The Experimental Study Of Allogeneic Bone Marrow Mesenchymalstem Cells Transplantation For The Treatment Of Vascular Dementia Rats Model

【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.