)布時間：2018-12-18 10:43

【摘要】：第一章大鼠BMSCs體外分離、培養(yǎng)、鑒定和標記 [目的]探索SD大鼠骨髓間充質干細胞(Bone Marrow Mesenchymal Stem Cells, BMSCs)體外分離、培養(yǎng)、鑒定及標記的方法。為下一步的移植研究提供充足的BMSCs。 [方法]應用全骨髓貼壁改良法分離培養(yǎng)SD大鼠BMSCs,顯微鏡觀察細胞形態(tài),繪制細胞生長曲線。使用流式細胞儀檢測細胞表面標記物及茜素紅成骨誘導染色對第三代細胞進行鑒定。并對第3代細胞進行BrdU標記。 [結果]體外培養(yǎng)的原代BMSCs生長旺盛,以梭形為主,細胞集落呈螺旋狀。第3代BMSCs幾乎均一表達CD29,CD90,而CD34,CD45幾乎不表達。BMSCs經(jīng)誘導能分化為成骨細胞,茜素紅染色出現(xiàn)紅色鈣化結節(jié)。BrdU免疫組化結果顯示,BMSCs標記率高達85.29%。 [結論]改良的全骨髓貼壁法體外分離、培養(yǎng)及傳代的SD大鼠BMSCs純度高,活力強,性狀穩(wěn)定。BrdU標記法安全、高效。 第二章血管性癡呆大鼠模型的制備 [目的]探討VD大鼠模型理想的制備方法,為下一步BMSCs移植研究提供VD大鼠模型。 [方法]采用間隔3天永久性結扎雙側頸總動脈法制備VD大鼠模型。假手術組不結扎雙側頸總動脈,其余手術操作步驟相同。術后4周,采用Morris水迷宮試驗測定模型大鼠的行為學表現(xiàn)。 [結果]假手術組術后因飼養(yǎng)不當死亡1只,余9只。模型組大鼠存活59只,死亡18只,1只出現(xiàn)眼球變白、萎縮,3只未達VD標準。本實驗成功制備55只VD大鼠。模型組大鼠造模后水迷宮的逃避潛伏期較假手術組明顯延長,平臺象限滯留時間比假手術組明顯縮短(p0.05)。模型組大鼠的存活率為76.62%,成模率為93.22%。 [結論]改良雙血管法具有死亡率低,成模率高,簡單易行等優(yōu)點,是制備VD大鼠模型的理想方法。 第三章甘露醇對骨髓間充質干細胞靜脈移植治療血管性癡呆大鼠模型的療效觀察 [目的]探討甘露醇預處理對骨髓間充質干細胞移植治療VD大鼠模型療效及海馬CA3區(qū)突觸素表達水平的影響。 [方法]造模4周后將VD大鼠隨機分為空白對照組、培養(yǎng)基組、甘露醇組、BMSCs組、甘露醇預處理BMSCs組,分別給予相應的實驗干預措施。觀察干預4周后實驗大鼠的行為學表現(xiàn)及應用免疫組化法檢測海馬CA3區(qū)突觸素的表達水平。 [結果]甘露醇預處理BMSCs組行為學較BMSCs組、培養(yǎng)基組、甘露醇組、空白對照組有明顯改善(P0.05),其逃避潛伏期縮短(P0.05),平臺象限滯留時間延長(40.75±6.29s,P0.05),其海馬CA3區(qū)突觸素表達水平(39624±7797)亦比BMSCs組、培養(yǎng)基組、甘露醇組、空白對照組明顯提高(27060±4667,19955±4243、18293±6445,18946±4953,P0.05)。 [結論]甘露醇預處理后靜脈移植BMSCs顯著改善VD大鼠行為學表現(xiàn),并使其海馬CA3區(qū)突觸素表達顯著增加。甘露醇預處理明顯提高靜脈移植BMSCs治療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 鹵6.29 s). The level of synaptophysin expression in hippocampal CA3 region (39624 鹵7797) was significantly higher than that in BMSCs group, medium group, mannitol group and blank control group (27060 鹵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.
【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

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