| ObjectiveBone marrow mesenchymal stem cells(BMSCs) is coming from bone marrow fluid, derived from the mesoderm, and possessed more potential to differentiate adult stem cells. In certain conditions it can induce mesoderm from a variety of cells, such as osteoblasts, adipocytes, muscle cells. Recent years, with in-depth study in BMSCs, we found that BMSCs also have cross-germ layer differentiation potential. BMSCs transplanted into the central nervous system of living can survive and can differentiate into neurons, glial cells. External experiments have confirmed that under certain conditions, BMSCs have been induced to neurons, glial cells transformation, can be improved to varying degrees traumatic brain injury, stroke and other neurological diseases in the defects state.So, BMSCs is a very good transplantation treatment of neurological disorders of cells is of great clinical value and prospects. Purpose of this study is to explore the using by comparing the two methods:①density gradient centrifugation and fetal bovine serum adherent culture and②extracting the whole bone marrow fluid and fetal bovine serum adherent culture through extracting and culturing the Sprague Dawley rats,BMSCs. Observation of cell morphology, biological characteristics, phenotype of cell etc. proliferation and biological activity in various aspects of whether the differences to evaluate the advantages and disadvantages of both methods, In the hope of finding the better extracting and culturing the bone marrow mesenchymal stem cells methods.MethodBy density gradient centrifugation, and total bone were extracted from the left and right lower limb 4-6 week-old SD rats, of BMSCs in sterile conditions, Then with 10% fetal bovine serum LG-DMEM (low glucose DMEM) BMSCs cultured and amplified,Extracts by density gradient centrifugation and then by BMSCs in primary culture of fetal bovine serum adherent obtained after passage over a large number of amplification can be purified BMSCs cells (cells obtained A), Obtained under sterile conditions in the whole bone marrow adherent culture in fetal calf serum medium was changed after the through passage, also get a lot of BMSCs (get the cell to B), Flow cytometry density gradient centrifugation, two methods of extraction and whole bone marrow cells cultured CD29 +, CD44-, CD45-expression, identification of the cultured BMSCs, BMSCs to draw the growth curve and cell morphology were compared BMSCs , BMSCs cryopreservation survival rate and growth status.Result1.The A group and the B group are more stable biological traits BMSCs, cultured cells are all first round, stable cells were passaged BMSCs typical spindle, and whirlpool arrangement growth; a typical characteristic of adherent growth;2.By flow cytometry the A group and the B group, BMSCs cell surface markers: CD29+,CD44-,CD45-, the results of the A group CD29+(93.24%),CD44-(94.90%),CD45-(94.22%); the B group CD29+(92.89%),CD44-(96.70%),CD45-(93.10%), are in line with the characteristics of BMSCs;3.The A group and the B group were adherent BMSCs growth of primary cells was detected by trypan blue staining activity of group A was 99.1% adherent (B) was 99.7%, using the statistical software SPSS16.0 statistical analysis, the difference were significant; 4.The A group of cell growth characteristics: BMSCs adherent growth in primary culture for 10 days reached 90% confluence, after passage of the BMSCs proliferation rate than the primary (P0) develop faster, third-generation (P3) of BMSCs has been basically purified, BMSCs were Typical spindle-shaped arrangement of vortex growth, with the passage, reduced cell proliferation and differentiation; the B cells is different from the A group: P0 cells for the whole bone marrow cells, medium was changed, the process is substantially purified by passage the main method of the fourth generation (P4) cells were observed only meet the basic purification, but the resulting cells activity was stronger than the A group.Conclusion1.Density gradient centrifugation and cultured whole bone marrow adherent both fetal bovine serum extracted, cultured SD rat bone marrow mesenchymal stem cells can be obtained more purified BMSCs, and both have the biological characteristics of BMSCs; 2.Density gradient centrifugation of mesenchymal stem cells extracted cell activity than whole bone marrow method (statistical difference), this difference may be related to the process of centrifugation and washing repeated loss and damage caused to the cells and other reasons;3.The bone marrow extraction method and density gradient centrifugation method than simple, low cost, density gradient centrifugation short period of time required for extraction of high purity mesenchymal stem cells. |
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