| PART IObjective To investigate the role of mTOR 、Tc1/ Tc17 cells response and inflammatory cytokines secretion in pulmonary emphysema mice induced by Chronic cigarette smokeMethods 20 healthy male C57BL/6 mice were randomly divided into two groups according to the random number table: air-control mice,cigarette smoke mice which is under Chronic cigarette smoke exposure for 24 weeks.After24weeks,the mice were sacrificed by cervical dislocation and take out the lung and spleen.Cut 20 mg of the up left lung with the rest together to frozen in the refrigerator for detecting expression level of mRNA and inflammatory cytokines by RT-PCR and ELISA,respectively.The down left lung was to observe the pathological changes by HE stained.Furthermore all spleen and the right lung were to determined Tc1/Tc17 T cells and mTOR in CD8+T cells of each groupby flow cytometric.Observing the pathological changes of the lung by HE stained and evaluated the MLI(Mean linear intercept).T cells produced cytokines IFN-γ and IL-17 and this study investigated the percentage of CD8+IFN-γ+T(Tc1)cells and CD8+IL-17+T(Tc17)cells and explore the variation of them by flow cytometry.IFN-γ mRNA,RORγt mRNA and mTOR mRNA were detected by RT-PCR.The expression level of inflammatory cytokines IFN-γ,IL-17,TNF-α in the lung were measured by ELISA respectively.Results The pathological changes of lung in different group of mice were stained by H&E and quantitative analysis about the lung airspace was shown with MLI.We observed that alveolar structure in lung of air-control mice present well,there was no damages obviously.In contrast,it had significantly destruction of alveolar in cigarette smoke mice which performed pulmonary emphysema,as the alveolar wall became thinner and the alveolar spaces is enlarged,cilia lodging,inflammatory cells infiltration,alveolar fracture.It has significant difference between air-control mice and cigarette smoke mice in MLI values and quantitative analysis is P﹤0.05(P=0.000).Tc1/Tc17 cells based on the representative flow cytometric dot plots in spleen and lung of experimental mouse shown different.Study showed that Tc1 cells expressed higher in cigarette smoke mice than air-control mice in spleen and lung and the quantitative analysis between them has significance,P﹤0.05(P=0.000,P=0.000)as same as Tc17 cells P﹤0.05(P=0.000,P=0.001).Flow cytometric also showed that mTOR in CD8+T cells in cigarette smoke mice expressed more than air-control mice,P ﹤ 0.05(P=0.000)Furthermore,RT-PCR showed that the relative expression levels of IFN-γ mRNA、RORγt mRNA and mTOR mRNA in cigarette smoke mice upregulated and expressed more comparing withair-control mice,p﹤0.05(P=0.006,P=0.000,P=0.003).Inflammatory cytokines IFN-γ,IL-17 and TNF-α which were measured by ELISA.The levels of them in lung of cigarette smoke mice secreted much more than that in air-control mice P﹤0.05(P=0.000,P=0.000,P=0.000).Conclusion Tobacco smoke stimulates high expression of mTOR in lung tissue of mice,and is positively correlated with Tc1/Tc17 cells and IFN-γ,IL-17 and TNF-α.Also,mTOR plays an important role in chronic obstructive pulmonary inflammation and immune imbalance,and it may be an important treatment target in the future.PART IIBackground and objective Long-term chronic cigarette smoke exposure could induce serious small airway inflammation,emphysema and immune disorder.Rapamycin could play immunosuppressive role to inhibit CD8+T cells and modulate immune disorder in tumor and transplantation.But whether rapamycin could inhibit CD8+T cells and modulate inflammation,immune disorder in emphysema was unknown.This study is to investigate CD8+IFNγ+T(Tc1)/CD8+IL-17+T(Tc17)cells response and inflammatory cytokines secretion in pulmonary emphysema mice induced by Chronic cigarette smoke when treatment with rapamycin.Methods 40 healthy male C57BL/6 mice were randomly divided into four groups according to the random number table: air-control mice,cigarette smoke mice which is under Chronic cigarette smoke exposure for 24weeks;cigarette smoke + three-months-rapamycin mice which is treated by gavage with 1mg/kg rapamycin beginning at the 12 weeks,three times a week for 12 weeks until 24 weeks totally.And cigarette smoke + six-months-rapamycin mice which is treated by gavage with 1mg/kg rapamycin beginning at the first week,three times a week for 24 weeks totally.There were 10 mince in each group.After24 weeks,the mice were sacrificed by cervical dislocation and take out the lung and spleen.Cut 20 mg of the up left lung with the rest together to frozen in the refrigerator for detecting expression level of m RNA and inflammatory cytokines by RT-PCR and ELISA,respectively.The down left lung was to observe the pathological changes by HE stained.Furthermore all spleen and the right lung were to determined Tc1/Tc17 T cells and m TOR in CD8+T cells of each group by flow cytometric.Observing the pathological changes of the lung by HE stained and evaluated the MLI(Mean linear intercept).T cells produced cytokines IFN-γ and IL-17 and this study investigated the percentage of CD8+IFN-γ+T(Tc1)cells and CD8+IL-17+T(Tc17)cells and explore the variation of them by flow cytometry.IFN-γ m RNA,RORγt m RNA and m TOR m RNA were detected by RT-PCR.The expression level of inflammatory cytokines IFN-γ,IL-17,TNF-α in the lung were measured by ELISA respectively.Results The pathological changes of lung in different group of mice were stained by H&E and quantitative analysis about the lung airspace was shown with MLI.We observed that alveolar structure in lung of air-control mice present well,there was no damages obviously.In contrast,it had significantly destruction of alveolar in cigarette smoke mice which performed pulmonary emphysema,as the alveolar wall became thinner and the alveolar spaces is enlarged,cilia lodging,inflammatory cells infiltration,alveolar fracture.It has significant difference between air-control mice and cigarette smoke mice in MLI values and quantitative analysis is P ﹤ 0.05(P=0.000).Pathological damages in cigarette smoke + three-months-rapamycin mice and cigarette smoke +six-months-rapamycin mice became better,lung destruction mitigate,as alveolar fracture mitigate and the alveolar spaces became decreased when compared with the cigarette smoke mice,and quantitative analysis of MLI has significance,P ﹤ 0.05(P=0.000,p=0.000).At the same time,there was no significance between the above three and six-months-rapamycin mice,p ﹥0.05(p=0.175).Tc1/Tc17 cells based on the representative flow cytometric dot plots in spleen and lung of experimental mouse shown different.Study showed that Tc1 cells expressed higher in cigarette smoke mice than air-control mice in spleen and lung and the quantitative analysis between them has significance,P﹤0.05(P=0.000,P=0.000)as same as Tc17 cells P ﹤ 0.05(P=0.000,P=0.001).Oppositely,the percentage and number of Tc1 cells were decreased significantly when treatment with rapamycin three and six months in spleen and lung comparing with cigarette smoke mice and the quantitative analysis between them also has significance,P﹤0.05(P=0.000,P=0.002),(P=0.000,P=0.001),as same as Tc17 cells P ﹤ 0.05(P=0.000,P=0.013),(P=0.000,P=0.009)and furthermore Tc1/Tc17 cells in spleen and lung had no significance between the above two rapamycin mice,p﹥0.05(P=0.999,P=0.870)/(P=0.956,P=0.959).Flow cytometric also showed that m TOR in CD8+T cells in cigarette smoke mice expressed more than air-control mice and three and six-months-rapamycin mice,the quantitative analysis between them also has significance,P ﹤0.05(P=0.000,P=0.000,P=0.000)and there was no significance between the above two rapamycin mice,p﹥0.05(P=0.678).Furthermore,RT-PCR showed that the relative expression levels of IFN-γ m RNA 、 RORγt m RNA and m TOR m RNA in cigarette smoke mice upregulated and expressed more comparing with air-control mice,p﹤0.05(P=0.006,P=0.000,P=0.003)as same as three and six-months-rapamycin mice,p﹤0.05(P=0.025,P=0.009,P=0.030),(P=0.036,P=0.001,P=0.033).All m RNA expression had no significance between the two rapamycin mice,p﹥0.05(P=0.838,P=0.230,P=0.957).Inflammatory cytokines IFN-γ,IL-17 and TNF-α which were measured by ELISA.The levels of them in lung of cigarette smoke mice secreted much more than that in air-control mice P﹤0.05(P=0.000,P=0.000,P=0.000),but they expressed less after treatment with three and six-months-rapamycin comparing with cigarette smoke mice,P ﹤0.05(P=0.005,P=0.006,P=0.000),(P=0.001,P=0.003,P=0.036).While all the inflammatory cytokines has no significance between the three and six-months-rapamycin mice,p﹥0.05(P=0.549,P=0.809,P=0.157).Conclusion Rapamycin improves emphysema and acquired immune imbalance by inhibiting m TOR channels in emphysema mice which exposures to Chronic cigarette smoke,and it may be of great significance in the future treatment of COPD. |
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