| Objective In this study,we compared the effects of different concentrations of dexamethasone on bone marrow cells,and observed the therapeutic effect of bone marrow cells on acute lung injury,and explored the appropriate concentration of dexamethasone.Methods 1 In vitro experiments:Normal bone marrow group:The bone marrow cells of normal fluorescent mice were divided into 5 groups,and the number of bone marrow cells in each group was 5×10~6.The 4 groups were treated with dexamethasone,2ng/ml,4ng/ml,10ng/ml,1ug/ml,and the fifth group was not treated with dexamethasone as the control group.All the cells were added to the culture medium,and the culture medium was 1ml,then centrifuged and suspended after 24h cultivation.The obtained normal bone marrow cells were placed in lung tissue cells(5 x 10~6cells per group)of normal healthy C57BL/6 mice stimulated by LPS,and after 48 hours of culture,the cell morphology was recorded under inverted phase contrast microscope,and the apoptosis rate of lung tissue cells was detected by flow cytometry by adding antibody and 7-AAD reagent.Sepsis bone marrow group:Bone marrow cells were harvested by cecal ligation and perforation,and the patients were treated with normal bone marrow.All the cells were added to the culture medium,and the culture medium was 1ml,then centrifuged and suspended after24h cultivation.The resulting bone marrow cells were injected into the lung cells of normal healthy C57BL/6 mice stimulated by LPS(5 x 10~6cells per group),Each group was treated with 48h after treatment with normal bone marrow group.Simple control group:The single lung tissue cell group(10~7lung tissue cells)without bone marrow cells was treated with 48h after treatment with normal bone marrow group.2 In vivo experiments:A total of 11 groups,5 groups of normal bone marrow group:Normal bone marrow group without dexamethasone treatment and normal fluorescent mice bone marrow cells(5 x 10~6cells per group)were treated with different concentrations of dexamethasone(2ng/ml,4ng/ml,10ng/ml and 1ug/ml).5 groups of sepsis bone marrow group:Transfusion without dexamethasone treatment of sepsis in mice bone marrow cells and fluorescence after different concentrations of dexamethasone(2ng/ml,4ng/ml,10ng/ml,1ug/ml)in the treatment of sepsis by mouse bone marrow cells(5 x 10~6cells per group).Simple control group:Reinfusion of physiological saline.Each experimental group by intraperitoneal injection method to enter normal C57BL/6 mice in vivo,the mice were sacrificed after 24h,and removing of the lung tissue,grinding,centrifugation,resuspended respectively after recording cell morphology,number and fluorescence under the inverted microscope.And adding antibody and 7-AAD reagent,the lung tissue cell apoptosis detected by flow rate cytometry.3 Mortality test:220 normal C57BL/6mice were obtained,each group of 20,a total of 11 groups,the acute lung damage model was made by using the LPS nasal drip method,the five groups of normal fluorescent bone marrow cells(5 x 10~6cells per group)treated with different concentrations of dexamethasone(0ng/ml,2ng/ml,4ng/ml,10ng/ml,1ug/ml),the five groups of sepsis fluorescent bone marrow cells(5 x 10~6cells per group)were processed by different concentrations of the different concentrations of dexamethasone(0ng/ml,2ng/ml,4ng/ml,10ng/ml,1ug/ml).One control group:Reinfusion of physiological saline.Fast,the water continues to feed 48h and watch mortality.Results 1 In vitro experiments:The number and morphology of the cells were changed.In the normal bone marrow group by contrast,can be found in the experimental group and the simple set of lung tissue cells,compared to the number,and the experimental group treated with dexamethasone 2 ng/ml cell count the most,and vision can be found within the tissue mass.The number of cells in each experimental group was basically the same,and no tissue was found.In the sepsis group,the number of cells in each experimental group was lower than that in the lung tissue group,and the cell morphology was poor and the cell debris was more.The comparison between the experimental group and the cell morphology was basically the same.The apoptosis rate of each group of normal bone marrow cells was lower than that of sepsis test groups(P<0.05).Compared with the control group,the apoptotic rate of the normal bone marrow group treated with dexamethasone and the normal bone marrow group treated with 2ng/ml dexamethasone was lower than that of the control group(P<0.05).The apoptotic rate of each sepsis group was higher than that of the control group(P<0.05).2 In vivo experiments:It can be found that the number of cells in each experimental group has changed.In the normal bone marrow group,the number of cells in each group was higher,especially after 2ng/ml dexamethasone treatment of normal bone marrow group,the largest number of cells,can be found in the microscopic tissue mass;In the sepsis bone marrow group,almost all of the experimental group in the microscope field of view can not find the cell structure of the living cells,and can be found to have apoptotic cleavage of cell debris.And the lung tissue control group under the microscope visual field observation of its cell morphology is very poor,the number of cells is very small.Flow cytometry showed that the apoptosis rate of each group was lower than that of sepsis group(P<0.05).Compared with the control group,the apoptotic rate of normal bone marrow cells was lower,and the apoptotic rate was the lowest in the normal bone marrow group treated with 2ng/ml dexamethasone(P<0.05).And all the apoptosis rate of sepsis bone marrow group were higher than that of the control group(P<0.05).3 Mortality test:It was found that the mortality rate of each normal bone marrow cells group were lower than that of the control groups(P<0.05),while the mortality rate of the sepsis bone marrow cells groups were not different from that of the control groups(P>0.05).Groups of different concentrations of normal bone marrow cells each comparison between the experimental groups,treated with 2 ng/ml dexamethasone groups of normal bone marrow cells of the lowest mortality(P<0.05)Conclusions 1 Dexamethasone can stimulate the protection of normal bone marrow to damage lung tissue.2 Dexamethasone is unable to mobilize the bone marrow cells in the body of sepsis.3 The concentration of dexamethasone has a certain impact on the protective effect of mobilization of bone marrow cells,2ng/ml dexamethasone the best protective effect on mobilization of bone marrow cells,the higher dose may inhibit the mobilization,thereby inhibiting the immune function.Figure 10;Table 8;Reference 101... |
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