| Part One Changes of CD31CD45-Sca-1+cells quantity and Shh signaling pathway in lung of emphysema mouse induced by CSE intraperitoneal injectionObjective:to investigate the quantity of CD31’CD45-Sca-1+cells and expression level of Shh signaling pathway in lung of emphysema mouse induced by CSE intraperitoneal injection.Methods:using a random number table,40BALB/c inbred male mice were divided randomly into two groups (PBS group and CSE group) with20mice in each group. At day1,11and22, PBS group mice were injected intraperitoneally with PBS (0.01M, PH7.2-7.4) and CSE group mice were injected intraperitoneally with CSE (0.3ml). The animals were sacrificed at the28th day of the experiment. Airway resistance (Raw) and dynamic lung compliance (Cdyn) were recorded by the Small-animal Pulmonary Function Instrument. After the preparation of lung histological sections stained with hematoxylin-eosin (HE), the mean liner intercepts (MLI, μm) and destructive index (DI,%) of the lung were measured to assess the severity of emphysema. After using enzyme digestion method to prepare lung sing cell suspension, the percentage of lung CD31"CD45-Sca-1+cells was detected by Flow cytometry. The mRNA expression of major component of Shh signaling pathway Shh, Ptchl and Gli1in lung was detected by real time RT-PCR, and their protein expression in lung by Western-blot assay. The distribution and expression of Shh, Ptchl and Glil in lung was detected by histochemistry assay. Statistical analysis was performed using SPSS statistical softmware package, version18.0(SPSS Inc, Chicago, IL). Data were presented as Mean±SD and analyzed using independent samples t-test. Statistical significance was defined as P<0.05.Results:(1) Airway resistance (Raw) was higher in the CSE group than in the PBS group [(0.48±0.28) cmH2O. mL-1. min-1vs.(0.24±0.14) cmH2O. mL-1. min-1, P<0.05], dynamic lung compliance (Cdyn) was lower in the CSE group than in the PBS group [(0.054±0.019) ml/cmH2O vs.(0.074±0.030) ml/cmH2O, P<0.05).(2)Lung tissue quantitative pathologic analysis:the mean liner intercepts (MLI) and destructive index (DI) of the lung were higher in the CSE group mice than in the PBS group [(42.0±4.2)μm vs.(23.8±0.8)μm,38.2%±2.1%vs.10.7%±1.3%, P<0.05].(3) The percentage of lung CD31-CD45-Sca-1+cells was lower in the CSE group mice than in the PBS group (0.87%±0.16%vs.1.26%±0.18%, P<0.05).(4) There were no statistical significances for the mRNA relative expression level of Shh, Ptchl and Glil in lung tissue between CSE group mice compared and the PBS group (0.11±0.02vs.0.38±0.15,0.85±0.28vs.1.06±0.34,1.01±0.16vs.1.21±0.14,P>0.05). (5) The protein relative expression levels of Shh, Ptchl and Glil in lung tissue were lower in CSE group than in the PBS group (0.28±0.15vs.0.52±0.02,0.12±0.05vs.0.36±0.05,0.17±0.04vs.0.45±0.11, P<0.05).(6) The distribution and expression of Shh, Ptch1and Gli1in lung by histochemistry assay:a. distribution:Shh and Ptchl were mainly expressed in epithelial cells of airway other than pulmonary alveolar, few expressed in pulmonary alveolar epithelial cells, Shh and Ptchl were subcellularly located in the cytoplasm and at the cell member, Gli1in the cytoplasm nucleas. b. expression:the optical density of Glil were lower in CSE group than in the PBS group (6455±2606vs.13866±6364, P<0.05), but there is no statistical significances for the optical density of Shh and Ptchl between CSE group mice compared and the PBS group (8426±3646vs.9730±3959,4129±2320vs.6442±3496, P>0.05)Conclusions:(1) CSE intrperitoneal injection successfully induced emphysema mice.(2) the quantity of lung CD31-CD45-Sca-1+cells was decreased in CSE intrperitoneal injection induced emphysema mice.(3) the activation of Shh signaling pathway was inhibited in CSE intrperitoneal injection induced emphysema mice. Part Two The effects of acute adenovirus infection on CD31-CD45-Sca-1+cells quantity and Shh signaling pathway in lung of emphysema mouse induced by CSE intraperitoneal injectionObjective:to investigate the effects of acute adenovirus infection on CD31-CD45-Sca-1+cells quantity and Shh signaling pathway in lung of emphysema mouse induced by CSE intraperitoneal injection.Methods:using a random number table,60BALB/c inbred male mice were divided randomly into three groups (ie. PBS group, CSE group, and CSE+Adenovirus group) with20mice in each group. At day1,11and22, PBS group mice were injected intraperitoneally with PBS (0.01M, PH7.2-7.4), CSE group mice and CSE+Adenovirus group mice injected intraperitoneally with CSE (0.3ml) respectively. At day15th, PBS group mice and CSE group mice were dripped intratracheally with PBS (0.01M, PH7.2-7.4) respectively, CSE+Adenovirus group mice were dripped intratracheally with adenovirus (0.3ml,1010ifu/ml). The animals were sacrificed at the28th day of the experiment. Airway resistance (Raw) and dynamic lung compliance (Cdyn) were recorded by the Small-animal Pulmonary Function Instrument. After the preparation of lung histological sections stained with hematoxylin-eosin (HE), the mean liner intercepts (MLI, μm) and destructive index (DI,%) of the lung were measured to assess the severity of emphysema, and the inflammation of airway and vessel were semi-quantitatively scored. After using enzyme digestion method to prepare lung sing cell suspension, the percentage of lung CD31-CD45-Sca-1+cells was detected by Flow cytometry. The mRNA expression of major component of Shh signaling pathway Shh, Ptchl and Gli1in lung was detected by real time RT-PCR, and their protein expression in lung by Western-blot assay. The distribution and expression of Shh, Ptchl and Gli1in lung was detected by histochemistry assay. Statistical analysis was performed using SPSS statistical softmware package, version18.0(SPSS Inc, Chicago, IL). Data were presented as Mean±SD and analyzed using one-way ANOVA (LSD-t). Statistical significance was defined as P<0.05.Results:(1) Airway resistance (Raw) was higher in the CSE group than in the PBS group [(0.48±0.28) cmH2O. mL-1. min-1vs.(0.24±0.14) cmH2O. mL-1. min-1, P<0.05], Raw was also higher in the CSE+Adenovirus group than in PBS group[(0.49±0.24) cmH2O.mL-1. min-1vs.(0.24±0.14) cmH20. mL-1. min-1, P<0.05), dynamic lung compliance (Cdyn) was lower in the CSE group than in the PBS group [(0.054±0.019)ml/cmH2O vs.(0.074±0.030)ml/cmH2O, P<0.05), cdyn was also lower in the CSE+Adenovirus group than in PBS group [(0.052±0.014)ml/cmH2O vs.(0.074±0.030)ml/cmH2O, P<0.05], but the difference of Raw and Cdyn between CSE+Adenovirus group and CSE group has no statistical significant[(0.49±0.24)cmH2O. mL-1. min-1vs.(0.48±0.28)cmH2O. mL-1. min-1,(0.052±0.014)ml/cmH2O vs.(0.054±0.019)ml/cmH2O,P<0.05)(2) Lung tissue quantitative pathologic analysis:the mean liner intercepts (MLI) and destructive index (DI) of the lung were higher in the CSE group mice than in the PBS group [(42.0±4.2)μm vs.(23.8±0.8)μm, 38.2%±2.1%vs.10.7%±1.3%, P<0.05], MLI and DI were aso higher in the CSE+Adenovirus group than in CSE group [(38.1±2.1)μm vs.(23.8±0.8)μm,39.1%±2.6%vs.10.7%±1.3%, P<0.05].(3)①the semi-quantitative score of airway inflammation:There were no statistical significances between CSE+Adenovirus group and CSE group (0.18±0.08vs.0.12±0.15, P>0.05), the semi-quantitative score was higher in CSE+Adenovirus group than in the CSE group (1.81±0.30vs.0.18±0.08, P<0.05).②the semi-quantitative score of vessel inflammation:There were no statistical significances between CSE+Adenovirus group and CSE group (0.08±0.02vs.0.01±0.02, P>0.05), the semi-quantitative score was higher in CSE+Adenovirus group than in the CSE group (2.28±0.38vs.0.08±0.02, P<0.05).(4) The percentage of lung CD31-CD45’Sca-1+cells was lower in the CSE group mice than in the PBS group (0.87%±0.16%vs.1.26%±0.18%, P<0.05), higher in CSE+Adenovirus group than in the CSE group (1.30%±0.34%vs.0.87%±0.16%, P<0.05).(5) mRNA relative expression level of Shh, Ptchl and Glil in lung tissue: There were no statistical significances for Shh, Ptchl and Glil between CSE group mice compared and the PBS group (0.11±0.02vs.0.38±0.15,0.85±0.28vs.1.06±0.34,1.01±0.16vs.1.21±0.14, P>0.05). The mRNA relative expression level of Shh and Gli1were higher in CSE+Adenovirus group than in the CSE group (1.10±0.48vs.1.10±0.48,0.98±0.40vs.0.85±0.28,1.40±0.34vs.1.01±0.36, P<0.05), but the difference of Ptchl mRNA relative expression level between CSE+ Adenovirus group and CSE group was of no statistical significance (0.98±0.40vs.0.85±0.28, P>0.05).(6) The protein relative expression levels of Shh, Ptchl and Glil in lung tissue were lower in CSE group than in the PBS group (0.28±0.15vs.0.52±0.02,0.12±0.05vs.0.36±0.05,0.17±0.04vs.0.45±0.11, P<0.05), higher in CSE+Adenovirus group than in the CSE group (0.73±0.16vs.0.28±0.15,0.62±0.08vs.0.12±0.05,0.56±0.22vs.0.17±0.04, P<0.05).(7) The expression of Shh, Ptchl and Glil in lung by histochemistry assay:there is no statistical significances for the optical density of Shh and Ptchl between CSE group mice compared and the PBS group (8426±3646vs.9730±3959,4129±2320vs.6442±3496, P>0.05), the optical density of Glil were lower in CSE group than in the PBS group (6455±2606vs.13866±6364, P<0.05), the optical density of Shh, Ptchl and Glil were higher in CSE+Adenovirus group than in the CSE group (17490±7989vs.8426±3646,17347±7482vs.4129±2320,34418±7299vs.6455±2606, P<0.05).Conclusioins:In emphysema mice induced by CSE intrperitoneal injection:(1) acute adenovirus infection can upregulate the decreased lung CD31-CD45-Sca-1+cells quantity;(2) acute adenovirus infection can activate the Shh signaling pathway. |
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