| Objective:The cerebral vascular accident is one of three severe causes of death in the world and is also the first pathogeny of the adult disability.The year incidence of the cerebral vascular accident in our country is approximately 130-300 tenthousand, and 60-100 tenthousand are dead among them, and seventy-five percent survivors had some disability. Although great progress about the first aid and early healing to the cerebral vascular accident had been obtained, there were short of effective treatment for the later severely neurological deficiencies of accident. 21st century is the century of regeneration. The study of stem-cell is the focus of life medicine. Compare with the embryo-stem cell and the neuro-stem cell, BMSCs have many merits, including:①get and separate the material conveniently and easily, we can withdraw it from our own bone marrow.②Expands and increases in vitro rapidly and has multi-potential.③BMSCs are early cells from mesoblast, which can avoid tissue type and immuno-exclusion.④can avoid the ethical dispute and so on. More and more scholars had paid attention to the BMSCs. Because the transplanted BMSCs have more probability to translate to the neuro-glial cell than neuron-like cell in internal-entironment, so if we can translate the BMSCs to neuron-like cell in vitro, we can have twice the result with half the effort to transplant the neuron-like cells.By establishing the cell-culturing system in vitro of hBMSCs and neuron-like cell from hBMSCs early and observing the biological properties, hBMSCs and the neuron-like cells differentiated from hBMSCs are considered to be good resources of cell transplant and the cell treatment. The objective of this study is to explore a new method for the isolation, purification and amplification in vitro of hBMSCs from human bone marrow. We observe their biological character and directional differentiate to neuron- type cell, and observed the survival, migration, differentiation and the recovery of neurological deficits after transplantation of hBMSCs neuron- type cell into ischemic rats by stereotactic operation. Then we explored the effects of hBMSCs neuron- type cell transplantation on the repair of neurological functions and possible mechanism, so that theoretical assistance for clinical therapy of cerebral ischemia with hBMSCs neuron- type cell transplantation could be provided.Method:(1) BMSCs were isolated and purified from the bone marrow of human by density gradient centrifugation and by adhering to the culture plastic. After successive subculture and amplification, the growth curve was drawn, the morphology and growth characteristics were observed under phase contrast microscope , and the cell surface antigens were examined by immunocytochemistry and image analysis in vitro; (2) BMSCs were co-cultured with T lymphocytes with 3H-TdR were mixed to analysis its immunogenicity.(3) BMSCs were induced towards neuro- cells with all-trans-retinoic acid (ATRA) and butylated hydroxyanisole(BHA), then identified with immunocytochemistry. (4) Focal cerebral ischemia was induced by transient MCA with a monofilament suture in adult Wistar rats. The cerebral ischemia rats were divided into three groups including group PBS, group hBMSCs and group of neuron-like cells from BMSC randomly. We transplant the hBMSCs marked by PBS and BrdU and neuron-like cells from BMSC marked by BrdU into rats'striatum by stereotactic operation. We used the NSS, BBT, EBST, SDPAT and WMT to evaluate the rats at pro-operation and 1st, 3rd,6th,8th week post-operation. These rats were killed at 8th week after cerebral ischemia. In the same time, HE staining, immunohistochemical staining for BrdU, and immunohistochemical doublestaining for BrdU/NSE and BrdU/GFAP were processed to observe the survival, migration and differentiation of grafted BMSCs.Result: (1) Human bone marrow mesenchymal stem cells can generate with large quantity in vitro and primary culture require 2~3 weeks under conventional culture, the growth velocity speed up after passage. We can get a number of cells adhering to the culture plastic.Uiform fibroblast-like cells were got and the surface antigen of MSCs detected by flow cytometry show it is positive for CD44,CD29,CD105 and negative for CD45,CD106,CD34,HLA-DR. (2) When BMSCs were cocultured with T lymphocytes, the proliferation of T cells was not found. It suggested the low immunogenicity of the BMSCs. (3) BMSCs could be induced to neuron-like cells, which developed rounded cell bodies with multiple neuron-like extension as well as several neuronal proteins such as neuron specific enolased and astrocyte marker glial fibrillary acid protein. (4) Left hemiplegia and right Honner's sign were observed after transient right MCA with a monofilament suture. TTC and HE staining showed that ischemic regions were located in the right striatum and frontoparietal cortex. The position and area were relatively stable each time. The NSS, BBT,EBST of group hBMSCs are much better than the group PBS at the 1st,3rd,6th,8th week post-operation (P<0.01), and better than the group of neuron-like cells from hBMSCs at 1st,3rd week post-operation(P<0.05), but there is no statistical significance between the group hBMSCs and group of neuron-like cells from hBMSCs(P>0.05); The NSS, BBT,EBST of group of neuron-like cells from hBMSCs are much better than the group PBS at the 3rd,6th,8th week post-operation (P<0.01), is lower than the group hBMSCs at 1st,3rd week post-operation(P<0.05), and there is no statistical significance between the two groups at the 6th ,8th week (P>0.05) .The SDPAT and WMT of the two groups of neuron-like cells and hBMSCs are much better than the control group, and the effection of group hBMSCs is worse than the group of neuron-like cells from hBMSCs (P<0.05). After the 8th week of transplantation, numerous cells labeled with BrdU were present at both of the group hBMSCs and group neuron-like cells from BMSCs, which were present in the injecting points and surrounding areas, and had the trend to migration to the ischemia areas. There were no significance glial-cell proliferation and lymphocyte infiltration. Among all the Brdu reactive cells of group hBMSCs, 11.5-19.3% were reactive for the astrocyte marker glial fibrillary acidic protein(GFAP), 1.5-4.8% expressed the neuronal markers neuron specific enolase(NSE). Among all the Brdu reactive cells of group of neuron-like cells from hBMSCs, 23.5-39.1% were reactive for the neuronal markers neuron specific enolase(NSE), 9.8-17.6% expressed the astrocyte marker glial fibrillary acidic protein(GFAP).Conclusion:hBMSCs are mono-neuclear cells in the bone marrow,and have much charactistics such as low inmunogenicity, good plasticity, strong generation et.al , and we can isolate and purify the hBMSCs by density gradient centrifugation combined with adhering to the culture plastic effectively . The cultured cells can induced by the all-trans-retinoic acid (ATRA) and butylated hydroxyanisole(BHA) to nerve cells, and can be induced to neuron-like cells from hBMSCs wich expressed the neuron-spesific protein—neuron specific enolased. The two groups of hBMSCs and neuron-like cells from hBMSCs both can improve the neurological function of the focal cerebral ischemia rats effectively, but hBMSCs have faster effection than another. The transplantion effection on improving study and memory of group of neuron-like cells from hBMSCs is better than another group. The most of neuron-like cells from hBMSCs in vivo are expressed NSE which is better than the group of hBMSCs, and there are no tumors appear and inflammation responds on local regions. The intra-cerebral transplantation of the neuron-like cells from hBMSCs would be one of measures for treatment of local cerebral ischemia. |
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