The Effect Of The Mobilization Of Autologous Bone Marrow-Derived Cells By G-CSF On Mice Lung Injury Induced By Bleomycin

Bone marrow-derieved cells (BMDCs) are an important source of various types of marrow stem cells. Granulocyte colony-stimulating factor (G-CSF) has been reported to show an effect of mobilization of BMDCs, and the BMDCs can contribute to the formation of differentiated cell types in other tissues. The aim of this study was to investigate the contribution of mobilized BMDCs to lung repair after lung injury induced by bleomycin, and the mechanisms of any protective effects conferred by BMDCs. Part 1Objective In order to prepare for the tracer study in vivo in the follow-up experiments, BMDCs were labeled with GFP (green fluorescent protein).Methods 20 C57BL/6J mice were randomly divided into two groups, and there were 10 animals in each group. Group A: transplantation group (total body irradiation + bone marrow transplantation). Group B: control group (total body irradiation). Two groups were given lethal irradiation, then bone marrow transplantation for group A. GFP transgenic mice served as bone marrow donors. Observation including: (1). Weight and survival time; (2). The amount of peripheral leucocytes; (3). The expression of GFP in bone marrow on day 28.Results (1).The weights of mice in group B decreased significantly, while the mice in group A only had a slight weight loss. (2). All mice in group B died within the next 11 days after irradiation, but only two mice died in group A. (3). The amount of peripheral leucocytes of mice in group B decreased during the observation and reduced to zero in the fifth day, but the mice in group A had a slight loss in the initial several days. On day 28, the amount of peripheral leucocytes of mice in group A recovered to normal. (4). The amount of GFP-positive BMDCs in mice of group A was evaluated by flow cytometry on day 28 and was same approximately to GFP transgenic mice.Conclusion we successed to establish the mice models of marrow reconstruction, and the survival rate was 80%. BMDCs labeled with GFP can be used in the follow-up experiments.Part 2Objective To observe mobilization of BMDCs by G-CSF to peripheral blood and its effect on mice lung injury induced by bleomycin.Methods 60 marrow-reconstructed mice were randomly divided into two groups including group A: mobilization group (bleomycin+G-CSF) and group B: control group (bleomycin+ normal saline). In Group A and B, each had 30 mice. Each group was divided into three subgroups, which were sacrificed respectively on days 3,7 and 28. Testing items including: (1). The pathological changes of lung tissue by macroscopic observation; (2). Hematoxylin-Eosin (HE) stain was performed on the lung tissue slices to observe the extent of alveolitis and fibrosis; (3). Masson stain was performed on the lung tissue slices to observe the extent of collagen deposition; (4). Cytokines including transforming growth factorβ1(TGF-β1),interferon-gamma(IFN-γ),matrix metalloproteinase-9(MMP-9)and tissue inhibitor of metalloproteinase-1 ( TIMP-1 ) were detected by immunohistochemistry; (5). Spectrophotometric method for the determination of hydroxyproline (HYP) to estimate the collagen content in lung tissue; (6). Ratio of GFP-positive cell was evaluated by flow cytometry in lung cell suspension and the expression of GFP was observed by laser scanning confocal microscope in lung tissue frozen section.Results (1). There was no obvious difference in the pathological change of lung tissue between two groups by macroscopic observation; (2). HE stain: On day 7, more severe inflammation was shown in the histological examination of the lung tissue slices of group B. However, in the group A, inflammation intensity were attenuated compared with group B (p<0.05). On days 3 and 14, there were no statistical difference among two groups; (3). Masson stain: Compared with group B, the extent of collagen deposition was slightly lower on day 14 and significantly lower on days 3 and 7 in group A (p<0.05); (4). TGF-β1: Compared with group B, the content of TGF-β1 in lung tissue was slightly lower on day 14 and significantly lower on days 3 and 7 in group A (p<0.05); (5). IFN-γ: Compared with group B, the content of IFN-γin lung tissue was slightly lower on days 7 and 14 and significantly lower on day 3 in group A (p<0.05); (6). MMP-9: Compared with group B, the content of MMP-9 in lung tissue was slightly lower on days 3 and significantly lower on days 7 and 14 in group A (p<0.05); (7). TIMP-1: There was no statistical difference between two groups; (8). HYP : On day 7, the hydroxyproline content of group A was much more lower than that of group B, and there was statistical significance between them; (9). flow cytometry: On day 7, the percentage of GFP-positive cell in lung cell suspension of group A was the highest and there was statistical difference between group A and group B.; (10). laser scanning confocal microscope: On days 3 and 7, GFP-positive cells can be observed in lung tissue frozen section in group A. However, there were no obvious GFP-positive cells in lung tissue frozen section in group B at every observation time. Conclusion Mobilization of BMDCs by G-CSF has a protective effect on mice lung injury induced by bleomycin. The effect may be associated with the amount of BMDCs and down-regulating the level of fibrosis cytokines.Part 3Objective To investigate the effect of mobilization of BMDCs by G-CSF on mice lung injury induced by bleomycin and survey on the mechanism. Methods 75 C57BL/6J mice were randomly divided into three groups including group C, n=30, mobilization group (bleomycin+G-CSF); group D, n=30, control group (bleomycin+ normal saline) and group N, n=15, normal group (normal saline only). Every group was divided into three subgroups, which were sacrificed respectively on days 3,7 and 28. Testing items including: (1). The pathological changes of lung tissue by macroscopic observation; (2). Hematoxylin-Eosin (HE) stain was performed on the lung tissue slices to observe the extent of alveolitis and fibrosis; (3). Masson stain was performed on the lung tissue slices to observe the extent of collagen deposition; (4). Cytokines were detected by immunohistochemistry; (5). Spectrophotometric method for the determination of hydroxyproline (HYP) to estimate the collagen content in lung tissue; (6). Pulmonary permeability index (LPI):protein content of bronchoalveolar lavage fluid / protein content of serum. Another 20 C57BL/6J mice were randomly divided into two groups including group E: mobilization group, n=10, (bleomycin+G-CSF) and group F: control group, n=10, (bleomycin+ normal saline). The survival time of each mouse was observed without end point.Results (1). There was no obvious difference in the pathological change of lung tissue between group C and group D by macroscopic observation; (2). HE stain: On day 7, the inflammation of group C were attenuated compared with group D in the histological examination of the lung tissue slices (p<0.05). (3). Masson stain: In group C, the extent of collagen deposition was significantly lower than group D on day 7 (p<0.05); (4). TGF-β1: In group C, the content of TGF-β1 in lung tissue was significantly lower than group D on days 3,7 and 14 (p<0.05); (5). IFN-γ: In group C, the content of IFN-γin lung tissue was significantly lower than group D on days 3 and 7 (p<0.05); (6). MMP-9: In group C, the content of MMP-9 in lung tissue was significantly lower than group D on day 7 (p<0.05); (7). TIMP-1: There was no statistical difference between group C and group D; (8). HYP: On day 7, the hydroxyproline content of group C was much more lower than that of group D (p<0.05); (9). LPI: In group C, LPI was significantly lower than group D on days 3 and 7 (p<0.05); (10). There was no statistical difference between group E and group F on survival time.Conclusion Mobilization of BMDCs by G-CSF has a protective effect on mice lung injury induced by bleomycin, but has no significantly influence on survival time.