| Object:Irreversible nerve damage makes the potential for stem cell transplantation for treatment of more and more attention from researchers. And a stable, safe, and effective source of cells from the body also appears to be particularly important. Mesenchymal Stem Cells have unique features include easy drawn, purification and cultivation technology is more mature, less mmunogenic, multipotent differentiation capacity and strong proliferative capacity, and many other advantages to become a research hotspot in recent years, tissue engineering seed cells. The content of the bone marrow BMSCs limited to neural-like cells uncertainty differentiation mechanism needs further deepened and so limit the clinical application. Therefore, this experiment students affect the application of bFGF on BMSCs to observe the growth process, BMSCs proliferation of bFGF in the course of the role. And neural-like cells by bone marrow mesenchymal stem cells during differentiation arising morphological identification and use of immunocytochemistry express specific markers of neuronal cells, RT-PCR technology to detect other, whereby explore bFGF in BMSCs proliferation and nerve-like cells induced role in the process to provide experimental evidence and theoretical foundation for BMSCs soon be able to clinical services.Method:Birth SPF Wistar rats using aseptic bone marrow cells were isolated and cultured extracted using adherent screening separation and purification of BMSCs, cell surface antigens by flow cytometry to detect markers identified when the third passage, the application of immunity cytochemical methods for cell CD90, CD71-positive cells were identified. At the third-generation (F3) cells continued to be cultured (10%FBS, DMEM/F12), and the cell population is divided into intervention group and control group, the experimental group were added to different concentrations of bFGF. The control group continued using normal culture medium. Conducted when the cells reached80%confluence digestion, passage, train nine days.Difference in the use of optical microscopy methods were observed daily for stem cells, taking pictures, recording growth and proliferation activity and differentiation of BMSCs. Nine days later, the use of technology to detect specific staining of immune cells such as nerve cell marker nestin (Nestin), neuron-specific enolase (NSE), etc.; application of RT-PCR method for neuron-specific enolase (NSE), nestin (Nestin), β-tubulin Ⅲ (β-Ⅲtubulin) three markers for semi-quantitative detection of mRNA expression level. Take the intervention group and control group cells were treated with serum-free DMEM/F12medium induced2h, two cell orphological changes, and after using immunocytochemistry to detect the intervention group and the control group induced NSE, Nestin of expression. While using RT-PCR method neuron-specific enolase (NSE), nestin (Nestin), β-tubulin Ⅲ (β-Ⅲtubulin) three markers for semi-quantitative detection of mRNA expression level.Result:After three passages of the F3generation of adherent cells by flow cytometry CD71, CD90, CD29expression was positive, CD45negative, immunohistochemical staining method for identifying markers CD90, CD71positive expression. After adding bFGF intervention can be observed cell proliferation rate has accelerated,2-3d passaged once, cell morphology was observed under light microscope can be found in the cell body becomes more slender, the refractive index increases. Expression by immunohistochemistry staining in the intervention group and the control group-specific neural markers, microscope, different concentrations of nerve intervention group after induction NSE, nestin expression, the positive rate was no significant difference between the intervention groups. The control group NSE, nestin-positive rate is low not statistically significant. Total RNA was extracted in the intervention group and the control group, using RT-PCR technique to detect cells in each group can be detected nestin p III-tubulin expression, NSE mRNA, but the expression level of the intervention group was significantly higher than the control group, no significant difference in expression levels between the intervention group group each concentration group. BMSCs in the intervention group after using serum-free medium can be observed1h soma shortened refraction increases, the formation of a number of prominent, and with the induction time gradually become irregular in shape, was bipolar or multipolar cell morphology after2hours basically similar to neural-like cells, whereas the control group and intervention group compared to the group using cell morphology changes in serum-free medium after time later, after changing the cell shape is similar to the control group, but the proportion of neural-like cells than the intervention group is low. For serum-induced cell intervention group and the control group after immunohistochemical staining detected nerve-like cells found in each group after the induction of neural cell markers were nestin, NSE expression. Total RNA was extracted cells in serum-free medium in the intervention group and the control group after induction of differentiation lines by RT-PCR nestin β Ⅲ-tubulin expression, the cells can be detected in each group, NSE mRNA, but the expression level was significantly higher in the intervention group in the control group, the intervention group no significant group differences in expression levels between the various concentrations.Discussion:The experimental application of adherent culture,24h after controlling for initial medium was changed and a variety of methods such as digestion of rat BMSCs more effective separation and culture,3rd generation BMSCs by flow cytometry and immunofluorescence cytochemistry cellular and molecular detection of CD90, CD71, CD29was positive, but negative for CD4S cell analysis confirmed after the isolation and culture of primitive bone marrow cells eventually become more homogeneous BMSCs. Serum-free culture medium using methods to induce the control group and intervention group BMSCs to differentiate into neuron-like cells with nerve cell morphology, immunofluorescence staining and RT-PCR results show that both the control group or the intervention group were there β Ⅲ-tubulin nestin expression as a nerve cell-specific markers, NSE, indicating nerve-like cells. It also describes the process of bFGF on BMSCs differentiation into neuron-like cell proliferation and induce played a role in improving the BMSCs induced to differentiate into neuron-like cells in the conversion rate, providing a theoretical basis for further clinical applications. |
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