| Radiotherapy is the effective treatment to thoracic tumor. Radiation induced lung injury (RILI) is the frequent complication during and/or after radiotherapy. RILI includes acute injury and delayed injury which occurs three months after irradiation. It not only severely affects the patient's life quality but also causes death sometimes. The high dose glucocorticoid is the most common therapy for acute lung injury, which may result in diabetes or femoral head necrosis. The delayed lung injury occurs torpidity relatively. The symptoms often show up3-6months after radiotherapy, when lung fibrosis has been established and the best time for treatment has been missed. The early prediction would allow immediate therapies and thus reduce the incidence of RILI. So it is urgently needed to have effective and low-toxic therapy as well as feasible predictive markers to improve the prognosis of RILI.Mesenchymal stem cells (MSC) are an important member of stem cells which can be obtained from different tissues of mice including bone marrow, murine embryonic lung and dorsal aorta area as well as umbilical cord and amnionic membrane of human beings. Under specific conditions MSCs can differentiate into adipocyte, cartilage, myocardial cells and lung epithelial cells in vivo or in vitro. Meanwhile much attention has been paid on its immune regulation functions.In this study, MSCs from different sources (both from human being and mice) were isolated, cultured and identified. Then we transferred these MSCs into RILI mouse model to evaluate their effects on lung injury.1. Isolation, culture and transplantation of MSCs from mouse bone marrow, murine embryo lung and dorsal aorta area (DA area).25experimental mice were divided into5groups:normal control (group N), radiation control (group R), radiation and transplantation MSC derived from bone marrow (group BM), radiation and transplantation MSC derived from embryo lung (group EL), radiation and transplantation MSC derived from dorsal aorta (group DA). Each mouse received a total dose of18Gy/1fraction irradiation on thorax. The results of mice appearance and pathological score of lung tissue were evaluated at five different time point, that is4,9,13,17,23weeks after irradiation. The appearance scores for group N, DA, EL, BM, R was10±0,8±0.71,7.6±0.57,6.1±1.75,4.8±0.84, respectively. Statistics showed significant difference between group R and group DA or EL. No difference was found between the MSCs transplantation groups. The pathological scores for lung injury of group N, DA, EL, BM, R was0.12±0.16.0.35±0.18,0.22±0.1,0.33±0.18,0.54±0.27, respectively. The percent of alveolar interstitial in pathological slice by computer image analysis for group N was64.3±1.6, group DA was76.8±3.3,group EL was76.1±2.8,group BM was77.2±2.7, group R was80.8±3.5. The appearance score was significantly related to the pathological score(p=0.040) and computer image analysis score (p=0.039) by statistics. Conclusion:transplantation of MSCs derived from bone marrow, DA or embryo lung are effective in reducing radiation-induced lung injury.2. The effect of MSCs transplantation on RILI was confirmed and some related items were detected in this part. There were40mice randomly arranged to4groups:normal control (group N), radiation control (group R), radiations followed EL MSCs transplantation (group EL), and radiation followed DA MSCs transplantation (group DA). Each mouse received a total dose of18Gy/1fraction irradiation on thorax. The grading of mice appearance and histological examination of the lung was done at the time point of4and8weeks after treatment. The plasma level of TGF-β,IL-6and hydroxyproline content in lung tissue were detected. Results:for appearance score, significant difference was found at8weeks after radiation, but not at4weeks. The score of group R (6.6±0.5) was lower than that of other groups(N9.6±0.9, DA7.6±0.9, EL7.8±0.4, P<0.01). The score of group DA was similar with that of group EL (p=0.374). Histological score of the lung injury at4weeks after radiation was as follow:The score of group N(0±0) was lower than that of group DA(0.19±0.05, p<0.01), group EL(0.29±0.22, p=0.019) and group R(0.38±0.35, p<0.001). The score at8weeks after radiation of group N (0.06±0.08) was lower than group DA(0.26±0.17, p=0.044), group EL (0.23±0.10, p=0.016) and group R (0.30±0.18, p=0.025). No significant difference was found among group EL, DA and group R at two time points.The IL-6level (pg/ml) of group N was489.3±75.3at4weeks and421.2±80.1at8weeks. The data was437.9±87.1and398.1±116.3for group R. There was no significant difference between any groups.The TGF-β1level (pg/ml) of group N was20.2±12.8at4weeks and16.3±4.6at8weeks. The data of group DA was27.5±13.5and12.4±2.6respectively (p=0.039). For group EL, it was15.5±8.4and28.3±18.4(p=0.194). and for group R it was9.0±2.8and31.5±27.9(p=0.110).There was no significant difference for the content of hydroxyproline in lung tissue at the same time. But the content of each group at4weeks was lower than that at8weeks. The content of group N at4weeks was812.9±68.4and1080.8±189.5at8weeks, p=0.018. The content of group DA at4weeks was823.3±14.6and1266.4±76.9at8weeks, p<0.001. The content of group EL at4weeks was796.2±62.7and1234±153.9at8weeks, p<0.01. The content of group R at4weeks was839.0±17.0and1260.9±218.0at8weeks, p=0.003.Conclusion:MSC derived from different tissue of mouse is useful to radiation induced lung injury. The difference of the score is significant among each group at8weeks after radiation. Histological score of the lung injury is similar among group DA, group EL and group R. The plasma level of TGF-β,IL-6should be detected further more. The content of hydroxyproline in lung tissue turns to increase by time. That reflects the lung fibrosis in the later time of the injury.3. The amnion MSC (AMSC) and umbilical cord MSC (UMSC) from human being were isolated, cultured and characterized in this part. Then AMSC and UMSC were transplanted into lung-radiated mice to investigate their effect on lung injury. Total46C57BL/6mice of8-10weeks old were randomly arranged to four groups including normal control (group N), radiation control (group R), radiations followed AMSC transplantation (group AMSC), and radiation followed UMSC transplantation (group UMSC). The mice were sacrificed at7weeks (n=21) and11weeks (n=25) after radiation. Radiation dose changed to20Gy/1fraction. The degree of radiation induced injury was evaluated by grading of mice appearance and histological examination of the lung. Several plasma cytokines including IL6,IL-10, MCP-1,INF-γ,TNF-α,IL-12p70were measured by Cytometric Bead Array (CBA) method at two time point. Results:No accidental death occurred on the transplanted mice. The group N had significant high appearance scores at any time points (9.8±0.4/10±0)than other groups (group R8.2±0.4/8.0±0, AMSC8±0.6/7.8±0.5, UMSC8.3±0.5/8.0±0.5). No difference was found among group R, group AMSC or group UMSC. No significant difference was found in histological score of the lung injury in both time points(group N0.06±0.05/0.12±0.05, group R0.18±0.06/0.19±0.08, group UMSC0.11±0.01/0.12±0.04, group AMSC0.14±0.09/0.14±0.06in).The plasma level of all six cytokines was similar between two time points in group N. The level of these cytokines changed significantly lower at11weeks than that at7weeks in other three groups. There was no significant difference among all groups at11weeks after radiation. The change of cytokines level showed that the inflammatory reaction occurred after radiation.In a word, MSC derived from mouse could definitely relieve the degree of RILI. Appearance score, histological score and plasma cytokines could be took as the method of evaluation radiation injury. MSCs derived from human being were safety to mice and Its effect need more study.
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