【作者】 莫雪安；

【导师】 赖永榕；

【作者基本信息】 广西医科大学 ， 内科学， 2011， 博士

【摘要】 【目的】探讨大鼠骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells, BMSCs)体外分离、培养、纯化、传代、鉴定和标记的方法,为进行BMSCs移植治疗血管性痴呆(Vascular dementia, VD)大鼠模型提供功能稳定的种子细胞。【方法】采用全骨髓细胞培养贴壁筛选法在体外分离培养大鼠BMSCs,相差显微镜观察BMSCs细胞形态,透射电镜观察BMSCs超微结构并绘制生长曲线,使用地塞米松等物质诱导其向成骨细胞和脂肪细胞两个方向分化。用BrdU对BMSCs进行体外标记,通过免疫组化染色观察并计算BrdU阳性细胞标记率。①选取3-4W龄SD大鼠,从其股骨中取材,在无菌条件下操作,用剪刀剪去股骨的两端,暴露骨髓腔,用含15%胎牛血清(FBS)的DMEM-LG完全培养基从骨髓腔中冲出骨髓于平皿中,并用同样含15%FBS的DMEM-LG完全培养基培养,用消化控制和全骨髓细胞贴壁分离法分离纯化BMSCs,观察其生长状态,相差显微镜观察BMSCs的细胞形态,透射电镜观察BMSCs超微结构并绘制生长曲线。②取生长旺盛的第3代细胞,用流式细胞仪检测BMSCs表面抗原CD34(PE标记)、CD90 (FITC标记)、CD44 (FITC标记)及同型对照小鼠抗大鼠IgG1型单克隆抗体(分别为PE和FITC标记)。并分别以诱导其向骨细胞分化及向脂肪细胞分化的方法对分离扩增的BMSCs进行鉴定。③用5-溴-2’-脱氧尿嘧啶核苷(BrdU)免疫组化染色方法对第3代细胞BMSCs进行体外标记,通过免疫组化染色观察并计算BrdU阳性细胞标记率。【结果】①原代培养的SD大鼠BMSCs为大小不等圆形细胞。纯化、扩增后BMSCs呈均匀一致的长梭型,传代后的BMSCs形态趋于均一,排列有序,呈鱼群样或漩涡状排列。BMSCs超微结构见SD大鼠BMSCs细胞体积较小,核质比例较大,细胞核呈椭圆形,也可见不规则形,核仁1-2个大而明显,核染色质较疏松,细胞表面可见有较多微绒毛,胞质中可见较丰富的细胞器,如线粒体、粗面内质网和高尔基体。传代周期为8-9d,细胞接种后1～2 d为滞留期,3d后达对数生长期,第6天进入平台期。②流式细胞仪检测第3代细胞示,CD44阳性率为89.4%、CD90为99.2%,CD34为1.82%。经诱导BMSCs向骨细胞分化后,茜素红S染色可见钙结节阳性。经诱导BMSCs向脂肪细胞分化后,油红O染色,苏木素复染,细胞内可见圆形脂滴空泡,被油红O染成特异性橘红色。③10μmol/LBrdU体外孵育48h后BrdU阳性细胞呈棕黄或黄褐色,阳性反应物位于细胞核,标记率为89%～92%。【结论】体外BMSCs易于分离、培养和扩增。体外培养的BMSCs有独特的超微结构特点。BMSCs可通过诱导向成骨细胞和脂肪细胞分化。用BrdU标记BMSCs的浓度为10μmol/L,时间为48h。【目的】研究立体定位海马移植同种异体BMSCs,对VD大鼠模型的学习记忆功能障碍改善的疗效、脑组织病理学改变、脑组织中移植BMSCs的存活迁移情况等的影响。【方法】①用2-VO法制作血管性痴呆大鼠模型,将11～13月龄的SD大鼠随机分成3组：假手术组10只,暴露双侧颈总动脉但不结扎；培养组基对照15只,结扎双侧颈总动脉(2-VO),30d后脑立体定位将10μl PBS注入大鼠海马内；BMSCs移植组15只,2-VO 30d后脑立体定位将1×106BMSCs移植入大鼠海马内。②饲养实验大鼠4w后以Morris水迷宫作定位航行试验和空间探索实验检测各组大鼠空间学习记忆能力。③HE染色后光镜下观察各组大鼠海马区和额叶皮质的病理变化。④免疫组织化学染色观察BrdU标记的BMSCs在VD大鼠脑组织中的存活及迁移情况进行研究。【结果】①造模前各组大鼠Morris水迷宫检测成绩差别无统计学意义,海马定位移植4w后,模型对照组与假手术组比较,平均逃避潜伏期长(P<0.01),且60s内在平台象限滞留时间短(P<0.01)。BMSCs移植组逃避潜伏期比模型对照组明显缩短(P<0.01), BMSCs移植组在平台象限滞留时间较模型对照组长(P<0.05)。但都达不到假手术组的水平(P<0.01)。②光镜下可见模型对照组大鼠海马及额叶皮质的细胞排列不齐,细胞核固缩,胞质密度增高,细胞脱失。而BMSCs移植组上述病理改变较轻。③BMSCs移植组经海马定位移植BMSCs 4w后,可在实验大鼠海马观察到呈棕黄或黄褐色的BrdU标记BMSCs,有局灶聚集现象。额叶也见少量分散的BrdU阳性细胞。【结论】①BMSCs海马定位移植治疗使VD模型大鼠空间学习记忆能力得到改善,但未达到假手术组水平。②BrdU免疫组化染色证实海马定位移植的BMSCs能在海马存活,并且能够在移植大鼠脑内迁移。【目的】研究甘露醇预处理对同种异体BMSCs静脉移植治疗VD模型大鼠学习记忆功能障碍改善疗效的影响,通过观察实验大鼠脑组织脑源性神经营养因子(BDNF)含量、血管内皮生长因子(VEGF)及海马胆碱能系统活性,探讨甘露醇预处理后BMSCs静脉移植治疗VD模型大鼠疗效的机制。【方法】①造模成功后将VD模型大鼠随机分成模型对照组8只(注射1ml无血清培养基)、BMSCs移植组11只(注射1×106个BMSCslml)及甘露醇预处理BMSCs移植组9只(注射1.5g/kg甘露醇后,在10-30分钟之内注射1×106个BMSCslml)。还设假手术组7只,暴露双侧颈总动脉,但不结扎即缝合,不进行任何干预。②移植后笼养实验大鼠4w后,Morris水迷宫作定位航行试验和空间探索实验检测各组大鼠空间学习记忆能力。③HE染色观察大脑病理结构的变化。④ELISA法检测各组实验大鼠脑组织BDNF含量变化。⑤ELISA法检测各组实验大鼠脑组织VEGF的含量变化。⑥检测实验大鼠海马胆碱乙酰转移酶(ChAT)和乙酰胆碱酯酶(AChE)的活性。【结果】①BMSCs静脉移植4w后,大鼠空间学习记忆能力测试结果显示,甘露醇预处理后BMSCs移植组和BMSCs移植组的逃避潜伏期均较培养基对照组明显缩短(分别p<0.01,p<0.05),平台象限平均滞留时间均延长(分别p<0.01,p<0.05)；甘露醇预处理后BMSCs移植组的逃避潜伏期比BMSCs移植组更明显缩短,平台象限平均滞留时间更长(p<0.001)；甘露醇预处理后BMSCs移植组的的逃避潜伏期和平台象限平均滞留时间与假手术组的差异无统计学意义(P>0.05)。②BMSCs移植组额叶皮质组织HE染色病理片显示其病理损伤较培养基对照组轻,神经元细胞数量较多,神经元肿胀、脱失及核固缩现象减少,而以甘露醇预处理后BMSCs移植组的病理损伤更轻。③BMSCs移植组海马和额叶组织BDNF含量高于培养基对照组(P<0.05)；甘露醇预处理BMSCs移植组海马和额叶BNDF含量不但高于培养基对照组(P＜0.05),而且比BMSCs移植组更高(P＜0.05)；但是,尚未达到假手术组的水平(P<0.05)。④甘露醇预处理BMSCs移植组、BMSCs移植组额叶组织VEGF含量均值比培养基对照组显著增高(P=0.000)；甘露醇预处理BMSCs移植组额叶VEGF含量比BMSCs移植组更高(P=0.000)；与假手术对照组比较,培养基对照组大鼠额叶VEGF含量也升高(p=0.006)。⑤培养基组大鼠海马组织AChE和ChAT活性较假手术组明显减弱(P<0.05); BMSCs移植组大鼠海马组织AChE和ChAT活性较培养基组有明显增强(P<0.05)；甘露醇预处理BMSCs移植组大鼠海马组织AChE和ChAT活性不仅比培养基组有更显著增强(P<0.05),而且比BMSCs移植组也显增强(P<0.05)。[结论]用甘露醇预处理后静脉移植同种异体BMSCs能使VD模型大鼠的学习及记忆能力的改善比单纯静脉移植BMSCs更明显。BMSCs静脉移植可减轻大脑的病理性损伤,而甘露醇预处理后行BMSCs静脉移植的病理损伤更轻。提示甘露醇预处理可能增加了BBB通透性,使更多BMSCs进入脑内发挥作用。静脉移植BMSCs4w时所获的疗效与脑内BDNF和VEGF表达升高,胆碱能神经系统活性增强有关。更多还原

【Abstract】 【Objective】To explore the way of isolation, culture, purification, passage, identification, and labeling of rat bone marrow mesenchymal stem cells (BMSCs), providing seed cells with stabilized functions for transplantation in vascular dementia (VD) rat models.【Methods】BMSCs were harvested from rats by whole marrow method and adherent culture method in vitro, cell morphology was observed by phase contrast microscopy, and BMSCs ultrastructure was observed by transmission electron microscope. BMSCs cellular growth curve was drawn, induced to differentiate into osteoblasts, adipocytes by substances such as dexamethasone. Labeled by BrdU in vitro BMSCs were stained immunohistochemical and calculated the rate of BrdU positive cells.①SD rats aged 3-4 weeks were selected. and cells were collected from their femur bone marrow. All operations were conducted under sterile conditions, during which both ends of the femur were cut off using scissors to expose the bone marrow cavity, and the bone marrow cavity was then washed using 15% fetal bovine serum (FBS) in DMEM-LG complete medium in a petri dish, and cultured with the same complete medium. BMSCs were purified by whole marrow method and adherent culture method. The cells’growth state was observed with a naked, cell morphology by phase contrast microscopy, BMSCs ultrastructure by transmission electron microscope, and the BMSCs cellular growth curve was drawn.②The expression of cell surface antigens of the thriving 3th-generation BMSCs were analyzed by flow cytometry including CD34 (PE labeled), CD90 (FITC labeled), CD44 (FITC labeled) and compared with control mice anti-rat IgG1 monoclonal antibody (labeled with PE and FITC, respectively). After isolated and expended, BMSCs were identified by inducement to differentiate into osteoblasts and adipocytes.③The 3th-generation BMSCs were labeled by BrdU in vitro, stained immunohistochemical and calculated the rate of BrdU-positive cells.【Results】①In primary rat culture, cells were round cells of varying sizes. Purified and expended BMSCs were spindle shape, typically uniform in shape, arranged in order in fish-like or whirlpool-like arrangement. SD rat BMSCs ultrastructural features are as follows:BMSCs were small, nuclear-cytoplasmic ratio was a larger proportion, the nucleus was oval, irregular shape can also be found with 1 to 2 large and obvious nucleolus. The chromatins were loosing. A lot of microvillus were found on the surface of cells. Abundant organelles can be found in cytoplasm, such as mitochondria, rough endoplasmic reticulum and Golgi apparatus. Passage cycle was 8～9d, the 1～2d after passage was latency, convened into the exponential phase after 3d and entered into the beginning of platform growth after 6d.②The results of flow cytometry analysis indicated the positive rate of CD44 was 89.4%, CD90 was 99.2%, CD34 was 1.82%. After osteogenic induction, Alizarin red staining showed the formation of calcium nodules. After adipocytes induction, followed by oil red O dyeing and Hematoxylin staining, specific orange-red round droplets of fat were revealed, stained by oil red O.③48h after culture in 10μmol/L BrdU medium in vitro, BrdU-positive cells were brown or tawny, positive production located in the nucleus, with a labeled rate of 90%.[Conclusion] The culture method combining the whole medulloculture and digestion control is simply performed in need of little condition. The cultured BMSCs are active in high purity with stable biological character. BMSCs of rat cultured in vitro have unique ultrastructural features. BMSCs can be induced to differentiate into osteoblasts and adipocytes. The suitable concentration for BrdU labeling BMSCs is 10μmol/L, over a period of 48h. 【Objective】To research the influence of transplanting BMSCs stereotactically into hippocampus about The improvement of the learning and memory, the histopathological change of brain tissue, the migration and survival of transplanted BMSCs and the expression of BDNF in brain.【Methods】①SD Rats of 11-13 months old received permament bilateral carotid arteries ligation to establish VD models and randomly divided into three groups. sham group (n=10):only bilateral carotid arteries exposed but not ligated. culture medium control group (n=15):lOul serum-free medium was transplanted stereotactically into hippocampus at the Thirty day after bilateral carotid arteries ligation。BMSCs group(n=15):1×106 BMSCs were transplanted stereotactically into hippocampus at the same time.②Fifth week after transplantion, The learning and memory ability of three group rats were tested by Morris water maze.③The histopathological change of hippocampus and frontal lobe were observed by light microscope after HE staining.④Observing and analyzing the survival of BrdU labeled BMSCs in VD rats brain tissue by immunohistochemical staining in the rat brain of three group.【Results】①The learning and memory ability of three group rats was not statistically significant, before ligationo At the fifth week after transplantation, Compared with sham group, The average escape latency of PBS control group rats was lengthened obviously(P<0.01) and the retention time quadrant were receded distinctly(P<0.01); Compared with PBS control group, The average escape latency of BMSCs group was reduced distinctly(P<0.01) the retention time quadrant was lengthened obviously(P<0.01); Compared with BMSCs group,the ethology of sham group was better Performanced (P<0.01).②Sham group cell nuclei become pyknotic, cytoplasmic density increase and cell depigmentation in light microscope.③At the fifth week after transplantation, BrdU positive cells were found in the hippocampus tissue of BMSCs group, in the brain tissue of rats in the treatment group by immunohistochemical assessment. there was a focal gathered phenomenon. A few scattered BrdU positive cells were observeded in the frontal tissue.【Conclusions】①The learning and memory ability of BMSCs group rats was improved at the fifth week after transplantation, but not as well as the sham group.②BrdU immunohistochemical staining confirmed that BMSCs transplanted stereotactically into hippocampus can survival and migrate in brain. 【Objective】To research the influence of Mannitol pretreatment to improve the learning and memory functions after intravenous transplantation allogeneic BMSCs in the rat model of VD. To discuss the mechanism of the effect about intravenous transplantation allogeneic BMSCs and BMSCs transplantation of Mannitol pretreatment in the VD rat model by observeding BDNF expression, VEGF and the hippocampal cholinergic system activity.【Methods】①VD model rats were randomly divided into three groups. culture media control group (n=8):1ml serum-free medium was injected through tail vein at the Thirty day after bilateral carotid arteries ligation。BMSCs group (n=11):1×106 BMSCs in lml PBS were transplanted by tail vein injection。Mannitol with BMSCs group(n=9):intravenous injection of Mannitol at a dose of 1.5 g/kg,10-30 minutes later, intravenous injection of 1×106 BMSCs in lml PBS. Moreover, a sham group (n=10):no ligation and no intravenous injection.②Morris water maze test including training trail and probe trail to detect the spatial learning and memory in rats at the fifth week after transplantation.③Brain tissue pathological change was observeded by HE staining.④BDNF and VEGFcontent in brain tissue were detected by ELISA experiments.⑤The activity of ChAT and AchE in hippocampus were detected respectively ChAT and AchE kit.【Results】①At the fifth week after transplantation, the result of Morris water maze test showed:Compared with PBS control group The average escape latency of Mannitol with BMSCs group and BMSCs group rats was reduced obviously(respectively p<0.01, p<0.05) and the retention time quadrant were lengthened distinctly(respectively p<0.01, p<0.05); Compared with BMSCs group,The average escape latency of Mannitol with BMSCs group was reduced more obviously and the retention time quadrant were lengthened distinctly (p＜0.001); There was no statistical difference between Mannitol with BMSCs group and sham group about the result of Morris water maze test (P>0.05)②The HE staining of Frontal cortex showed:Compared with PBS group, the phenomenon of neuronal swelling、neurons drop out and pyknosis of neurons was reduced in BMSCs group. the phenomenon was reduced more in Mannitol with BMSCs group③Compared with PBS group, BDNF content of Hippocampus and frontal increased obviously in BMSCs group (P<0.05) BDNF content increased more obviously than PBS group and BMSCs group (P <0.05),but There was no statistical difference between Mannitol with BMSCs group and sham group (P>0.05)④Compared with PBS group, the VEGF content of Mannitol with BMSCs group and BMSCs group increased obviously (P=0.000), the VEGF content of Mannitol with BMSCs group increased more obviously than BMSCs group (P=0.000), the VEGF content of PBS group increased obviously than sham group (p=0.006)。⑤The activity of ChAT and AchE in hippocampus of BMSCs group increased obviously than PBS group (P <0.05), The activity of ChAT and AchE of Mannitol with BMSCs group not only increased obviously than BMSCs group,but also increased more obviously than PBS group (P<0.05)【Conclusions】Intravenous transplantation BMSCs can improve spatial learning and memory ability in VD rats. Mannitol pretreatment with intravenous transplantation BMSCs can improve spatial learning and memory ability even more. Intravenous transplantation BMSCs can reduce the pathological damage in VD rats, intravenous transplantation BMSCs of Mannitol pretreatment can reduce the pathological damage even more. The result suggest that Mannitol pretreatment may increase the BBB permeability and more BMSCs got into the brain tissue, and the Efficacy of intravenous transplantation BMSCs associated with the increased expression of BDNF and VEGF and Cholinergic activity in brain, at the fifth week after transplantation.更多还原