Experiments on the effect of drugs on the proliferation of lymphocytes in animals in vivo
Experiments on the effect of drugs on the proliferation of lymphocytes in animals in vivo
The experiment on the effect of drugs on the proliferation of lymphocytes in animals is to study the effect of drugs on the proliferation of tumor-infiltrating lymphocytes and anti-tumor sparks in mice in vivo, and to identify them by means of proliferation kinetics and pathology.
Operation method
Cell Culture Technology
Principle
TIL is the second generation of antitumor effector cells after LAK, and it is one of the hot spots in recent experimental research and clinical application. Currently, IL-2 is routinely added to TIL culture, but the proliferative and antitumor activities of TIL decreased with the extension of culture time.
Materials and Instruments
Hormonal Mouse Move 1. Isolation and culture of TIL Common Problems I. Discussion One of the main problems in treating human malignant tumors with tumor-specific T cells is how to obtain T lymphocytes with tumor-specific killing power and capable of expanding in sufficient numbers in vitro. Currently, most laboratories prepare TIL by using a three-step method of mechanical separation, enzymatic digestion, and discontinuous density gradient centrifugation of solid tumor tissues, which can obtain a larger number of TILs, but the proliferation rate and activity of the cells This method can obtain a higher quantity of TIL, but the proliferation rate and activity of the cells are lower. For more product details, please visit Aladdin Scientific website.
RPMI 1640 culture medium Calf serum
Scissors Tubes Centrifuge
(1) Take 20 mice with loaded tumors, excise the subcutaneous tumor mass under aseptic conditions after execution, place it in RPMI 1640 culture medium, remove the necrotic tumor tissue and surface connective tissue, and then cut it up and pass it through the cell sieve to collect single cell suspension.
(2) The equal volume of specific gravity 1.088 and 1.075 lymphocyte isolate and cell suspension were successively placed in the centrifuge tube for discontinuous density gradient centrifugation, and after centrifugation at 1800 r/min for 20 min, the interface of 1.075 was enriched with tumor cell suspension, and the interface between 1.088 and 1.075 was enriched with TIL suspension.
(3) TIL was collected, and the cell concentration was adjusted to 5×105/ml by dilution with culture medium containing 10% calf serum, IL-2100 u/ml RPMI1640 to culture in two groups.
(4) One group added cryotumor seedling every 5 days at a concentration of effector/tumor cells (E/T) = 50/1, and the other group was cultured routinely without cryotumor seedling.
(5) The culture conditions were 37°C, 5% CO2, counting every 5 days, and maintaining cell concentration of 5×105/ml.
2. Preparation of cryotumor seedling
(1) Take the tumor cell suspension and dilute it to 1×105/ml with RPMI 1640 culture medium in a cryopreservation tube.
(2) Freeze and thaw with liquid nitrogen for 3 freeze-thaw cycles, place in -20℃ refrigerator for storage and standby.
3. Preparation of a tumor-bearing mouse model
(1) Ascites of ascites-type Hepa breed mice were extracted, diluted with saline to 1×106/ml of tumor cells, and 0.2 ml was inoculated subcutaneously in the right axilla of the mice.
(2) After 1 week, the tumor grew to about 0.5 cm in diameter, and then a mouse tumor model was made for testing the anti-tumor effect of TIL.
(3) When the tumor grows to a diameter of 1~2 cm, it will be used to isolate TIL.
4. The anti-tumor effect of cultured TIL was divided into 3 groups of 20 mice each. In the control group, 0.2 ml of saline was injected into the tumor; in the experiment 1 group, 1×106 conventional cultured TIL was injected into the tumor; in the experiment 2 group, 1×106 cultured TIL was injected into the tumor with the addition of frozen tumor seedling. Injections were made once every 3 days for a total of 6 times.
5. Pathological examination of mice was executed, tumor size was measured, and the results were expressed as the average of the diameter in the vertical direction. Tumor tissues were sent for pathological examination and routinely stained with HE. The degree of lymphocyte infiltration was classified into 4 grades according to the criteria of Anneroth et al. Grade 0 was no infiltration; grade I was lymphocyte infiltration limited to the periphery of the tumor foci, with an area of no more than 25% of the tumor; grade II was lymphocyte infiltration toward the center of the tumor foci, with an area of 25%-50% of the tumor; and grade III was lymphocyte infiltration with an area of more than 50% of the tumor.
6. Statistical methods used were t-tests and X2 tests.
In this study, we used the direct isolation method, which eliminated the step of enzyme digestion, and the operation was simple, saving enzyme and time. Although the number of isolated TIL was lower, the proliferation speed and activity were high, and there was no difference in the anti-tumor activity of cells obtained in the later stage of culture between the two methods. Therefore, in tumor specimens
Freshly isolated TIL showed no or only a slight proliferative response to mitogen, with minimal or absent killing of autologous, allogeneic and other tumor cell lines, and the addition of IL-2 to in vitro culture altered the inhibitory state of TIL.
In this study, it was observed that the proliferation of TIL in conventional culture only started after a resting period of 4~5 d, which was related to the gradual improvement of the low activity state of TIL.The expression of IL-2 receptor by T cells required constant stimulation by antigen to maintain, and after removal of the stimulus, the expression of IL-2 receptor was gradually reduced, and the responsiveness and proliferative capacity of the T cells to IL-2 were reduced as a result.
The residual tumor cells in TIL culture have the effect of continued sensitization and stimulation of proliferation of T lymphocytes in vitro, which converts TIL into specific T killer cells.The gradual death and disappearance of tumor cells at the late stage of IL-2 culture of TIL deprives the proliferation of TIL of its probable stimulant, and decreases its proliferative power. Therefore, the weak proliferative force and short survival time of cells in the conventional TIL culture group in this study are related to this.
Freezing not only kills tumor cells, but also makes the freeze-damaged tumor tissues become antigenic stimulators and stimulates anti-tumor immunity, and the amount of antigen released from tumor cells inactivated by freezing is higher than that released from cancer cells inactivated by radiation and laser irradiation. In this experiment, it was observed that TIL began to proliferate after 3~4 d stationary period after adding frozen tumor seedling, which took shorter time than conventional culture TIL, and could be cultured for a long time, with a large amount of expansion, and the survival time was significantly longer than that of conventional culture TIL, which was consistent with the above theory.
In this study, the gross pathology shows that the two groups of TIL treated tumors grow slowly, and the diameter is smaller than that of the saline treated group, and the difference is significant, which indicates that TIL has an anti-tumor effect, and to a certain extent, it can slow down the growth of tumors. There was shrinkage and regression of the tumor after TIL treatment by cryotumor seedling stimulation, while there was none in the conventional culture TIL treatment group, suggesting that the antitumor effect of the former was stronger than that of the latter, and that there was a qualitative difference.
Under the microscope, it was observed that: the tumor of frozen tumor seedling stimulated culture TIL treatment group had complete necrosis phenomenon, and those with a large number of lymphocyte infiltration were tumor shrinkage or stable tumor-bearing mice; while the conventional culture TIL treatment group did not have the phenomenon of complete tumor necrosis, and only had a small amount of lymphocyte infiltration to a medium amount of lymphocyte infiltration, which indicated that the antitumor effect of the former group was stronger than that of the latter group, and the antitumor effect was related to the lymphocyte infiltration degree, and those with a large number of lymphocytes had strong antitumor effect. The anti-tumor effect is related to the degree of lymphocyte infiltration, and the one with more lymphocytes has a strong anti-tumor effect.
In contrast, the tumors in the control group were focally necrotic, with no or only a small number of lymphocyte infiltration and congestion and hemorrhage of small blood vessels, suggesting a tendency to deterioration. In addition, the proliferation of fibroblasts in the tumor periphery also indicated that the tumor spread was limited after treatment with TIL.
One of the characteristics of biological therapy is that the time of effect is slow, usually need to be observed for a longer period of time to see obvious therapeutic effect, therefore, in this experiment, after the frozen tumor seedling stimulation of TIL treatment, the tumor of the mice gradually receded or remained stable, and the changes in the general outlook were not significant, but under the microscope, it could be seen that all the tumors were necrotic or only a small number of them remained, and it takes a process for the necrotic tissues to be absorbed by the body, so that if we further prolonged the observation time, more tumors would be eliminated. If the observation time is further prolonged, more tumors will subside or even disappear completely and be cured.
The results of this study showed that the frozen tumor seedling can maintain the proliferation ability of cultured TIL and improve the anti-tumor effect of TIL in mice, which provides an important experimental basis for the clinical application of TIL in the treatment of tumors.