The Effects Of Bone Marrow-derived Mesenchmal Stem Cells Engraftment On Inflammatory Response And Tissue Repair In Smoke Inhalation Injury

Objective:To explore a method of isolation, purification and culture of bone marrow-derived mesenchymal stem cells (MSCs) of rabbits in vitro for the sake of a further study.Methods:Bone marrow tissue was harvested from bilateral posterior iliac crest and tibias bone of young New Zealand white rabbits. MSCs were isolated and proliferated by the method of whole marrow culture. Growth curve of 2nd,3rd,4th,5th,6th generation MSCs were drawn by MTT method. CD34, CD44, CD45, CD 105 antigen of MSCs were identified by flow cytometry.The purified MSCs were labelled with BrdU and Immunohistochemistry was performed to calculate and evaluate the labeling rate and determine the optimal labeling time and labeling concentration.Results:Primary cultured MSCs start adhering to plates 12 hours after seeding and spindle-shaped MSCs were observed under microscopy and growing at a rapid speed.Confluence of MSCs reach to 80% 7-8days after seeding. From the MSCs growth curve, which was S shape, it was known that MSCs grew in a rapid stage at 4th-8th day after seeding, and MSCs of 3rd-5th generation possess higher activity. CD34(-), CD45(-) and CD44(+), CD105(+) were detected by flow cytometry, which confirmed finally that the cells we cultured was MSCs. The positive rate of MSCs labeled by BrdU was 85-90%.40μmol/L and 72h were the optimal labeling time and labeling concentration respectively.Conclusion:In this study, by our culture protocol which is characteristic of simple operation, high efficiency and is economic and practical, MSCs were isolated and proliferated stably and rapidly.Cultured MSCs possessed high activity of growing and amplification and were appropriate for further research. Application of BrdU labeling for MSCs in vitro is safe and reliable. Objective:To establish model of smoke inhalation injury in rabbit.Methods:Model was established by using of home-made smoke inhalation injury generator. Desiccated pine wood chip and kerosene were used to produce smoke. Rabbits inhaled smoke in air tight container for 10 min and repeated at 2 min interval. After injury, clinical manifestation was observed and blood gas at every time point was recorded. After 24h, rabbits were killed and Lung histopathologic slide was analyzed and evaluated.Results:(1) After injury, breath frequency of rabbits increased obviously and appeared dyspnea and distressed. Dry rales in lung could be heared. After 24h, breath was improved. (2) PaO2 decreased from pre-injury 90.20±18.44mmHg to 63.48±12.90mmHg,56.96±10.23mmHg and 65.76±12.55mmHg at post-injury 10min,2h and 4h respectively and the difference is significant (P<0.05). At post-injury 24h, PaO2 returned to normal; PaO2 increased from pre-injury 32.10±5.48mmHg to 41.72±6.33mmHg,43.12±5.42mmHg and 39.11±6.91mmHg at post-injury 10min,2h and 4h respectively and the difference is significant (P<0.05). At post-injury 24h, PaCO2 returned to normal. (3) Histopathology observation: Morphologically, injuried lung seemed to be pale and coated tension and large hemorrhage could be observed. Histopathologically, congestion in pulmonary blood vessel, edema and hemorrhage in alveoli, interstitial edema and inflammatory cell infiltration.Conclusion:Rabbit model of smoke inhalation injury recommended in this article is a convenient, useful, stable, economic and reduplicated animal model and refers to research of smoke inhalation injury. Objectives:To investigate the effect of bone marrow-derived mesenchmal stem cells engraftment on inflammatory response in smoke inhalation injury rabbits, evaluate the therapeutic effect and reveal its possible mechanism initially.Method:64 healthy New Zealand rabbits were divided into two groups randomly via Number table:smoke inhalation injury group (S group) and MSCs treatment group (M group). As control group, baseline was recorded in another 8 rabbits.10ml PBS was injected via ear marginal vein in control group rabbits; 10ml PBS was injected via ear marginal vein immediately on injury in S group rabbits; The third generation MSCs labeled by BrdU whose concentration was 1×107/10ml PBS was injected via ear marginal vein immediately on injury in M group rabbits. Main observation index and methods:①TNF-α, IL-1β, IL-6, IL-10 and vascular endothelial growth factor (VEGF) in peripheral blood and lung tissue were detected by ELISA at 2h,4h and 6h after injection in S group and M group and baseline in control group respectively and analyzed.②mRNA expression of TNF-α, IL-1β, IL-6, IL-10 in lung tissue were detected by RT-PCR at 2h,4h and 6h after injection respectively and analyzed in S group and M group.③Lung water mass fraction were measured at 6h and 24h after injection in S group and M group and baseline in control group respectively and analyzed.④Change of lung tissue and bronchial tissue were observed morphologically and histopathologically at 2h,4h,6h,24h after injection respectively and analyzed in three groups.Results:(1) Pro-inflammatory cytokines:Concentration of IL-1β, IL-6 and TNF-αin peripheral blood and lung tissue at 2h,4h and 6h after injection in M group decreased significantly when compared to values at corresponding time point in S group (P<0.05). Concentration of IL-1β, IL-6 and TNF-αin peripheral blood and lung tissue at 2h,4h and 6h after injection in S group increased significantly when compared to baseline (P<0.05). Concentration of IL-1βin peripheral blood and lungⅨ tissue at 2h,4h and 6h after injection in M group had no significance when compared to baseline (P>0.05). Concentration of TNF-αin peripheral blood and lung tissue at 2h,4h and 6h after injection in M group increased significantly when compared to baseline (P<0.05). Concentration of IL-6 in lung tissue at 2h,4h and 6h after injection in M group increased significantly when compared to baseline (P<0.05) but had no significance in peripheral blood (P>0.05). (2) Anti-inflammatory cytokines:Concentration of IL-10 in peripheral blood at 2h,4h and 6h after injection in M group increased significantly when compared to values at corresponding time point in S group (P<0.05). Concentration of IL-10 in peripheral blood at 2h,4h and 6h after injection in S group and M group increased significantly when compared to baseline (P<0.05). Concentration of IL-10 in lung tissue at 4h and 6h but 2h after injection in M group increased significantly when compared to values at corresponding time point in S group (P<0.05). Concentration of IL-10 in lung tissue at 2h,4h and 6h after injection in M group had no significance when compared to baseline (P>0.05). Concentration of IL-10 in lung tissue at 4h and 6h but 2h after injection in M group increased significantly when compared to baseline (P<0.05). (3) VEGF:Concentration of VEGF in peripheral blood and lung tissue at 2h,4h and 6h after injury in S group increased significantly compared to baseline (P<0.05). Concentration of VEGF in peripheral blood at 2h,4h,6h after injury appeared increase trend (compared to value at Oh, P<0.05) in M group but decreased obviously compared to values at corresponding time point in S group (P<0.05) meanwhile Concentration of VEGF in lung tissue at 2h,4h,6h after injury appeared decrease trend (compared to baseline, P<0.05) in M group but increased obviously compared to values at corresponding time point in S group (P<0.05). (4) mRNA expression of main cytokines:mRNA relative expression of TNF-α,IL-1β,IL-6 at 4h and 6h in M group were less significantly than values at corresponding time point in S group (P<0.05), but there was no significance when mRNA relative expression of IL-6 at 2h in M group compared to value at corresponding time point in S group (P>0.05); mRNA relative expression of IL-10 at every time point in M group were more significantly than that at corresponding time point in S group (P<0.05). (5) Lung water mass fraction in M group decreased significantly compared to value in S group at 6h and 24h after injection (P<0.05). Values at 6h and 24h in S group and M group increased significantly when compared to baseline (P<0.05). (6) Histopathological observation:Morphologically, comprehensive observation from several aspects including color, coated tension, smoothness, congestive condition, the size of hemorrhage and necrosis and secretions etc. showed that lung and bronchus improved significantly at every time points in M group compared to those in S group meanwhile lung and bronchus were normal in control group and U group. Histopathologically, comprehensive observation from several aspects including epithelial loss, congestion, hemorrhage, exudation, alveolar septa case, pulmonary edema, atelectasis, emphysema,inflammatory cell infiltration, fibroblast proliferation etc. showed that lung and bronchus improved significantly at every time points in M group compared to those in S group meanwhile structure of lung and bronchus were normal in control group and U group.Conclusion:MSCs engraftment intravenously into smoke inhalation injury could significantly decrease systematic and local pro-inflammatory cytokines and increase systematic and local anti-inflammatory cytokines, decrease extravascular lung water, improve systematic inflammatory response and alleviate lung and bronchial injury, which could show protective effect on smoke inhalation injury. Effect of anti-inflammatory and immunomodulation could be another mechanism of MSCs engraftment on therapeutic effect of smoke inhalation injury. Objectives:To investigate the effect of bone marrow-derived mesenchmal stem cells engraftment on tissue repair in smoke inhalation injury rabbits.Method:32 healthy New Zealand rabbits were divided into four groups randomly via Number table:control group (C group), control group+MSCs treatment group (U group), smoke inhalation injury group (S group) and MSCs treatment group (M group). There were 8 rabbits in every group.10ml PBS was injected via ear marginal vein in C group rabbits; 10ml PBS was injected via ear marginal vein immediately on injury in S group rabbits; The third generation MSCs labeled by BrdU whose concentration was 1×107/10ml PBS was injected via ear marginal vein immediately on injury in M group rabbits. The third generation MSCs labeled by BrdU whose concentration was 1×107/10ml PBS was injected via ear marginal vein in U group rabbits. There were two observation points:7th day and 28th day and 4 rabbits in every observation point. Main observation index and methods:①Change of lung tissue and bronchial tissue were observed morphologically and histopathologically at 7th day and 28th day after injection respectively and analyzed in four groups.②Homing of MSCs in vivo was observed at 7th days after injection by immunohistochemistry in four groups; Differentiation of MSCs in lung tissue and bronchial tissue observed at 28th days after injection by immunohistochemical double staining in four groups.Results:①Results from immunohistochemistry showed that MSCs labeled by BrdU in M group could migrate into and locate within injuried lung and bronchial tissue meanwhile MSCs labeled by BrdU in C group, U group and S group could not or rare to be detected.②Results from immunohistochemical double staining showed that there were positive cells of aquaporin-5 (AQP-5) and BrdU, which could suggest that MSCs could differentiate to become alveolar epithelial cells typeⅠin lung; there were positive cells of alkaline phosphatase (AKP) and BrdU, which could suggest that MSCs could differentiate to become alveolar epithelial cells typeⅡin lung; there were positive cells of CD34 and BrdU, which could suggest that MSCs could differentiate to become pulmonary vascular endothelial cells in lung. However, there was no above-mentioned positive cells in C group, U group and S group.Conclusion:MSCs which were engrafted into smoke inhalation injury rabbits could migrate to homing or accumulate within the inflammatory and injuried site and differentiate to alveolar epithelial cells typeⅠandⅡand pulmonary vascular endothelial cells, reduce tissue injury, which suggested MSCs engraftment could participate in and accelerate the process of tissue repair of smoke inhalation injury.