哮喘大鼠模型,分离T淋巴细胞 z-bo 2020-10-14 424

　　Grouping and establishment of rat asthma model Grouping of experimental rats: According to the principle of randomization, 24 male Wistar rats (weight 190-250 g) were numbered according to their body weight, and a random list method was used. I use it and divide it into the following four groups.

　　(1) Normal control group

　　(2) Asthma 1 week group (excited 1 week)

　　(3) Asthma 2 weeks group (excited 2 weeks)

　　(4) Asthma group for 4 weeks (excited for 4 weeks or longer).

　　Establishing an asthma model: On the first day, each rat in the asthma group was subcutaneously injected 1 ml of OVA suspension (OVA 10 mg + aluminum hydroxide 200 mg + 0.9% 1% normal saline) into the inner thigh of each rat. At the same time, intraperitoneal injection was used to inactivate whooping cough, and 1ml of Bacillus suspension (about 6 * 109 strains) was used as an adjuvant. The sensitization was repeated on the 8th day and sensitization on the 15th day. Put it in a self-made glass container, use 402 ultrasonic nebulizer (Shanghai Silin Medical Equipment Factory) every other day, inject 50 ml of 2% OVA medium haze suspension 50 times a week for 50 minutes. Group stimulation for 1 week, 2 weeks and 4 weeks or longer. After stimulating the OVA suspension, the rats showed symptoms such as irritation, coughing, shortness of breath, slow movement or tendency, and histopathological sections of the lung showed infiltration of eosinophils and lymphocytes. The constriction of small airways and small blood vessels triggers asthma. success. The normal control group was injected intraperitoneally with an equivalent amount of 1ml PBS instead of OVA suspension, sprayed after 2 weeks, and inhaled with 50ml PBS solution in a mist for 30 minutes once a day for 15 days. .. T lymphocyte isolation and in vitro culture. Aseptically puncture the subgranular vein, collect 10ml of blood from each group of rats, divide it into centrifuge tubes, and dilute with the same amount of PBS. , Slowly add the same amount of lymphocytes. A layered interface is formed on the separation liquid.

　　Centrifuge at 2200pm for 20 minutes to see the layered interface. Aspirate a layer of milky white liquid in the middle of the centrifuge tube, add 0.85% ammonium chloride to destroy the red blood cells, leave it at room temperature for 2 minutes, and then stop the reaction with PBS. Centrifuge at 1800 pm for 10 minutes at room temperature to remove the supernatant.

　　Continue to wash twice with PBS diluent and centrifuge at 1600pm for 15 minutes.

　　The supernatant was removed, and the precipitate was adjusted to 1 ml with RPMI-1640 medium containing 10% fetal bovine serum, and mixed gently with a pipette. Place the cell suspension in a 37°C, 5% CO2 incubator for 1 hour;

　　Incubate the cell suspension in a sterile nylon cotton column in a 37°C, 5% CO2 incubator for 1 hour to isolate T cells. Test live cells with ≥90% tripan blue;

　　Use RPMI-1640 medium containing 10% FBS and 1 to adjust the T cell concentration to 2 * 106/ml on a 24-well culture plate. Inject 1 ml of 5% CO2 per well in a 37°C incubator. in.