The Effect And Mechanism Of Endothelial Progenitor Cells On Cigarette Smoking-induced Chronic Obstructive Pulmonary Disease Model Of Mice

Objective To discuss the cultivation and identification methods of endothelial progenitor cells (EPCs) and observe the differences between whole bone marrow culture method and density gradient centrifugation method in isolating and cultivating EPCs.Methods Bone marrow obtained from healthy C57BL/6J mice were cultivated by whole bone marrow culture method and density gradient centrifugation method respectively in ECM-2MV medium. The morphologic features of cells were observed under inverted microscope, and the abilities of DiI-acLDL uptaking and FITC-UEA-I binding were detected through fluorescence stain. Immunofluorescence was used to investigate the expression of von Willebrand factor (vWF). Immunocytochemistry was used to investigate the expression of endothelial nitric oxide synthase (eNOS). The cell surface markers were analysed by flow cytometry.Results(1) Primary cells culture with density gradient centrifugation method showed the characteristics of forming capillary structure and "cobblestone" morphology. Under the method of whole bone marrow culture, most adhere cells were long fusiform-shaped and grown in spreading style.(2) The double-positive rate in uptaking DiI-acLDL and binding FITC-UEA-I of adhere cells in density gradient centrifugation method [(95.3±1.8)%] was higher than that in whole bone marrow culture method [(59.7±8.6)%] (P<0.05).(3) The expression rate of vWF and eNOS of adhere cells in density gradient centrifugation method [(92.9±1.5)%,(92.9±1.5)%] were higher than that in whole bone marrow culture method [(51.6±3.5)%, (45.8±1.6)%](all P<0.05).(4) More CD34+Flk-1+and CD34+CD133+Flk-1+cells were detected in density gradient centrifugation method than whole bone marrow culture method [(26.09±1.62)% vs. (7.58±0.70)%, (18.41±0.97)%vs. (5.00±0.19)%, all P<0.05], but the ratio of CD34+CD133+cells were higher in whole bone marrow culture method than in density gradient centrifugation method [(49.51±1.79)% vs. (72.68±5.30)%, P<0.05].(5) Under the method of density gradient centrifugaion, the ratio of CD34+CD133+, CD34+Flk-1+and CD34+CD133+Flk-1+were increased during the culture time (all P<0.05)., and reached to (45.34±2.12)%, (11.65±1.01)% and (9.513±0.87)% in day 14 respectively.Conclusion (1) EPCs could be obtained from mice bone marrow under density gradient centrifugation method and whole bone marrow method under the culture system of EGM-2MV. (2) Compared with whole bone marrow culture method, density gradient centrifugation method was better for higher purity of EPCs. Objective To set and evaluate a mouse model of COPD induced by cigarette smoke (CS) and observe the systemic manifest.Methods Adult (n=8,18-20g body wt) male C57BL/6J mice (COPD group) were exposed 4 times per day, whole body, to CS from six cigarettes for continuous 90 days and then to observe for another 30 days. The control group (n=8) was sham-smoked. BAL with venous indwelling catheter was used to collected bronchoalveolar lavage fluid (BALF). Pulmonary function was measured by PLY 3211 small animal pulmonary function testing system. The total cell count and classification of BALF were measured, and morphology manifests of lung tissues were evaluated by H&E stain. Systemic manifests of mice were detected, including body weight, morphology manifests in H&E stain and apoptosis of skeletal muscles by TUNEL, and total antioxidant capacity (T-AOC) in serum measured by colorimetric technique.Results(1) Compared with the normal control group, higher Raw[(1.91±0.58) cmH2O·mL-1·min-1 vs. (0.22±0.12) cmH2O·mL-1·min-1, P<0.05], lower Cdyn [(1.019±0.004) mL/cmH20 vs. (3.100±1.367) mL/cmH2O, P <0.05] were found in COPD group. (2) Compared with the normal control group, COPD group owned more total cell count [(5.85±0.67)×108/L vs. (1.47±0.24)×108/L], AM [(4.45±0.63)×108/L vs. 1.34±0.14)×108/L], N [(7.76±0.92)×107/L vs. (0.77±0.09)×107/L] and higher N%[(13.7±2.4)% vs. (8.7±1.0)%] in BALF. (3) Morphology detection of lung tissues showed significant enlarged airspace, disruption of alveolar septum and formation of emphysema in COPD group. (4) Quantitative morphological analysis showed:compared with the normal control group, MLI and DI [(51.8±7.3)μm vs.(33.3±3.2)μm, (38.9±4.3)% vs. (12.9±3.2)% respectively, all P<0.05] were higher in COPD group, while MAST [(4.3±0.6)μm vs.(8.1±1.0)μm, P<0.05] was lower in COPD group. (5) Morphology detection of peripheral skeletal muscle showed random loose arrangement of fibers, inhomogenous stain or vacuole forming in endochylema, lessen of nuclei or even "nuclei ingression" in quadriceps femoris of COPD group. Compared with the normal control group, COPD group showed higher apoptosis index [(20.6±4.5)% vs.(2.9±0.6)%, P<0.05] in peripheral skeletal muscle. Lower total antioxidant capacity (T-AOC) in serum was detected in COPD group than that in normal control. (6) Body weight was positively correlated with T-AOC (r=0.815, P<0.05), and was negatively correlated with the apoptosis of skeletal muscle (r=-0.852, P<0.05). The apoptosis of skeletal muscle was negatively correlated with T-AOC (r=-0.941, P<0.05).Conclusions Cigarette Smoke could establish COPD model stably in mice, this mouse model owned high similarity to human COPD. Objective This study was designed to determine the effect of intratracheal allogenic EPCs transplantation on cigarette smoke-induced COPD model of mice and its mechanism.Methods (1) Isolated mononuclear cells from C57BL/6J mice bone marrow were cultured in EGM-2MV medium for 10-12 days, yielding EPCs. (2) Forty male C57BL/6J mice were randomly divided into five groups:normal control group, PBS early treated group, PBS late treated group, EPCs early treated group and EPCs late treated group. All groups except the normal control group which was sham-smoked were exposed passively to cigarette smoke (CS) (6 cigarettes for 15 minutes,4 times per day for continuous 90 days). At the 30th-day and 90th-day after cigarette smoke exposure, allogenic EPCs (105 cells in 30μL PBS) were administered into the trachea of mice in EPCs early treated group and EPCs late treated group respectively. Correspondingly, the PBS early treated group and PBS late treated group were treated with 30μL PBS at the 30th-day and 90th-day respectively. (3) 30 days after the final cigarette smoke exposure, mice were anesthetized to measure lung function by PLY 3211 small animal pulmonary function testing system, then sacrificed by intracardiac bloodletting, and the samples of serum, BALF and lung tissues were collected. Lung paraffin sections were stained with hematoxylin and eosin (HE), and measured mean alveolar septal thickness (MAST), mean linear intercept (MLI) and destructive index (DI) to evaluate the degree of emphysema. Frozen sections of lung were stained with immunofluorescence to located Pan-cytokeratin. The total cell and classification were counted in BALF. The total antioxidant capacity (T-AOC) in serum and BALF were measured by colorimetric technique, while the levels of MMP-2, MMP-9 and TIMP-1 in BALF were detected by ELISA. TUNEL was performed to observe the DNA damaged cells in lung and the expressions of MMP-2 and MMP-9 were determined by immunohistochemisty. MMP-2 and MMP-9 activities were investigated by gelatin zymography. And mRNA levels of MMP-2, MMP-9 and TIMP-1 were measured by reverse transcriptase PCR (RT-PCR).Results(1) Mononuclear cells from C57BL/6J mice bone marrow cultured in EGM-2MV medium had EC-liked shape and were identified as EPCs.(2) EPCs could immigrate to the airway, alveolar septum and pulmonary blood vessels after intratracheal transplantation.(3) The lung function showed higher airway resistance (Raw) and lower dynamic lung compliance (Cdyn) were detected in PBS or EPCs early and late treated groups than those of the control group (All P<0.05). And EPCs early and late treated groups owned lower Raw and higher Cdyn than corresponding PBS treated groups respectively (All P<0.05). Also, EPCs early treated group owned lower Raw and higher Cdyn than EPCs late treated group (All P<0.05). The differences among groups in PEF were of no significance (All P>0.05).(4) Morphology detection (HE stain) showed significant enlarged airspace, disruption of alveolar septum and formation of emphysema in PBS early and late treated groups, and the degree of alveolar destruction in EPCs early and late treated groups were relieved compared with those of corresponding PBS treated groups respectively. The MLI and DI were significantly increased and the MAST was decreased in the PBS early and late treated groups compared with the control group (All P<0.05). However, The MLI and DI were markedly decreased while the MAST was increased in EPCs treated groups (especially in EPCs early treated group) compared with corresponding PBS treated groups (All P<0.05).(5) The frozen sections stained with immunofluorescence manifest the positive areas in green fluorescence of FITC. Together with the red fluorescence of CM-DiI, some areas that were double stained by FITC and CM-DiI showed yellow or orange fluorenscence.(6) The number of total cells, macrophages, neutrophils and ratio of neutrophils in BALF were increased markedly in PBS early and late treated groups in comparison to the control group, and EPCs early treated group showed fewer total cells, macrophages and neutrophils in BALF than corresponding PBS treated group (All P<0.05). But there were no statistical difference between EPCs late treated group and PBS late treated group (All P>0.05).(7) The total antioxidant capacities (T-AOC) in serum and BALF were significantly decreased in PBS early and late treated groups in comparison with the control group (All P<0.05). After EPCs treatments, the decrease of T-AOC in serum and BALF was inhibited partly compared with corresponding PBS treated group (All P<0.05). And the EPCs early treated group owned higher level of T-AOC in serum and BALF compared with EPCs late treated group (All P<0.05).(8) Compared with the control group, PBS early and late treated groups showed higher level of MMP-2 and MMP-9, and lower level of TIMP-1 in BALF (All P<0.05). The EPCs treatments inhibited the increased level of MMP-2 and MMP-9 and decreased level of TIMP-1 in mice exposed to CS (All P<0.05). And the effects were more obviously in EPCs early treated group than EPCs late treated group (All P<0.05).(9) The TUNEL-positive cells were markedly distributed in alveolar septum and pulmonary blood vessels of the emphysematous lungs of mice in PBS early and late treated groups. The apoptosis index (AI) in both alveolar septum and pulmonary blood vessels were significantly higher in PBS early and late treated groups than that of the control group (All P<0.05). And the AI was reduced in EPCs treated groups, especially in EPCs early treated group (All P<0.05).(10) More MMP-2 and MMP-9 positive cells were distributed in the emphysematous lungs of PBS early and late treated groups comparing with those in the lung of the control group. The expressions of MMP-2 and MMP-9 were apparently reduced in EPCs early and late treated groups compared with those of PBS early and late treated groups respectively (All P<0.05). And the expressions of MMP-2 and MMP-9 in EPCs early treated group were lower than that of EPCs late treated groups (All P<0.05).(11) Mice in PBS early and late groups showed significantly increased MMP-2 and MMP-9 activities in lung homogenates compared with those of the control group(All P<0.05). Expectedly, MMP-2 and MMP-9 activities were reduced significantly in both EPCs early and late treated groups compared with corresponding PBS treated groups (All P<0.05). Also, EPCs early treatment showed more beneficial in inhibiting MMPs activities than EPCs late treatment (All P<0.05).(12) RT-PCR detected increase in mRNA levels of MMP-2 and MMP-9 and decrease in mRNA level of TIMP-lin lungs of PBS early and late treated groups in comparison to those of the control group, and EPCs treated groups (especially EPCs early treated group) owned lower mRNA levels of MMP-2 and MMP-9, and higher mRNA level of TIMP-lthan those of corresponding PBS treated group (All P<0.05).Conclusions(1) We concluded that intratracheal transplantation of EPCs protected against the development of emphysema and improved lung function probably by alleviating inflammatory infiltration, decelerating apoptosis of endothelial cells and epithelial cells, inhibiting proteolytic enzyme activity and expression, and enhancing local and systemic antioxidant activity. (2) EPCs early treatment was more beneficial than EPCs late treatment. EPCs might represent a new therapeutic option in the treatment of emphysema in humans.