Experiments on the effect of drugs on the activity of NK cells in animals
Experiments on the effect of drugs on the activity of NK cells in animals
The effect of drugs on the activity of NK cells in animals experiment, study the effect of drugs on the activity of NK cells in animals, explore explore the role of NK cells anti-tumor, anti-infection, immunomodulation.
Operation method
Subcutaneous inoculation with tumor cell suspension
Principle
Lung cancer is one of the most common malignant tumors that seriously threaten human health and life, and low immune function is closely related to the development of lung cancer.
Materials and Instruments
Mouse Move 1. Grouping and medication of animals Common Problems I. Discussion NK cells are an important class of immunomodulatory cells, which have anti-tumor, anti-infection, and immunomodulatory mechanisms: For more product details, please visit Aladdin Scientific website.
RPMI1640 culture medium Stabilizing solution Hank. s Trilatone X-100
ELISA 96-well 24-well flat-bottomed plates Centrifugal precipitator
(1) Forty mice were randomly divided into 10 normal control group and 30 hormonal group.
(2) The normal group was injected subcutaneously with saline 0.2 ml/pc in the right axilla, and the tumor group was injected subcutaneously with tumor cell suspension 0.2 ml/pc in the right axilla.
(3) The loaded tumor group was randomly divided into 3 groups and localized hair removal at the lung yu and inoculation (refer to Lin Wen ed. 5 Experimental Acupuncture and Moxibustion 6 for locating the acupoints), and the treatment was started on the following day.
(4) The model control group was plastered with drug-free hard paste.
(5) The localized paste group applied anticancer paste to the inoculated area.
(6) The acupoint application group applied anticancer paste on the lung Yu point and the inoculation localization, and each group replaced the paste every other day for a total of 14 days of treatment.
(7) The normal control group was not treated with drugs. The mice were executed on the 15th day, the eyeballs were removed to get blood, and the spleens were taken.
2. Detection method: Splenic NK cell activity assay.
(1) Preparation of splenic NK cells
①Aseptically take the mouse spleen, make a suspension of splenocytes, centrifuge and take the sedimentary splenic lymphocytes
② Transfer the cells into 100 ml cell culture flasks with 1640 complete culture medium containing 10% FCS.
③Cultivate at 37℃ and 5% CO2 for 4 h, adjust the cell concentration to 6×106/ml, and set aside.
(2) Target cells: YAC-1 cells in passaged culture were changed one day before the experiment and the concentration was adjusted to 3×105/ml.
(3) Measurement of NK cell killing activity
(1) Use a 96-well cell culture plate.
②Set up each sample
a: effector cell spontaneous release well (effector splenocytes 0.1 ml, complete culture medium 0.1 ml)
b: target cell spontaneous release wells (target cells 0.1 ml, culture medium 0.1 ml)
c: killing experiment wells (target cells 0.1 ml, effector splenocytes 0.1 ml)
d: Maximum killing experimental wells are the same as c. Set up 3 replicate wells for each well, centrifuge, discard 0.1 ml of supernatant and add 0.1 ml of 2% X-100.
(iii) Cultured at 37℃, 5% CO2 for 24 h, centrifuged (1000 rpm, 5 min).
④ Discard the supernatant, add 100 ul of NAG enzyme reactant solution to each well, and incubate at 37℃ for 40 min in a wet box.
⑤ Add 100 ul of suspension solution to each well, and measure the A value at 410 nm in enzyme-linked detector, which was expressed as a, b, c and d, respectively.
⑥ Calculate the NK cell killing activity according to the following formula: cytotoxicity index (CI) = (a + b) - c (a + b) - d × 100%.
3. IL-activity assay:
(1) Splenocyte preparation as above.
(2) Induction of IL-2
(1) Add 6×106/ml splenocyte suspension into 24-well culture plate and adjust the cell concentration to 10 ug/ml.
② The supernatant was collected by incubation at 37℃ and 5% CO2 for 48 h (1000 rpm, 10 min).
③ Sterile plastic-tipped tubes were the samples to be tested, and the assay activity was stored at -20℃.
(3) Activity Measurement
①The IL-2-dependent cell line CTLL-2 was used as the target cell, and the concentration was adjusted to 2. 5×105/ml with 1640 culture medium with 20% FCS, and added to 96-well culture plate with 100 ul per well.
②Another 100 ul of the sample to be tested was added, and three replicate wells were set up for each sample, and negative control wells were set up.
③Centrifuge (2000 rpm, 10 min) after 48 h of incubation, and discard the supernatant.
④ Add 100 ul of NAG enzyme reaction substrate, place the wet box at 37 ℃ and incubate for 40 min, then add 100 ul of suspending solution to each well, and measure the A value at 410 nm in the enzyme-linked detector.
⑤ Calculate the proliferation rate of CTLL-2 according to the following formula, indicating IL-2 activity. Proliferation rate (%) = sample A value - negative control well A value negative control well A value × 100%.
4. The t-test was used for statistical methods.
(1) through its surface receptors, recognize the target structure on the tumor cells to kill them directly, or through the release of soluble mediators to kill tumor cells.
(2) NK cells have FcCR receptors on their surface, which can bind to the Fc segment of the antibody covering the tumor cells and exert a cytotoxic effect on the tumor cells.
(3) NK cells can release cytokines such as interferon C (IFN-C), I-l1, and IL-2 to enhance anti-tumor effects.
NK cells are in the first line of defense of the body against tumors. It is important for inhibiting tumor growth, development, and metastasis.IL-2 also has an important role in tumor immunity, and its mechanism of action is:
(1) Stimulate the growth of NK cells and enhance the cytolytic activity of NK cells.
(2) Inducing the differentiation and effector function of CTL, LAK and other tumor killer cells.
(3) Inducing tumor killer cells to produce cytokines such as IFN-C, TNF-a, etc., enhancing anti-tumor effects.
NK cell surface has IL-2 receptor, under the stimulation of IL-2, cell proliferation reaction can occur, and produce IFN-a and other cytokines, IL-2, IFN-a, IFN-C can synergistically improve the activity of NK cells, with the most prominent role of IL-2. NK cells and lL-2 promote each other and play a tumor-killing effect.
This treatment is aimed at the evidence of lung cancer, with the principle of supporting the positive and dispelling the evil, benefiting qi and nourishing yin, clearing heat and removing toxins, softening hardness and dispersing blood stasis as the principle of treatment, selecting American ginseng to benefit the qi and nourish yin, Astragalus to tonify the qi and elevate the yang and benefit the lungs and strengthen the spleen, and antlers to tonify liver and kidneys and nourish the essence and blood.
The combination of all the medicines can achieve the effect of correcting and combating cancer. Anti-cancer cream is applied to Lung Yu acupoint because Lung Yu acupoint is the place where the meridian qi of lung is infused, and its position corresponds to the actual part of lung, so it is the key acupoint for the treatment of lung diseases, and from the point of view of the skin characteristics of the acupoint, the skin of the back is thinner than that of the four limbs, so it is easy for the drugs to penetrate into the subcutaneous tissues to exert the therapeutic effect, so the Lung Yu acupoint is the best point for the treatment of lung cancer by applying acupoints.
Research has proved that: astragalus in anti-cancer cream, its pharmacological component astragaloside can enhance cellular immune function, promote IL-2 production, and its aqueous decoction can promote NK cell activity, ginsenoside Rd contained in American ginseng, confirmed by in vivo animal experiments, can improve NK cell activity and interferon C production level, enhance immune function, enhance the rate of tumor suppression in mice, reduce tumor weight; antler gel contains antler essence and gelatin, which can increase heart, kidney and kidney function, and can increase heart, kidney and kidney function, and can increase heart and kidney function. Deer antler gum contains antler essence and colloid protein, which can increase heart and kidney function, and the combination of the three drugs can improve the immune function.
The three drugs together can improve the immune function of the body and reduce the weight of the tumor. Baihua snakeroot and shanzhi mushroom are heat-clearing and detoxicating drugs, which can inhibit or kill the tumor cells; leech, curcuma and other blood-activating and stasis-eliminating drugs can dilate blood vessels, improve the blood stasis of advanced lung cancer, inhibit thrombosis and reduce the metastasis of the tumor; seaweed, and Andrographis paniculata can elevate the leukocytes of the body, improve the immune function of the body, inhibit the growth of the tumor, and reduce the volume of tumor.
The mechanism of treating lung cancer by applying anti-cancer paste to lung acupoints in this therapy is: under the dual action of drugs and acupoints, it enhances the activity of NK cells and the ability of T cells to secrete IL-2, and raises the induced level of interferon, so as to improve the immune function of the organism. Tumor patients, especially those with progressive stage, have lower level of IL-2 secretion by T cells in peripheral blood than normal people, and the activity of NK cells is also lower than that of normal people. Therefore, applying anticancer ointment to lung acupoints can improve the immune function of the patients by increasing the activity of NK cells and the level of IL-2 induction, thus playing a therapeutic role in lung cancer.