BCG对哮喘大鼠Th1/Th2平衡调节与其对血管重塑影响

【Abstract】wWw.shuoshilunwen.com Objective: To evaluate the effect of BCG on the prevention of inflammatory and hyperresponsiveness of airway in asthmatic rats, and to investigate the influence of BCG on Th1/Th2 balance and vascular remodeling.Method: Forty cleaning SD rats (male, four weeks, weights from 50 to 100 grams) were randomly divided into four groups: control group, asthma group, multiple BCG vaccination group and BCG intervention group. The asthma group rats were sensitized on the first day and the 8th day with ovalbumin (100mg) and AL(OH)3(100mg) by intraperitoneal injection (mixed in NS 2ml). And then those rats were inhaled aerosolized 2﹪OVA 5ml in a not close tightly box from fifteen day. Rats were inhaled 30 minutes each time in every other day, 20 times together.The multiple BCG vaccination group rats in 7, 3, 1days before OVA sensitization were injected subcutaneously BCG 0.025mg. Other steps were same as asthma group.The BCG intervention group rats in 7 days before OVA stimulation were injected subcutaneously BCG 0.025mg. And then were injected the same dose 30 minutes before OVA stimulation on the 15th day. Other steps were same as asthma group.The control group rats were treated as asthma group with NS instead of OVA.After 24 hours of last stimulation, rats were anesthesiaed by 1% pentobarbitalin and measured pulmonary function. The number of total cells and eosinophils in the BALF were counted. Lung tissue was sliced and stained with HE. The expression of VEGF and Endostatin in airway tissues were observed by immunohistochemistry combined with the micro-image analysis, and mean optical density of VEGF and Endostatin were measured by image analysis software. The parameters such as thickness of airway wall and vascular density were measured by image analysis system. The level of IL-4, IFN-γ, VEGF and Endostatin in serum was measured by ELISA.Results:1 Result of pulmonary function: When rats were injected normal saline and Ach (6.25μg/kg), the erage expiratory peak was not significantly different among the four groups (F=2.204 P>0.1, F=1.453 P>0.1). When asthmatic rats were injected Ach (12.5μg/kg, 25μg/kg, 50μg/kg), the erage expiratory peak were lower than that of other three groups (F=10.660 P<0.01, F=25.531 P<0.01, F=44.125 P<0.01). The change of the expiratory peak in the multiple BCG vaccination group had no difference compared with the BCG intervention group (P>0.1). The two group were lower than the control group (P<0.01).2 BALF total cell and eosinophils: The number of total cells and eosinophils in the BALF in each group were as follows: Asthma group was (17.15±3.02)×106, (2.19±0.30)×106. Multiple BCG vaccination group was (8.78±2.00)×106, (1.19±0.31)×106. BCG intervention group was (11.87±2.31)×106, (1.49±0.30)×106. Control group was (5.40±1.52 )×106, (0.51±0.18)×106 . Asthma group was the highest among the four groups (P<0.01). Multiple BCG vaccination group was lower than the BCG intervention group (P<0.05), the both groups were higher than the control group (F=47.930 P<0.01, F=62.569 P<0.01).3 Pathological findings: The obvious inflammatory cells infiltration and goblet cells proliferation and hyperplasia of vascular, mucus and ooth muscle and airway wall thickness can be found in the lung tissue of asthmatic rats. After the treatment with BCG, the change of the above mentioned pathology character were observed.4 Result of image analysis: Observing the airway wall thickness and vascular density in these four groups showed that the asthma group (98.17±9.38, 203.88±14.15) was the highest among the four groups (P<0.01). Multiple BCG vaccination group (72.10±8.70, 138.13±9.35) was significantly lower than the BCG intervention group (80.34±9.23, 177.03±7.52) (P<0.05), the both groups were significantly higher than the control group (61.70±8.77, 97.78±7.27) (F= 168.435 P<0.01, F=152.663 P<0.01).5 Result of immunohistochemistry: The erage light density values of VEGF and Endostatin in the asthma group (0.41±0.02, 0.47±0.03) were the highest among the four groups (P<0.01). Multiple BCG vaccination group (0.24±0.02, 0.31±0.02) was significantly decreased compared with the BCG intervention group (0.33±0.02, 0.38±0.03) (P<0.01). The two group were significantly higher than the control group (0.17±0.02, 0.22±0.02) (F=168.435 P<0.01, F=152.663 P<0.01).6 Result of ELISA: The expression level of IL-4, IFN-γ, VEGF, Endostatin and VEGF/Endostatin in serum in each group were as follows: Asthma group was 33.98±2.35, 59.54±6.86, 3.54±0.53, 17.01±2.48. Control group was 11.43±2.72, 10.60±3.93, 1.04±0.37, 5.81±2.41. Multiple BCG vaccination group was 16.46±1.92, 21.33±2.88, 1.84±0.36, 9.26±1.72. BCG intervention group was 20.89±2.29, 37.79±6.33, 2.56±0.55, 13.62±2.50. Asthma group was the highest among the four groups (P<0.01). Multiple BCG vaccination group was lower than the BCG intervention group (P<0.01), the two group were significantly higher than the control group (F=136.418 P<0.01, F=131.25 P<0.01, F= 42.19 P<0.01, F=36.42 P<0.01).The level of IFN-γin the asthma group (10.92±1.93) was the lowest among the four groups. Multiple BCG vaccination group (22.66±2.05) was significantly higher than the BCG intervention group (18.46±1.48). The two group were significantly higher than the control group (30.36±3.65) (F= 90.064 P<0.01).7 Result of correlation analysis: There was a significant positive correlation between airway thickness, the level of IL-4, VEGF and VEGF/Endostatin and the vascular density in the asthma group (r=0.670 P<0.05, r=0.602 P<0.05, r=0.709 P<0.05, r=0.638 P<0.05). It was a significant negative correlation between the vascular density and IFN -γ(r=-0.593 P<0.05). Conclusion:1 BCG may significantly decrease the expression of IL-4 and increase the level of IFN-γ, it demonstrated that BCG may adjust the balance of Th1/Th2.2 BCG may significantly decrease the number of total cells and eosinophils in the BALF, it suggested that BCG may reduce airway inflammation by decreasing the level of IL-4 and increasing the level of IFN-γ. And the multiple BCG vaccination group were significantly decreased compared with the BCG intervention group.3 BCG may significantly reduce airway resistance, it indicated that BCG may reduce hyperresponsiveness of airway by decreasing the level of IL-4 and increasing the level of IFN-γ. There was no difference between the multiple BCG vaccination group and the BCG intervention group.4 BCG may significantly decrease the expression of VEGF and the ratio of VEGF/Endostatin in serum, and vascular density were positively correlated with the expression of IL-4, VEGF and the ratio of VEGF/Endostatin in asthmatic rats, and vascular density were negative correlated with IFN-γ. The results showed that BCG may decrease the ratio of VEGF/Endostatin through decreasing the level of IL-4 and increasing the level of IFN-γ, and then BCG may inhibit the vascular remodeling. And the multiple BCG vaccination group was better than the BCG intervention group.