| Burn injury caused by heat, electricity, chemistry and radioation is one of the medicine focuses. Systemic Inflammatory Response Syndrome(SIRS) and multiple organ dysfunction syndrome(MODS) complicated by serious burn exist high mortality. Although with the improvement of healthcare, the incidence of the complications, such as heart failure, pulmonary edema, gastrointestinal hemorrhagic stress ulcer and acute renal failure, were induced. The improvement of the brain injury is not obvious. Owing to lack of objective diagnostic criteria for the brain injury after burn, the brain injury is easily covered by the serious and vital complications. Large area and deep wound burn results in toxic encephalopathy, cerebral edema and convulsions, followed with sequelae of cerebral damage. Previous study revealed that after 7 hours, blood-brain barrier opened sustained several days, and inflammation reaction and high glucose metabolic syndrome damage the brain further. Currently, the studies about the brain injury damaged by burn is limit, the clinical treatment is mainly symptomatic support and control complications. Therefore, it is urgent to seek a new therapy.Stem cells, a kind of progenitor cell, are able to repair damage under certain conditions. At recent years, stem cell therapy has been widely applied in neural injury and neurodegenerative diseases, such as cerebral palsy, spinal cord injury, motor neuron disease, Parkinson’s disease, cerebral hemorrhage, sequel of cerebral infarction and post-traumatic brain sequel and so on. Bone marrow mesenchymal stem cells(BMSCs), a kind of pluripotent stem cells obtained easily, which posess high self-proliferative and expansion capacity, low immunogenicity and multipotent differentiation ability, and the ability to secret various cytokines in the damaged tissue after the transplantation in vivo, have made a great progress in clinical application. Our previous study showed that BMSCs could treat the injury of liver and intestines after burn. Aimed at the complexity of body injury and the occult of brain damage caused by serious burn, this study will discuss the therapeutic effect of BMSCs transplantation on the brain injury and explore the potential repair mechanisms. Objective:The objective was to explore whether transplantation of bone marrow mesenchymal stem cells(BMSCs) can repair brain injuries in severely burned rats and the possible mechanisms. Methods:In this study, BMSCs were cultured and purified by the whole marrow adherent method. BMSCs markers including CD44, CD45 and CD90 and cell cycle were analyzed by flow cytometry. The obtained cells were induced to differentiate into osteogenic and neural cell, which were used to identify the BMSCs. Wistar rats were randomly divided into control group, model group, cell therapy group. The 30% body surface area of rat back was immersed into 97±1℃ water for 18 seconds to prepare the rats model of burn. Pathological change of burned skin was examined by hematoxylin and eosin staining(HE) staining. On the 1st and 2nd days after burn, BMSCs suspension in 0.1ml PBS, containing 5×106 cells, labelled by CM-Dil was injected into the retro-orbital sinus, respectively. The changes of rat body weight and general status in each group were observed and recorded. one week later, cerebrum was excised, cerebral pathological change was observed by HE and Nissl staining, and immunohistochemical method was used to detect the expressions of MAP-2, GFAP, NSE, apoptotic related proteins Caspase-3, autophagy-related protein Beclin1 and LC3 B in cerebral nerve cells. Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling(TUNEL) was used to determine the cell apoptosis. The nerve cells markers such as NSE and GFAP, expressions of apoptosis-related proteins including p53, Bax, Bcl-2, BIM, cytochrome C, Caspase-3, autophagy-related proteins Beclin 1 and LC3 B in the brain tissue were detected by Western Blot. The expression of autophagy-related LC3 B protein in neurons was detected by double-immunofluoresence method. The laser scanning confocal microscope(LSCM) was used to observe the localization of BMSCs labelled by CM-Dil in brain tissue. Results:1. The culture and identification of BMSCs: cultured cells of the third passage presented in uniform spindle morphology. The laser scanning confocal microscope result showed that 88% cells remained in G1 stage, which implied the cultured cells posess strong proliferation potential. The positive percentages of CD44 and CD90 were 92.86% and 95.77%, respectively, however, that of CD45 was near to zero, which were accordance with the surface markers of BMSCs. The immunocytochemistry result showed that the differentiated cells from BMSCs expressed the neural markers MAP-2, and BMSCs could produce calcium nodules under specific induction conditions, which suggested the BMSCs differentiated into osteocyte.2. The preparation of the rat model of burn: in one week, the burned skin of rat became hard and black, the marginal of the burn skin started form the scab at 7th day, rats ware less food and drink with dispirited spirit. HE result revealed that the burned skin, at 6 hours after burning, appeared pathological changes. The attachment of the epidermis and the dermis and the cells were damaged, which can be determined as Ⅱ~Ⅲ degree burns. After one week, HE staining showed that hippocampal and cortical parts of the brain cells had different degrees of edema, and nuclei appeared condensation. Nissl staining indicated that the nissl bodies of the neuron of hippocampus and cortex reduced and disordered. The immunocytochemistry results showed that the expression of MAP-2 in the cerebra in model group was less than that in control group(P<0.05), and the glial cell marker GFAP increased significantly(P<0.05).3. The repair effect of BMSCs transplantation on serious burned rats: At one week after BMSCs transplantation, the rats of model group had less food and dispirited spirit, however, the rats in the therapy group were active and good intake. Compared with that in model group, in the cell therapy group, the average body weights increased and tended to rise slowly; HE staining indicated that the edema of cells in the cerebral cortex and the pyknosis of nucleus reduced; Nissl staining showed that the nissl bodies increased and regular arrangement in the rat cerebral cortex. In therapy group, the positive percentage of apoptosis cells was significantly lower than that in model group and significantly higher than that in control group(P<0.05). Immunohistochemistry and Western Blot results revealed that compared with the model group, the expressions of NSE, Bcl-2, Bcl-2/Bax increased significantly(P<0.05); the expressions of GFAP, p53, Bax, BIM, cytochrome C and Caspase-3 in the rats of therapy group were significantly lower than those in model group(P<0.05). Double immunofluorescence showed the expression of LC3 B in the NSE-positive cells in therapy group was less than that in the model group(P<0.05). Conclusions:Therefore, above results suggested that a severe burn can induce brain injury, and BMSCs transplantation can repair the damaged brain. It may be the mechanisms for repairing brain injury caused by serious burn with BMSCs that BMSCs can inhibit excessive autophagy and apoptosis. |
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