The Protection Of Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells (BMD-MSCs) In Phosgene-induced Acute Lung Injury

PART 1. SD rat bone marrow mesenchymal stem cells separation and purification, identification and differentiationObjective:To investigate SD rats BMD-MSCs in vitro amplification and observe identify their biological characteristics, surface markers and differentiation capacity. Methods:The whole bone marrow adherent separation separation SD rat bone marrow cells were passaged, inverted phase contrast microscope morphology, flow cytometry to identify cells. Results:The primary fusiform cells, swirling growth passaged multiple fibroblast cell shape,3rd cultured cell surface molecule CD29, CD90, CD44 expression was positive, but did not express CD34 and CD45. Conclusions:1 whole bone marrow cells adherent separation method can be successfully isolated and cultured stem cells were passaged up on behalf of the morphological change.2 identified by flow cytometry in passaged continuously improve the purity of the process.3 and successfully induced under certain conditions in vitro induced to differentiate into osteoblasts and adipocytes.PART 2. The Protection of Allogeneic Bone marrow-derived mesenchymal stem cells(BMD-MSCs) in phosgene-induced acute lung injuryObjective:BMD-MSCs to intervene if there is a protective effect on lung tissue by observing the changes of phosgene-induced lung injury in rats symptoms, inflammatory markers and markers of alveolar epithelial cells to explore. Method:1 Take 96 SD rats were randomly divided into four groups. K groups: normal control group was injected with lml PBS via the tail vein; PH group: exposure and concentration of 8.33g/m3 of phosgene poisoning; PM Group:after exposure, via the tail vein injection of 1×106 BMD-MSCs 1ml; KM groups:control group injected stem cells 1×106 BMD-MSCs 1ml via the tail vein; modeling 6h,24h,48h after the animals were sacrificed received specimens.2 aortic blood test, blood gas analysis, specimens from the supernatant after centrifugation into more than-80℃ to save the test. Take the next right lung lobe lung wet weight and dry weight, right middle lobe immediately frozen in liquid nitrogen, then transferred to -80℃ refrigerator to save the test. Leaves placed in paraformaldehyde fixed part of the right lung taken for histological observation, through the trachea into the lungs into the amount of OCT dilutions made frozen sections and observe carry stem cells transfected with GFP under a fluorescence microscope. Left lung specimens from bronchoalveolar lavage fluid to be measured. MRNA expression in lung wet to dry ratio, serum and bronchoalveolar lavage fluid TNF-a and IL-10 in lung tissue were measured AQP5 and SPC.3 Statistical analysis:SPSS22.0 statistical software for data processing, measurement information and data as mean ± standard deviation (x± s) said. Data between different treatment groups were compared preclude analysis of variance with the analysis of design information. Between the groups were compared using a completely random data between the different time points compared with the same point in time and between groups analysis of variance (one-way ANOVA method) to determine the difference between P 0.05< There was significant. Results: The general situation:PH group and the PM group of rats after exposure shortness of breath, weakness, drowsiness and other symptoms, but the PM group with mild symptoms and recover faster. W/D:PH group than in the K group was significantly higher than the wet-dry, and the difference was statistically significant (P<0.05), PM group than in the dry than wet PH group decreased significantly (P<0.05). Blood gas analysis:oxygen partial pressure PH group than in the low-K group, the difference was statistically significant (P<0.05), PM Group KM group as compared with the results; PM group than in the oxygen partial pressure PH group, the difference was statistically significant (P<0.05). Pathology:K group and the KM Group:clear structure of lung tissue, alveolar no inflammatory cell infiltration, alveolar wall is thin, no expansion of stromal vascular, bronchial epithelial integrity. PH group:extensive alveolar structure was destroyed, interstitial pulmonary edema, alveolar exudate large amount of red blood cells and inflammatory cell infiltration, gradually restored. PM Group:a small part of the alveolar structural damage, and interstitial lung tissue remaining structure is still intact, and some slightly widened alveolar septa, visible between mild pulmonary edema, a small amount of red blood cell and neutrophil infiltration. Homing:PM group and the group can be seen carrying GFP KM stem cells, but the PM group was significantly more than the KM group. Serum and BALF:1) TNF-a:TNF-a concentrations PH group was significantly higher than the K group, the difference was statistically significant (P<0.05), PM group compared with TNF-a concentrations PH group at the end, the difference was statistically significance (P <0.05).2) IL-10:PH group was significantly higher than that in IL-10 concentration of K group, the difference was statistically significant (P<0.05), PM Group KM group as compared with the results; PM group than IL 10-PH group of high concentration The difference was statistically significant (P<0.05). Lung tissue: AQP5 and mRNA levels of SPC PH group and PM group began to fall in six hours, 24 hours bottoming out, gradually increased, the difference in the group at different time points and between the same point in time with the PH group statistics PM significance (P<0.05). Conclusion:You can make the tail vein injection BMD-MSCs blood and bronchoalveolar lavage fluid in rats with anti-inflammatory cytokines IL-10 increased pro-inflammatory cytokines TNF-a decreased and slowed SPC and AQP5 decreased, and therefore recognize BMD-MSCs acute lung injury in rats induced pulmonary gas light has a protective effect.