The Isolation And Culture Of Bone Marrow Mesenchymal Stem Cells And The Influence Of Erebrospinal Fluid On Bone Marrow Mesenchymal Stem Cells

Objective: To establish the methods of isolation , purification and amplification of bone marrow mesenchymal stem cells (BMSCs) in vitro and explored the potentiality of BMSCs differentiating into neuron-like cells, and analyzed some of its phenotypic characteristics as well. To study the efection of cerebrospinal fluid(CSF) on bone marrow mesenchymal stem cells culture in vitro and to provide preliminary experimental evidence For BMSCs,Transplantation.Methods: 1. We selected the Clean SD rats, weighing 150g～200g. BMSCs were isolated and purified By density gradient centrifugation , adherent culture and the whole bone marrow culture, and BMSCs were amplified by passaged in vitro. The morphology of BMSCs was observed and the expression of cell surface antigens of BMSCs were analyzed with immunohistochemical method.2.The BMSCs were induced into neuron-like cells with antioxidants.The process of differentiation was observed. Specific markers of induced cells, such asβ-Ⅲ- Tubulin,Nestin and NSE which were expressed in nerve cells, were detected by immunofluorescence.3. The third and forth cells were cultured in cerebrospinal fluid to observe cells growth conditions, and analyzed some of its phenotypic characteristics as well. Cell growth curve would be made.Results: 1. BMSCs were mononuclear cells in the bone marrow. We had successfully and effectively isolated and purified BMSCs by density gradient centrifugation with adherent culture and the whole bone marrow culture, and stably passaged more than 20 passages . The results of the identification were positive of CD90, CD106, CD71, CD29,while CD45 was negative. 2. After neurogenic induced by bFGF and BHA ,the morphology of BMSCs had changed.BMSCs induced by bFGF for 24h had become polygon and rugosity from thin flat or Fusiform.The body of BMSCs induced by DMSO and BHA for 5h became round, and their refractive index was reinforced. BMSCs displayed long bipolar branching .The number of morphology changed cells increased progressively and some of them formed bipolar ,tripolar , and long branches. After 24h,the cells were more like nerve cells and interwoven into mesh. After neurogenic differentiation, immunohistochemically analysis showed that nerve cell surface marker were positive of Nestin,β-Ⅲ-Tubulin, NSE and CD106 which was surface marker of BMSCs. 3. Bone marrow mesenchymal stem cells could still prolifcrate in cerebrospinal fluid,thus it indicated that these cells possess of high adaptability to their growth environment.Their Characteristics and Phenotype was not impacted.Conclusion: This study has established a method of isolation, purification and amplification of BMSCs in vitro. BMSCs isolated by percoll lymphocyte separating solution can be quikly amplified and purified and stably cultured. BMSCs could be induced to differentiate into neuron-like cells with bFGF/BHA. Bone marrow mesenchymal stem cells could still be prolifcrated in cerebrospinal fluid, thus it indicats that these cells possess of high adaptability to their growth environment. Cerebrospinal fluid could become a good culture methods for BMSCs.