| Part 1 Comparative Study on cell culture of Circulating fibrocytes in normal and diabetic miceObjectives:Cell therapy on chronic wounds is one of the focus in the current research of traumatology, but is still barren of a stable and reliable cells. In this study, we cultured the peripheral blood of C57 mice and gene spontaneous mutations in diabetic db/db mice, to obtain circulating fibrocytes, and observing the proliferation activity in vitro 。 This aim is to explore a new methods for the treatment of wound cells and provide reliable cells, which can provide the basis for the follow-up study of animal experimental study on diabetic wound healing.Methods:Density gradient centrifugation to collect the C57 and diabetic db/db mice peripheral blood mononuclear cells (PBMCs), adjust the cell density ,inoculation in 20%fetal bovine serum with High glucose DMEM medium, Continuous observation and Cultivation, Cell was replacement fluid every 2-3 days, in addition to change the liquid and Rinse the cells with room temperature PBS,do not anything else treatment.under the inverted phase contrast microscope to observe cell proliferation and clonal morphological changes on the day after inoculation,3,5,1,10 days,through the digital camera to collect pictures.After the cell culture, observed the cell morphology and proliferation activity of C57 mice and db/db diabetic mice at the same time point, and the success rate, cFb obtain rate and the proliferative activity were observed.Results:In the process of cFb vitro isolation and culture we found that In this study,30 samples of heparin anticoagulant and 38 C57 mice were successfully incorporated into the experiment. The results of the success rate, cFb rate and the proliferation activity show that:Diabetic mice were isolated from PBMCs17 (57%) and obtained cFb10 (33%); While C57 was separated from PBMCs (73%), cFb18 (28) was obtained (47%); observed the proliferation activity of adherent spindle cell like cells obtained from cultured cells show that:The fusion rate of C57 mouse cFb in vitro cultured 5-7 days can reach about 60%, the proliferation activity of diabetic mice cFb was significantly delayed, need 10 to 14 days of cell fusion rate can reach about 50%, and under the phase contrast microscope, there was no significant difference in cell morphology between the two groups.Conclusions:1) The success rate and proliferation activity of cFb in peripheral blood of diabetic mice were lower than that in C57 mice;2) Both normal mice and diabetic mice, although in vitro amplification has been successful, but it is not stable, can not get the ideal number of circulating fiber cells.Part 2 Study on AMD3100 combined with G-CSF in the mobilization of circulating fibrocytes in Diabetic db/db MiceObjective:On the basis of previous research, To further explore the mobilization of circulating cells in vivo, observed and compared the number of peripheral fibrocytes in peripheral blood at different time points, to optimize the mobilization method and mobilization time, provide new direction and ideas for the treatment of chronic wounds, In order to provide stable and reliable cells in clinical treatment of chronic wounds, but also provide new ideas and new directions for further research.Methods:10-11 week old spontaneous mutant diabetic C57BL/KsJ (db/db) mice 60, Randomly divided into three groups, Group A (control group) 20, B group (AMD3100+NS group) 20, C group (AMD3100+G-CSFgroup) 20. A group, B group, C group each set 3d,5d,7d, 10d four time points,5 mice were randomly selected at each time point. Cardiac blood sampling after intraperitoneal injection, and flow cytometry of CD11b, a-SMA positive cells labeling rate. Results the average in x±s, statistical analysis and comparative analysis.Results:There was no significant difference in the double immunofluorescence staining positive cells in A group at four time points (p>0.05); The proportion of double immunofluorescence staining positive cells at the time points of 3d,5d,7d, 10d was increased in group B and group C after mobilization, and reached the peak at fifth days and then decreased gradually, and the proportion of B group and C group at each time point was higher than that of A group (p.< 0.05). The proportion of double positive cells was increased in group C compared with group B (p< 0.05);Conclusion:AMD3100+G-CSF and a single use of AMD3100 peripheral blood cFb were mobilized, The effect of combined mobilization is better than that of single mobilization, And fifth days after the mobilization of cFb reached a peak... |
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