Rat Model for Chronic Obstructive Pulmonary Disease (COPD)

1. Narcotize rats with ether at day 1 and day 14, expose its larynx, replace the tracheal tube with a venous cannula, inject 200μg/kg lipopolysaccharide (LPS) into rats trachea, then revolve rats vertically 10-20s to diffuse LPS at lung evenly.
2.At days 2-13 and days 15-28, fresh smoke was steadily provided to the closed glass box (70cm×60cm×60cm) by 15 cigarettes per day, to maintain smokescope about 100-120 mg/m3. After smoking, stimulate immediately with 5℃ cool air for 1 hour. End of modeling at day 28.

1.Observe activity, hair, appetite, breathe, weight of rats in each group. Weigh at 1 hour before feeding every week to observe the change of weight.
2.RT-qPCR Analysis
Collect rats PBMC and lung tissue, extract RNA, detect the level of Foxp3、RORγT by RT-PCR.
3.Western blot Analysis
Collect rats PBMC and lung tissue, extract protein, detect the level of TAZ by Western blot.
4. Flow cytometry for Peripheral blood T lymphocytes
Collect 2ml rat abdominal aortic blood in each group, extract peripheral blood mononuclear cells (PBMC) cell suspensions from rat peripheral blood lymphocytes.
(1)Detection of Th17: Add phorbol ester (50ng/ml), ionomycin (1μg/ml) and coban (2μmol/L) into the cell suspension. Incubate at 37℃, 5%CO2 for 6 hours, resuspend with 100μL PBS after washing 3 times. Add 10μL CD4 monoclonal antibody, mix it up, incubate at 4℃ without light for 30min. Wash, fix and break. Add 10μL IL-17 monoclonal antibody, incubate at 4℃ without light for 20min. Resuspend with 500μL PBS after washing repeatedly. Proportion of CD4+IL-17+ cells in CD4+T cells were detected by flow cytometry.
Detection of Treg cells: Add 10μl CD4 and CD25 monoclonal antibody into the cell suspension. After mixing, incubate at 4℃ without light for 30min. Wash, fix and break. Add 10μL FoxP3 monoclonal antibody, incubate at 4℃ without light for 20min. Resuspend with 500μL PBS after washed repeatedly. Proportion of CD4+CD25+FoxP3+ cells in CD4+T cells were detected by flow cytometry.

HE staining: The right lung lobe tissue was fixed on the embedded section, and the pathological changes of lung tissue were observed after staining.

ELISA Analysis
Detect the concentration of IL-17 and IL-10 in serum and bronchoalveolar lavage fluid (BALF).
BALF: After blood collection, open its chest, separate mediastina, inject 3ml saline from trachea to the lung. Every time lavage thrice, collect the douche. Centrifuged it at 4℃, 3000r/min for 15 min, take the supernatant.
Serum: Place whole blood samples collected in the serum separation tube at room temperature for 2 hours or 4℃ overnight. Centrifuge it at 1,000×g for 20 min, take the supernatant. Store the supernatant at -20℃ or -80℃, avoid repeated freezing and thawing.

SPSS software is used for statistical analysis, measurement data to mean ± standard deviation (x ±ｓ), using t test and single factor analysis of variance for group comparison , P<0.05 indicates there was a significant difference, P<0.01 indicates there are very significant differences.