| Objective:To investigate the isolation, culture amplification and identification of MSCs.To analyze the effect of inflammatory response and damage repair of lung in smoke inhalation injury rabbits after MSCs transplantation in order to analyze the possible mechanisms.Methods First, MSCs were isolated from rabbit born marrow and culture by the method of whole bone marrow culture in vitro and amplified into rabbit MSCs.Cell growth patterns were evaluated by MTT test.Phenotypes of MSCs was evaluated by flow cytometry. Secondly,To establish model of smoke inhalation injury in rabbit and transplante MSCs immediately after injury.72 healthy New Zealand rabbits were divided into three groups randomly:control group(C group,n=8), inject 10ml PBS by ear vein;smoke inhalation injury group (S group,n=32), inject 10ml PBS immediately by ear vein after injury and MSCs treatment group (M group,n=32) inject 10ml PBS containing the third generation of MSCs (1.0×10) immediately by ear vein after injury.S, M groups plasma were centrifugated by ear vein blood before and after injury 2h,4h,6h and frozen at-80℃refrigerator packaged in order to centralized test the concentration of plasma cytokines. C group as the control group. The levels of TNF-a, IL-1β, IL-6 and IL-10 on rabbit plasma and lung tissue homogenate were detected by enzyme-linked immunosorbent assay (ELISA) in S group and M group after injury 2h,4h,6h (8 for each time points) and C group.S, M groups rabbits were kill respectively before and after injury 2h,4h,6h,24h(8 for each time points). Separation of right middle lobe for the determination of lung water content; right lung lower lobe in 10% formalin fixed for pathological examination.Separation of left lung for preparation of homogenates. C group were measured lung water content and Pathology as a control group.Results 1,Primary cultured MSCs which observed under microscopy were show spindle-shaped at 4th after seeding and growing at a rapid speed.Confluence of MSCs reach to 80% 7-8days after seeding and subculture grew well.CD34(-), CD45(-) and CD44(+), CD105(+) were detected by flow cytometry, which confirmed finally that the cells we cultured was MSCs.2,Concentration of all pro-inflammatory cytokines at 2h,4h and 6h after smoke inhalation injury in M group decreased significantly when compared to values at corresponding time point in S group (P<0.05) in peripheral blood and lung homogenate. Concentration of anti-inflammatory cytokines (IL-10) of peripheral blood at 2h,4h and 6h while IL-10 of lung homogenate 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).3,Histopathological observation:Morphologically, comprehensive observation from several aspects including color,appearance,coated tension, congestive and hemorrhage condition,secretions and exudation 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. Histopathologically, comprehensive observation from several aspects including congestion and hemorrhage condition, alveolar septa case, pulmonary edema,epithelial loss inflammatory cell infiltration etc. showed that lung improved significantly at every time points in M group compared to those in S group meanwhile structure of lung were normal in control group.Conclusions 1,MSCs engraftment could significantly decrease proinflamm-atory cytokines and increase anti-inflammatory cytokines in the early stages of smoke inhalation injury,improve inflammatory response,which could show protective effect on smoke inhalation injury.2,After treatment and intervention of bone marrow mesenchymal stem cells for smoke inhalation injury rabbits has been to reduce the pathological changes.3,Immunomodulation may be another important mechanism of MSCs to improve lung injury. |
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