| From the 1960s, pulsed electromagnetic fields(PEMFs) is used to treat many kinds of bone diseases such as delayed union, nonunion and osteoporosis without any incursive damage. PEMFs can improve the proliferation and differentiation of live cells, stimulate the appearance of the local factors, and meliorate the bone density and biomechanics trait of thighbone. But the mechanism of electromagnetic fields on bone is unclear. The frequency, stimulation time, intensity and pulse shape of PEMFs can influence the therapeutic in different degree. Mesenchymal stem cells (MSCs) is one kind of tissue stem cells, they indwelled in marrow stroma system. MSCs have the ability to update themselves and the potential to transform into many kinds of other cells. And they can transform into osteoblast under specifical conditions. Furthermore, adult stem cells put up their chemotaxis when they were injected into one's body. In other words they concentrate in where is injured. Under the local microenvironment, adult stem cells turn into certain tissue cells which is urgently need to repair the injured tissue. And then, the injured tissue repaired. This offer the possibility that one will be cured with his own cells even with xenogenous cells.This experiment is to observe the effects of PEMFs on MSCs of SD rat in vitro. This experiment is to observe the effects of PEMFs on osteoblast of SD rat in vitro. MTT assay, Alkaline phosphatatase- specific activity assay, tetracycline fluorescence labeling assay,calcium nodal stain were used to observe the effects of PEMFs on MSCs'proliferation, differentiation and bone tissue-like formation. We design some experiments to observe the effect of PEMFs'frequency and intensity on MSCs'proliferation, differentiation. The experiment results offer us some evidence to treat bone diseases such as delayed union, nonunion and osteoporosis.The work can be summarized as follows:1. MSCs isolated and culturedOBJECTIVE: To study a simple and effective method to isolate MSCs form rat. METHTHODS: The mesenchymal stem cells were obtained by using whole marrow attachment method and differential speed attachment method was used to pure them. Draw the growth carve of MSCs of primary generation and third generation. Periodic acid-Schiff's stain, oil red-O stain and alkaline phosphatase stain were practiced. RESULTS: The configuration of MSCs is favorable, the growth carve show typical"S".The results of stains are all negative, it means the cells can's excrete hepatin, include no lipocyte and osteoblast. CONCLUSION: The method can obtained MSCs and it's simple and effective. It's a preparation for the next experiment.2. The effects of pulsed electromagnetic fields rat mesenchymal stem cellOBJECTIVE: To study the effects of pulsed electromagnetic fields (PEMFs) with different frequency and intensity on mesenchymal stem cells of rat. METHTHODS: The mesenchymal stem cells were obtained by using whole marrow attachment method. The third generation cells were irradiated by use of pulsed electromagnetic fields with 15Hz,0.2mT; 15Hz,0.4mT; 15Hz,0.8mT; 1Hz,0.2mT; 1Hz,0.4mT; 1Hz,0.8mT. The method of MTT was employed to evaluate the levels of proliferation. The parameters of the cells cycle were detected with the flow cytometer (FCM). RESULTS: After 5 days of irradiation by pulsed electromagnetic fields (once a day, 3h each time), the proliferation of the mesenchymal stem cells in rat was accelerated. The result of MTT is chaned significantly(P<0.01). The group of 1Hz ,0.4mT are most are most effective(3d,0.323±0.051;5d,0.714±0.058), they are increased significantly compare with the sham group(3d,0.246±0.044;5d,0.487±0.064)(P<0.01). Effective and the percentage of cells at the (S+G2/M) phase was increased significantly (P<0.05). And the group of 1Hz ,0.4mT are most effective(G0/G1:75.3±0.17, S : 8.5±0.12, G2/M : 16.4±0.20, PI : 24.9), they are increased significantly compare with the sham group (G0/G1:83.3±0.33,S:7.0±0.29, G2/M:9.8±0.23, PI : 16.8) (P<0.01). CONCLUSION: Irradiation of pulsed electromagnetic fields could increase the proliferation of mesenchymal stem cells of rat in vitro, and the results could be affected by the parameters of PEMFs.3. The effects of pulsed electromagnetic fields on inducing rat mesenchymal stem cells into osteoblastOBJECTIVE: To study the effects of pulsed electromagnetic fields (PEMFs) on inducing the mesenchymal stem cells (MSCs) into osteoblast. METHTHODS: The rat mesenchymal stem cells were obtained by using whole marrow attachment method, and then selected by the adhesive method. The third generation cells were irradiated by PEMFs of 1Hz, 0.4mT, then the activity of Alkaline phosphatase (ALP) was measured, tetracycline fluorescence labeling assay and bone nodule formation were used. RESULTS: After being exposed in PEMFs (once a day,3h each time), activity of ALP was increased significantly (P<0.05), and tetracycline fluorescence labeling assay and bone nodule formation were positive. CONCLUSION: Exposing in PEMFs could induce rat mesenchymal stem cells into osteoblast.
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