| Background and Objective:Granuloma caused by Mycobacterium tuberculosis (Mtb) is the hallmark of the tuberculosis (TB), of which the occurrence, development and outcome are closely related with the formation, mature and liquefaction of the granuloma. It is the major cytological basis of granuloma that macrophage activates and lymphocyte infiltrates. TNF-α、IFN-γ、IL-12 and CCL2 et al.participate in the formation of granuloma by regulating macrophage and lymphocyte. Although there are a large number of reports that about the relationship between granuloma and lymphocytes, macrophages and cytokines, of which the mechanism is still not fully unclear. Our experiment was to study the role of Mannan binding lectin-associated serine protease 2 (MASP-2) in the formation of granuloma damage and revealed the mechanism, then it may establish a new target which will serve in the immune theropy.Methods:We adopted the BLOK-iTTM expression system, and carried the Gateway technology to make the homologous recombination, then transported the production into the HEK 293A cells for packaging, at last the rAd-hMASP-2 were amplified in the cells. To establish the pulmonary tuberculous granuloma animal model, BCG (5×107 CFU per mouse) was used to infect the BALB/c mice through the trachea.The animals for study were divided into four groups including rAd-hMASP-2 recombinant adenovirus intervention group (BCG+rAd-hMASP-2 group), rAd adenovirus vector control (BCG+rAd group), model control group (BCG group) and normal control group (PBS group). We injected BCG (5×107 CFU per mouse) by trachea, and given recombinant adenovirus (5×107 PFU per mouse) by intranasal way, at post-infection 21 day, animals were sacrificed. Many tests as followings: ① Lungs stained with hematoxylin-eosin, and the figures were collected from the optical microscopic imaging system. We calculated granuloma area by Image-Pro Plus 6.0, and analyzed the percentage through IBM SPSS Statistics 9 software. The acid-fast stain of Ziehl-Neelsen was used to define BCG. ② Lungs’ homogente of mice cultured on 7H10 solid medium at the temperature of 37℃ for 18-20 days, then counted the single colony of BCG. ③ Fluorescence-based quantitative real-time PCR (RT-qPCR) was made to detact the gene of hmasp, ccl2, ill2, il8 and il6. ④We detected CD3+, CD11b+ cells and the deposits of C4b by immunohistochemical method. ⑤ The number of CD3T cells, CD3+CD4+ T cells and CD3+CD8+T cells in lungs and spleens of mice were counted by the Flow cytometry.⑥ELISA detected the levels of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) in the lungs, spleens and blood. ⑦ We infected the mice with the high dose (1×108 CFU per mouse) of BCG, and observed the effect of rAd-hMASP-2 on the survival of mice.Results:1. The packaging of rAd-hMASP-2 was successfully verified by PCR. We amplified rAd-hMASP-2 in HEK293A cells, and the titer was detected to be 109-1010PFU/ml.2. The pulmonary tuberculous granuloma model of BALB/c mice has been established, with 5×107CFU per dose of BCG injected by the trachea, and typical granuloma formed at the day of 21.3. Compared with all of the three control groups, hmasp-2 mRNA was detected in the lungs of rAd-hMASP-2 intervened animals. Compared with BCG+rAd group (optical density value was 0.0022±0.0013), C4b deposition of BCG+rAd-hNASP-2 (optical density value was 0.0252±0.0129) increased significantly (P=0.003). So hMASP-2 can be expressed by rAd-hMASP-2 in the lung of mice cleavage C4.4. The survival of mice in BCG+rAd-hMASP-2 group was 80%, which was significantly higher than BCG+rAd group (50%). So hMASP-2 can decrease the mortality of mice infected with BCG.5. Compared with BCG+rAd group, the percentage of granuloma area in BCG+rAd-hMASP-2 group (30.2%±17.4%) was obviously increased (P=0.023), while the number of viable BCG bacilli (3.3×105±4.5×105 CFU/g) was obviously decreased (P=0.005). So hMASP-2 can promote the formation of granuloma and decrease the number of viable BCG bacilli in the lung of BCG infected mice.6. Compared with BCG+rAd group, in BCG+rAd-hMASP-2 group, the total lymphocytes (2.56×105±6.41×104), CD3T cells (1.94×104±1.93×103) and CD3+CD8+ T cells (2.64×103±1.08×103) of the lungs were all significantly increased (P<0.05). Both of CD3+(optical density value:0.063±0.0065) and CDllb+ cells (optical density value:0.0124±0.0032) were obviously increased (P<0.05). That’s to say hMASP-2 can promote infiltrating of lymphocyte.7.Compared with BCG+rAd group, in the blood of BCG+rAd-hMASP-2 group, the level of TNF-α (21.9pg/ml)significantly increased (P<0.05). Compared with BCG group, the lung level of TNF-α (521.2 pg/ml) obverously increased too (P<0.05). While the lung level of IFN-γ (158.1 pg/ml) was higher than PBS group (P<0.05).So hMASP-2 probably promote immune cells to secrete TNF-α and IFN-γ.8. Compared with BCG+rAd group, in BCG+rAd-hMASP-2 group, the relative expression of il6 mRNA (1.21), il12 mRNA (1.86) and ccl2 mRNA (2.07) increased, while il8 mRNA (0.57) decreased. So hMASP-2 can promote the mice to secrete IL-6, IL-12 and CCL2.Conclusion:1.The pulmonary tuberculous granuloma animal model has been established successfully by injection if 5×107 CFU BCG through trachea.2. rAd-hMASP-2 was constructed successfully, and we proved that the expression of hmasp-2 in the lung of mice infected with BCG and indirectly proofed that the activity of hMASP-2 and initiated the lection pathway.3. We proved that rAd-hMASP-2 can promote the formation of the granuloma in the lung of BCG infected mice, reduced the bacterium of lungs, and increased the survival rates of mice infected with BCG.4. Chemokine CCL2 promotes the proliferation and activation of CD1lb+macrophage, the secretion of IL-6 and IL-12 and stimulates the increment of T cells, which is one of the most important reasons for rAd-hMASP-2 could promote the formation of the granuloma, reduce the bacterium of lungs and increase the survival rate of BCG infected animals.5.There is another important reason for that rAd-hMASP-2 could promote the formation of the granuloma, reduce the bacterium of lungs and increase the survival rate of experimental animals.which was the proliferation and activation of CD3+T and CDS3+DD8+T cells stimulated the secretion of IFN-γ, TNF-α and IL-12. |
PDF Full Text Request