| Objective:To test the increasing restoration of MSC overexpressing CXCR7 in the injured lung, attenuation of inflammatory response and lung protective effect in LPS-induced ARDS mice.Methods:90 C57BL/6 mice were randomly divided into Control group (NS+PBS), ALI group (LPS+PBS), MSC group (LPS+MSC), MSC-GFP group (LPS+MSC-GFP) and CXCR7 group (LPS+MSC-CXCR7). Murine model of ARDS was generated by intratracheal instillation of 5mg/kg lipopolysaccharide (LPS) and cell suspension/PBS was injected into the tail vein according to subgroup 4h later. After different treatment the specimens were harvested at 30min,24h and 72h to perform:1)observation the homing of MSC overexpressing CXCR7 in the injured lung:near-infrared (NIR) fluorescence imaging of the ex vivo organs, fluorescence microscopy of the lung tissue and examination of adhesion factor VCAM-1 and COL-1 in injured site by ELISA were achieved to evaluate MSC-CXCR7 homing synthetically in different aspects including organ, tissue and molecules; 2)evaluation of the severity of injured lung in ARDS mice after MSC overexpressing CXCR7 treatment: obtaining lung specimens and observing the degree of lung injury generally were achieved after the mice were sacrificed. Lung edema was estimated by lung wet weight/body weight ratio (LWW/BW), what’s more, histopathological analysis of lung tissues were performed by H&E staining and quantified by using Smith lung injury scores; 3) evaluation of the effect of MSC overexpressing CXCR7 homing on inflammation in ARDS injured lung:the levels of anti-inflammatory factor IL-10 and pro-inflammatory factor TNF-a in lung homogenates were measured by ELISA to reflect the pulmonary inflammation.Results:1.There were 5 groups and 3 time points in each group(n=6). Histopathological examination was perforemed after the mice sacrificed, and the results displayed that hemorrhage, infiltration of inflammatory cells and formation of hyaline membrane were observed widely in pulmonary alveoli and interstitial, indicating the ARDS models were constrncted successfully.2.Comparison of MSC homing towards the ARDS injured lung in different groups:1) NIR fluorescence imaging of organs ex vivo showed that the MSC-GFP predominantly localized within the lung 30min after delivery and peaked at 24h compared with Control group, and then decreased at 72 h; the observation and quantitative analysis after MSC-CXCR7 treatment showed the fluorescent signal was significantly higher than MSC-GFP group(24h:301.62±187.12 vs.71.75±32.37 scaled counts/mm2, #p<0.05; 72h: 217.02±126.38 vs.67.08±26.44 scaled counts/mm2,#p<0.05).2) fluorescence microscopy of the lung tissue showed that MSC which was expressed green fluorescent protein(GFP) presented in injured lung at 24h after the engraftment of MSC-GFP and performed a decreasing trend at 72h; hoever the fluorescent signal in ARDS lung tissue was more intensive in MSC-CXCR7 group than in MSC-GFP group.3)the measurements of concentration of MSC-homing related adhesive factor by ELISA showed the level of VCAM-1 in injured lung tissue was low in Control group but increased at 24h and 72h after the LPS challenge, moreover, the concentration further increased after the engraftment of MSC-GFP and MSC-CXCR7, while it was significantly higher in MSC-CXCR7 group(24h:0.873±0.021 vs. 0.463±0.021ng/ml,&p<0.05; 72h:1.340±0.141 vs.0.512±0.038 ng/ml,&p<0.05). The variation of COL-1 concentration was similar to VCAM-1, which indicated the level of COL-1 in injured lung tissue was significantly higher at 24h and 72h after the MSC-CXCR7 treatment when comparing with MSC-GFP group(24h:1.738±0.247 vs.0.977±0.133ng/ml, &p<0.05; 72h:4.137±0.386 vs.3.597±0.197ng/ml,&p<0.05).3.Comparison of severity of ARDS lung injury in different groups:MSC-CXCR7 treatment could significantly improve lung histopathology involving decreasing hemorrhage, infiltration of inflammatory cells and formation of hyaline membrane. Moreover it reduced LWW/BW(24h:5.98±0.63 vs. 7.33±0.53mg/g,&p<0.05; 72h:7.37±0.85 vs.8.97±1.25mg/g,&p<0.05) and lung injury score(30min:10.20±0.40 vs.11.80±0.78,&p<0.05; 24h:8.33±0.67 vs.12.87±0.38,§p<0.001; 72h:10.00±0.26 vs.14.00±0.72,§p<0.001) more effectively when comparing with MSC-GFP treatment.4.Comparison of pulmonary inflammation in different groups:the level of pro-inflammatory factor TNF-a was higher in ARDS group mice than in Control goup, while the level of anti-inflammatory factor IL-10 was lower. The measurement of TNF-a in ARDS injured lung tissue showed the concentration was markedly decreased at 24h and 72h after theMSC-GFP and MSC-CXCR7 treatment, while the degree was more significant in MSC-CXCR7 group(24h:6.665±0.349 vs.9.963±0.382 ng/ml,§p<0.001; 72h:7.592±0.434 vs.10.718±0.769ng/ml,§p<0.001). On the contrast, the concentration of IL-10 was much higher in MSC-CXCR7 group than that in MSC-GFP group(24h:176.432±4.431 vs. 148.082±4.469ng/ml,§p<0.001; 72h:176.300±2.508 vs.143.947±8.179ng/ml,&p<0.05). What’s more, the concentration of TNF-a at 72h after MSC-CXCR7 treatment was much higher than that at 24h, which has significant difference (§p<0.05).Conclusion:MSC modified with the CXCR7 gene homing to the LPS-induced injured lung was markedly increased and provided an additional attenuation of lung inflammatory response compared with MSC alone, which was much favorable for promoting the protective effect of MSC on ARDS injured lung. |
PDF Full Text Request