【摘要】：第一章大鼠BMSCs體外分離、培養(yǎng)、鑒定和標(biāo)記 [目的]探索SD大鼠骨髓間充質(zhì)干細(xì)胞(Bone Marrow Mesenchymal Stem Cells, BMSCs)體外分離、培養(yǎng)、鑒定及標(biāo)記的方法。為下一步的移植研究提供充足的BMSCs。 [方法]應(yīng)用全骨髓貼壁改良法分離培養(yǎng)SD大鼠BMSCs,顯微鏡觀察細(xì)胞形態(tài),繪制細(xì)胞生長曲線。使用流式細(xì)胞儀檢測細(xì)胞表面標(biāo)記物及茜素紅成骨誘導(dǎo)染色對第三代細(xì)胞進(jìn)行鑒定。并對第3代細(xì)胞進(jìn)行BrdU標(biāo)記。 [結(jié)果]體外培養(yǎng)的原代BMSCs生長旺盛,以梭形為主,細(xì)胞集落呈螺旋狀。第3代BMSCs幾乎均一表達(dá)CD29,CD90,而CD34,CD45幾乎不表達(dá)。BMSCs經(jīng)誘導(dǎo)能分化為成骨細(xì)胞,茜素紅染色出現(xiàn)紅色鈣化結(jié)節(jié)。BrdU免疫組化結(jié)果顯示,BMSCs標(biāo)記率高達(dá)85.29%。 [結(jié)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(qiáng),性狀穩(wěn)定。BrdU標(biāo)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(jié)扎雙側(cè)頸總動(dòng)脈法制備VD大鼠模型。假手術(shù)組不結(jié)扎雙側(cè)頸總動(dòng)脈,其余手術(shù)操作步驟相同。術(shù)后4周,采用Morris水迷宮試驗(yàn)測定模型大鼠的行為學(xué)表現(xiàn)。 [結(jié)果]假手術(shù)組術(shù)后因飼養(yǎng)不當(dāng)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(dá)VD標(biāo)準(zhǔn)。本實(shí)驗(yàn)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(shù)組明顯延長,平臺(tái)象限滯留時(shí)間比假手術(shù)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(jié)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(diǎn),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(zhì)干細(xì)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(yù)處理對骨髓間充質(zhì)干細(xì)胞移植治療VD大鼠模型療效及海馬CA3區(qū)突觸素表達(dá)水平的影響。 [方法]造模4周后將VD大鼠隨機(jī)分為空白對照組、培養(yǎng)基組、甘露醇組、BMSCs組、甘露醇預(yù)處理BMSCs組,分別給予相應(yīng)的實(shí)驗(yàn)干預(yù)措施。觀察干預(yù)4周后實(shí)驗(yàn)大鼠的行為學(xué)表現(xiàn)及應(yīng)用免疫組化法檢測海馬CA3區(qū)突觸素的表達(dá)水平。 [結(jié)果]甘露醇預(yù)處理BMSCs組行為學(xué)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(tái)象限滯留時(shí)間延長(40.75±6.29s,P0.05),其海馬CA3區(qū)突觸素表達(dá)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(jié)論]甘露醇預(yù)處理后靜脈移植BMSCs顯著改善VD大鼠行為學(xué)表現(xiàn),并使其海馬CA3區(qū)突觸素表達(dá)顯著增加。甘露醇預(yù)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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.13

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 扈榮;鐘曉明;;血管性癡呆動(dòng)物模型的研究概況[J];浙江中醫(yī)藥大學(xué)學(xué)報(bào);2008年01期

2 張艷玲,李露斯,肖桃元,可金星;血管性癡呆大鼠的突觸超微結(jié)構(gòu)改變及結(jié)構(gòu)參數(shù)分析[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2002年03期

3 段峰;王茂強(qiáng);;干細(xì)胞標(biāo)記示蹤技術(shù)的研究進(jìn)展[J];國際醫(yī)學(xué)放射學(xué)雜志;2009年06期

4 辛洪啟;突觸素在神經(jīng)元發(fā)育過程中的作用[J];國外醫(yī)學(xué)(神經(jīng)病學(xué)神經(jīng)外科學(xué)分冊);2003年03期

5 楊龍秀;劉金萍;張?zhí)i;陳志;莫雪安;;甘露醇對骨髓間充質(zhì)干細(xì)胞治療血管性癡呆大鼠行為學(xué)的影響[J];廣西醫(yī)科大學(xué)學(xué)報(bào);2011年01期

6 康建磊;王寅千;于書卿;;藥物透過血腦屏障的研究進(jìn)展[J];國際神經(jīng)病學(xué)神經(jīng)外科學(xué)雜志;2012年04期

7 關(guān)思虞 ,曾秋棠;重組腺病毒介導(dǎo)血管內(nèi)皮生長因子基因在骨髓間充質(zhì)干細(xì)胞中的表達(dá)及其影響[J];華中醫(yī)學(xué)雜志;2005年03期

8 趙憲林,李東培,方秀斌,楊國瑞;血管性癡呆大鼠海馬神經(jīng)元超微結(jié)構(gòu)的研究[J];解剖科學(xué)進(jìn)展;2000年02期

9 秦大蓮,肖順漢;血管性癡呆相關(guān)檢測指標(biāo)的研究進(jìn)展[J];四川生理科學(xué)雜志;2005年03期

10 李濤;程焱;;NGF基因修飾的BMSCs對VaD大鼠海馬區(qū)細(xì)胞凋亡及NMDAR1表達(dá)的影響[J];山東醫(yī)藥;2009年21期

相關(guān)碩士學(xué)位論文 前2條

1 劉金萍;甘露醇在骨髓間充質(zhì)干細(xì)胞治療血管性癡呆大鼠模型中作用的研究[D];廣西醫(yī)科大學(xué);2011年

2 陳志;骨髓間充質(zhì)干細(xì)胞移植對血管性癡呆模型大鼠腦源性神經(jīng)生長因子的影響[D];廣西醫(yī)科大學(xué);2010年

，

本文編號(hào)：2385730