Animals
Twenty SD rats, all male, weighing 250–360 g, 8 weeks of age, were purchased from Shanghai Slark Laboratory Animal Co., Ltd. All relevant animal experiments were approved by the Animal Ethics Committee of Naval Military Medical University. Animal experiments were performed in accordance with NIH (1996) Guide for the Care and Use of Laboratory Animals.
Models
Twenty SD rats were randomly distributed into two groups, namely the blank control group and the COPD group. Model rats were intranasally administered 200 ul endotoxin (1 mg / ml LPS) on the first day and fourteenth day, and cigarette smoke was inhaled on days 2–13 and 15–30. The rats in the COPD group were placed in a self-made smoke chamber. The volume of the smoke chamber is 110 cm × 86 cm × 72 cm. Using the static inhalation method, 20 cigarettes were placed in the smoking hole, and the cigarettes were ignited. Each cigarette was inhaled once a minute for 30 seconds each time. The collected main stream and side stream fumes were introduced into the smoke box through a plastic hose. Every 50 seconds, the flow fan in the box was turned on for 10 seconds to make the gas distribution in the box uniform. Turn off the smoking system after 8 minutes. The rats were exposed to the smoke for 1 hour each time, and were removed after 1 hour. The animals can eat and drink freely in the box when they were exposed to cigarette smoke. The blank control group was placed in the smoke chamber 3 times a day using the same method. Except for inhaling fresh air, other conditions were same as those in the COPD group.
Isolation And Extraction Of Alveolar Macrophages
Rats were anesthetized by intraperitoneal injection of 300 mg / kg of 10% chloral hydrate and sacrificed. Alveolar macrophages were collected by bronchoalveolar lavage. Macrophages were washed twice with RPMI-1640 medium. Trypan blue staining was used to detect cell viability and cell number, and the cells were formulated to the required concentration. The viability of macrophages can reach more than 90%, and macrophages accounted for more than 90% of the total.
Bronchial Epithelial Cell Culture
The human bronchial epithelial cell line 16HBE was purchased from ATCC (Manassas, VA, USA). 16HBE cells were cultured in RPMI-164 medium containing 100 U / mL penicillin, 100 µg / mL streptomycin and 10% (v / v) inactivated fetal calf serum, and placed in a cell incubator for routine subculture. Set the incubator temperature to 37 ° C and contain 5% CO2. Cells were exchanged every other day, depending on the growth, and passaged once every 2–3 days. In this experiment, cells in logarithmic growth phase were used.
Co Culture Of Alveolar Macrophages And 16HBE
16HBE cells were plated in the upper chamber of a 24-well transwell chamber at 5.0 × 105 and cultured in RPMI-164 containing 10% FBS. After 24 hours, the serum-free medium was changed, and co-cultured with the above-mentioned alveolar macrophages for 8 hours, 12 hours, or 24 hours. 16 HBE cells in the upper chamber were collected for subsequent experiments.
Isolation And Identification Of Exosomes Derived From Alveolar Macrophages
Exosomes were extracted from alveolar macrophages via SBI kit (Sigma,USA) according to manufacturer's instructions. The exosomes were refrigerated at − 80℃ for long-term storage. The morphology of exosomes was observed with an electron microscope. The size and concentration of exosomes was performed upon resuspension using nanotracking analysis on a ZetaView Particle Metrix (PMX, Germany). Western blot was used to detect the expression of exosomal marker protein.
Cell Transfection
The cells were inoculated in a 10 mm dish, cultured at 37 ℃ and 5% CO2 for 18–24 hours to reach 60% fusion degree. According to the transfection steps of miR-380 mimic, CFTR-siRNA and Lipofectamine 2000 TM reagent instructions. Liposomes, antibiotic-free OPTI-MEM medium and plasmids (24 µg), or negative controls was mixed and added to the cell culture medium. The medium containing the liposome complex was replaced after 4–6 hours of incubation. The cells were collected for subsequent experiments after 48 hours of transfection.
Quantitative Real-time PCR
Total RNA was obtained from exosomes stored at -80 °C with Trizol reagent according to the manufacturer’s protocol (Takara, China). Reverse transcription: The first strand of cDNA was synthesized by using Takara's m-mlv reverse transcription kit. The first strand of cDNA was synthesized by reverse transcription using total RNA as template and random primers as reverse transcription primers. GAPDH was used as the internal reference gene. The total RNA of all samples was transcribed in the same batch, and the operation was carried out according to the instructions provided by the manufacturer.
Reaction conditions: 37 ° C, 15 min; 85 ° C, 5 sec; 4 ° C, 10 min. The reaction system is as follows:
Components | Volume (µl) |
5 × PrimeScript RT Master Mix | 4 µl |
Total RNA | 1 µL(1 µg/µl) |
RNase Free ddH2O | Up to 20 µl |
Real-time PCR was performed on a Stepone Plus system (Applied Biosystems, Foster city, CA). The reagent used for quantification was 2xSYBR Green real-time PCR master mix. Reaction conditions: 1.95 ℃, 10 min; 2. 95 ℃, 15 s; 3. 56 ℃, 55 s; 4. 72 ℃, 20 s. 2–4 total 35 cycles. The reaction system is as follows:
Components | Volume (µl) |
2 × SYBR Green real-time PCR Master Mix | 10 µl |
PCR Forward Primer (10 µM) | 0.8 µl |
PCR Reverse Primer (10 µM) | 0.8 µl |
DNA template (<100 ng) | 2 µl |
Sterile water | Up to 20 µl |
CCK8 Proliferation Experiment
Cell proliferation was analyzed by cell counting kit 8 (CCK8, Beyotime, Japan).16HBE cells were implanted into 96-well plates (the number of cells in each well was about 1 × 104), and cultured in CO2 incubators. Before each detection, the cells were added into 10 µL of CCK-8 reagent and cultured for 1.5 h. The cell activity was measured at 0 h, 24 h, 48 h, and 72 h. Finally, the optical density (OD) values were measured by microplate reader at the 450 nm wavelength.
Transwell Invasion Experiment
Cell culture: Collected the transfected cells, inoculated group cells and 300 µl of serum-free RPMI-164 suspension in the upper chamber (including Matrigel) of the transwell chamber.
Placed 500 µl of RPMI-164 medium containing 10% fetal bovine serum in the lower chamber. After the treatment was finished, the 24-well plate was placed in a cell incubator for 48 hours.
Fixation and staining: After 48 hours of culture, the 24 hole plate (transwell) was taken out and washed gently with PBS for 3 times. 4% paraformaldehyde was fixed at room temperature for 15 minutes. The fixative solution was removed by the rinse with PBS. Crystal violet staining solution was added to stain for 15 minutes and the stained cells of the upper chamber was removed with cotton swabs.
Counting statistics: Five high power visual fields were randomly selected for observation and statistical analysis of photographic counting under the microscope.
Clone Formation Test
The cells were inoculated at a density of 100 cells / 4L in a 6-well plate and were incubated for 10 days at 37 ° C in a 5% CO2 incubator. The supernatant was discarded after 3 weeks. The cells was washed twice with PBS and fixed with methanol for 15 min. The fixing solution was discarded and cells were dyed for 10–30 min with Giemsa staining solution. Wash the staining solution slowly with running water and dry it. The number of clones greater than 50 cells was counted under a microscope, and the colony formation rate was finally calculated. Colony formation rate = number of clones / number of seeded cells × 100%.
Western Blot
We assessed the expression of mucins and CFTR in 16HBE by western blot. Total protein in cells was extracted and measured by BCA analysis. Samples ran on SDS-PAGE gel. Gel was then transferred to nitrocellulose membrane using Tris-Glycine transfer buffer. Nitrocellulose membranes were then blocked with 5% milk/TBST solution for 1 hour at room temperature and incubated with primary antibody overnight at 4C. Membranes were washed 3x with TBST ant then incubated 1:5000 dilution with HRP conjugated secondary antibody. Membranes washed 3X as above, and then developed using ECL Chemiluminescent Substrate kit (Biological Engineering Co., Ltd. Shanghai, China). Antibodies used for detection are as follows: MUC5AC (Abcam, ab77576, 1: 5000), MUC5B (1: 5000; ab105460; Abcam), MUC2 (1: 10000; ab133555; Abcam), CFTR (1: 500, ab2784; Abcam), GAPDH (1: 2500, ab9485; Abcam). Band detection was performed using QUANTITY ONE software.
ELISA
Cell-free supernatants were collected and stored at -80 °C. After thawing, the expression of IL-6 and TNF-αwere analyzed with IL-6 and TNF-αELISA kit (Abcam) according to the manufacturer’s instructions.
Statistical Analysis
Software SPSS 19.0 (SPSS, Chicago, IL, USA) were used for statistical analysis, and all measurement data were expressed as the mean ± standard deviation (mean ± SD). Comparisons between two groups were analyzed by unpaired student’s t-test analysis. Comparisons between multiple groups were analyzed by one-way analysis of variance. P values < 0.05 were considered significantly different. Statistical graphs were completed by using Graphpad Prism 5 (GraphPad Software Inc, San Diego, CA, USA).