| Mycoplasma pneumoniae could induce acute respiratory tract infection with pneumonia and severe extrapulmonary complications(eg.digestive,nervous,and cardiovascular diseases,etc.).The study explores the immune protection effect of Mp live attenuated vaccine and the anti-Mp infection effect of afatinib,which provides scientific basis for the prevention and treatment of Mp infection.Part One Evaluation of immune effect of live attenuated Mycoplasma pneumoniae vaccineObjective:To construct a live attenuated Mycoplasma pneumoniae vaccine and evaluate its immune protection.Methods:1.The standard strain of Mycoplasma pneumoniae M129 was passaged in vitro for approximately 80 generations to obtain a mutant strain,which was named Mut129 after mutation detection;2.PBS,M129 and Mut129 intranasally challenged BALB/c mice by measuring the number of viable bacteria in lung homogenate,antigen content and lung histopathological sections were used to identify the virulence of Mut129;3.Mut129 was used as a live attenuated vaccine to immunize mice intranasally,and the serum levels of Ig G and Ig G1,Ig G2a subgroups were detected by indirect ELISA;4.Using Mycoplasma pneumoniae standard Strain M129 challenged mice on 42 days after immunization,the study evaluate the immune protection of live attenuated vaccine by serum antibody Ig G and Ig A level,viable bacteria count,lung tissue HE pathological section and cytokine content(IFN-γ,IL-4,IL-17A,TNF-α,IL-1β,IL-6,IL-13).Results:The standard strain of Mycoplasma pneumoniae M129 was passaged in vitro for about 80 generations;SNP/In Del mutation detection revealed that P1 adhesion protein,P40/90,terminal organelle protein(top J,hmw1,hmw2,hmw3)and lipoprotein genes have mutations,and cell adhesion and pathogenic functions of mutant strain have changed than M129,thus named it Mut129.After Mut129 challenged mice,the number of live Mp bacteria in the lung homogenate was not detected,the antigen content was not significantly different from that of the normal group of mice,and the lung tissue had no pathological changes.The levels of specific Ig G antibodies the serum of the immunized and combined immunization group increased significantly after immunizing,the titer of Ig G antibody in the combined immunization group could reach 1:6400,the subclass Ig G1 was significantly increased,and the Ig G2a was not significantly increased.After 7 days of challenging mice,the lung homogenate of immunized mice did not grow"fried egg-like"colonies on the PPLO plate,and the liquid medium could not change from red to yellow.The Real-time fluorescence quantitative PCR results showed that the Mp in the lung tissues of immunized mice was 546.04±96.71CFU/m L,it was significantly decreased than poison attack group(1.08×10~5±0.21×10~5CFU/m L).The levels of anti-Mp-specific Ig A,Ig G were significantly increased in immunized group(P<0.05).In the immunized group,the lung interstitial inflammation and inflammatory cell infiltration were significantly reduced than the poison attack group,but there was still inflammatory infiltration compared with the normal group.The expression of IL-4,IL-1β,IL-6 and IL-13 in the supernatant of lung homogenate of the immunized mice was not significantly changed,but the levels of IFN-γand IL-17A were significantly reduced,and the level of TNF-αincreased in comparison with infection group(P<0.05).Conclusion:The mutant strain of M129 was obtained by passage in vitro for about 80 generations and named Mut129.Mut129does not cause pathological changes in lung tissue after challenge,and can be used as a live attenuated vaccine.Live attenuated vaccine has a strong immune protection against M129 challenge,it can induce specific humoral immune response and inhibit the proliferation of M129 in lung tissues,but cannot reduce the inflammation infiltration of lung tissues.Part Two The anti-infective activity of afatinibObjective: To explore the antibacterial and anti-inflammatory effect of Afatinib against Mycoplasma pneumoniae infection.Methods: The color change unit(CCU)and the minimum inhibitory concentration were used to evaluate the effect of afatinib in inhibiting the proliferation of Mp in vitro;Then,we constructed the model of Mycoplasma pneumoniae infected BALB/c mice,and collected blood and lung samples after afatinib treatment.In this study,Inflammation index(NLR was neutrophils divided by lymphocytes,MLR was monocytes divided by lymphocytes Number)were calculated by blood cell count(white blood cells,neutrophil,lymphocyte,monocyte);the effect of afatinib on Mp proliferation in vivo was evaluated by the living Mp number in lung tissue;HE staining was used to estimate the anti-inflammatory effect of afatinib on Mp infected mice.In addition,We detected the level of intracellular reactive oxygen species(ROS)and cytokines(TNF-α,IL-1β and IL-6)in lung homogenate supernatant to analyze its anti-inflammatory mechanism.Results: The study showed that the color change units of the Mp group were 104 CCU/m L and 107 CCU/m L at 7 and 14 days in vitro,while the afatinib group had no color change after 14 days,and the minimum inhibitory concentration of afatinib was 16 μg/m L.The viable count in lung tissue of afatinib group was significantly lower than Mp group(P<0.05),and the WBC,NEUT,inflammation index(NLR,MLR)were also significantly lower than Mp group(P<0.05),besides,the inflammation of lung slices was significantly relieved in comparision with Mp group.In addition,the levels of ROS and TNF-ɑ in lung tissue of afatinib group were significantly lower than Mp group(P<0.05).Conclusion: The study confirmed that afatinib can inhibit Mp proliferation in vivo and in vitro,and reduce inflammation in mice after Mp infection.It may provides a potential research basis for developing new treatment strategies of Mp infection in clinical practice. |
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