CN116270736A - Gamma delta T cell preparation and application thereof - Google Patents

Abstract

The invention provides a gamma delta T cell preparation and application thereof, wherein the gamma delta T cell preparation comprises gamma delta T cell liquid, 100-800 mu mol/L ergoline maleate, 100-700 mu mol/L moringa seed element and/or 100-1000 mu mol/L bergenin. The research result shows that the combination of the ergoline maleate, the moringa seed, the bergenin and the gamma delta T cells is used for killing tumor cells, has good inhibition activity, and particularly has the inhibition rate of about 60 percent and 90 percent on lung cancer cells and thyroid cancer cells respectively. The invention has the advantages of clear and simple formula, convenient use and contribution to popularization and application.

Description

A kind of gamma delta T cell preparation and its application

technical field

The invention relates to the technical field of cell preparation preparation, in particular to a γδT cell preparation and application thereof.

Background technique

According to the surface antigen receptor (TCR), T cells can be divided into αβ T cells and γδ T cells. γδT cells recognize antigens through γδTCR complexes, and their cell subtypes, distribution locations, and host immunity are different from αβT cells. Studies have found that γδT cells are closely related to tumor immunity. γδT cells mainly exist in barrier tissues such as skin and mucous membranes, and only account for a small part of CD3 ⁺ T cells in peripheral circulation and tissues, and they can enter the activated state within a few minutes after being stimulated by antigens. Due to the rapid production of various cytokines after activation, γδT cells participate in the first line of defense against infection and tumor. In addition to the characteristics of innate immunity, γδT cells also have adaptive immune functions. Human γδT cells can recognize melanoma-associated antigenic peptides through the MHC pathway, but unlike αβT cells, not only antigens presented by MHC molecules can activate γδT cells through γδTCR, such as phosphoantigens (PAgs) expressed in tumors and multiple All kinds of cytokines can act on γδTCR to activate cells.

The inherent and adaptive immune properties of γδT cells make them the main immunotherapy cells, and γδT cells have higher safety and smaller off-target effects when used in tumor immunotherapy. Moreover, since the recognition of antigens is not restricted by MHC, γδT cells can be isolated and expanded from healthy people, and can also be combined with CAR-T therapy to generate sufficient cell subsets, which not only reduces by-products in the expansion process, but also reduces cost of treatment. γδT cells can not only kill autologous tumor cells, but also allogeneic tumor cells without toxicity to normal cells. It has been reported that γδT cells have killing activity against breast cancer, kidney cancer, pancreatic cancer, colon cancer, melanoma and other tumors.

Because γδT cells have strong direct cytotoxicity to tumor cells and can cooperate with other immune cells to exert their anti-tumor activity, they have become a new research hotspot in cellular immunotherapy. As γδT cell immunotherapy has come into people's attention and become a current research hotspot, related cell preparation products have also received attention. The current gamma delta T cell preparation products are mainly gamma delta T cell liquid, although it also plays a therapeutic role, but its killing activity on tumor cells is limited, and it cannot meet people's needs well.

Contents of the invention

In view of the deficiencies in the prior art, the purpose of the present invention is to provide a γδT cell preparation and its application.

Technical scheme of the present invention is as follows:

A γδT cell preparation, containing γδT cell fluid, at least one of 100-600 μmol/L ergonovine maleate, 100-700 μmol/L moringa seed, and 100-1000 μmol/L petracenin. It is recommended to mix it before use.

Further, the cell concentration in the γδT cell liquid is 2×10 ⁷ -10×10 ⁷ cells/ml. Adjust the cell concentration with RPMI-1640 medium.

Further, the γδT cell preparation contains γδT cell liquid, at least one of 600 μmol/L ergonovine maleate, 600 μmol/L moringa seed, and 600 μmol/L petracenin.

Further, the γδT cell preparation contains γδT cell liquid, 200 μmol/L ergonovine maleate, 200 μmol/L moringa seed and 200 μmol/L petracenin.

On the other hand, the present invention also provides the application of the γδT cell preparation in the preparation of antitumor drugs.

Further, the tumor includes lung cancer and thyroid cancer.

Compared with the prior art, the beneficial results of the present invention are:

The product of the invention comprises γδT cell liquid, ergometrine maleate, moringa seed and petogenin. The results of the study showed that the combination of ergometrine maleate, moringa seed, petracenin and γδT cells for killing tumor cells has good inhibitory activity, especially for lung cancer cells and thyroid cancer cells. 60% and 90% or so.

The formula of the product of the invention is clear and simple, convenient to use and beneficial to popularization and application.

Description of drawings

Figure 1: The killing rate of each group of cell preparations on tumor cells.

Detailed ways

In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

The γδT cells described in the following examples are derived from the peripheral blood of healthy humans and isolated according to conventional methods. Ergometrine Maleate (C ₂₃ H ₂₇ N ₃ O ₆ ), Moringa (CAS No. 73255-40-0), and Petgenin (CAS No. 477-90-7) are commercially available product.

Example 1

A γδT cell preparation, containing γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml), 600 μmol/L ergonovine maleate, 600 μmol/L moringa seed and 600 μmol/L petracenin.

Example 2

A γδT cell preparation, containing γδT cell fluid (2×10 ⁷ -10×10 ⁷ cells/ml), 600 μmol/L ergonovine maleate.

Example 3

A γδT cell preparation, containing γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml), 600 μmol/L moringa seed.

Example 4

A γδT cell preparation, which contains γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml) and 600 μmol/L petracenin.

Example 5

A γδT cell preparation, containing γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml), 200 μmol/L ergonovine maleate, 200 μmol/L moringa seed and 200 μmol/L petracenin.

Example 6

A γδT cell preparation, containing γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml), 100 μmol/L ergonovine maleate, and 100 μmol/L moringa seed.

Example 7

A γδT cell preparation, containing γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml), 700 μmol/L moringa seed, and 100 μmol/L petracenin.

Example 8

A γδT cell preparation, containing γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml), 600 μmol/L ergonovine maleate, and 100 μmol/L petracenin.

Example 9

A γδT cell preparation, which contains γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml), 500 μmol/L ergonovine maleate, 500 μmol/L moringa seed, and 1000 μmol/L petracenin.

Example 10

A γδT cell preparation, containing γδT cell liquid (2×10 ⁷ -10×10 ⁷ cells/ml), 500 μmol/L ergonovine maleate, 500 μmol/L moringa seed, and 500 μmol/L petracenin.

γδT cell preparation killing assay

Lung cancer cells A549 (1×10 ⁶ /mL) and thyroid cancer cells SW579 (1×10 ⁶ /mL) cultured to the logarithmic growth phase were respectively inoculated into 96-well cell culture plates as target cells, 100 μl/well. The γδT cell preparation was adjusted to 2×10 ⁷ /ml with RPMI-1640 medium, and added to the culture plate inoculated with target cells according to the following design, 100 μl/well, 6 replicate wells for each group, and the experiment was repeated three times.

Experimental group 1: 100 μl target cells + 100 μl γδT cell preparation (2×10 ⁷ /ml γδT cells)

Experimental group 2: 100 μl target cells + 100 μl γδT cell preparation (2×10 ⁷ /ml γδT cells + 600 μmol/L ergonovine maleate)

Experimental group 3: 100 μl target cells + 100 μl γδT cell preparation (2×10 ⁷ /ml γδT cells + 600 μmol/L moringa seed)

Experimental group 4: 100 μl target cells + 100 μl γδT cell preparation (2×10 ⁷ /ml γδT cells + 600 μmol/L petogenin)

Experimental group 5: 100 μl target cells + 100 μl γδT cell preparation (2×10 ⁷ /ml γδT cells + 200 μmol/L ergonovine maleate + 200 μmol/L moringa seed + 200 μmol/L petogenin)

Target cell group: 100 μl target cells

Negative control group: 100 μl γδT cell preparation (2×10 ⁷ /ml γδT cells)

The above groups of cells were cultured in an incubator at 37°C with 5% CO ₂ , and 20 μl/well MTT solution (5 mg/ml) was added after 24 hours, and the culture was stopped after 4 hours, the supernatant was carefully sucked off, and 150 μl/well dimethyl Sulfoxide, mix gently, and measure the OD value at a wavelength of 470nm with a microplate reader. Calculate the kill rate of each group. Analysis of variance was performed using SPSS software.

Killing rate=(OD value of target cell group+OD value of negative control group-OD value of experimental group)/OD value of target cell group×100%

Experimental results:

The experimental results are shown in the table below. The results showed that the killing rate of thyroid cancer cells in experimental group 2 to experimental group 5 was significantly higher than that in experimental group 1, indicating that ergometrine maleate, moringa seed, and petogenin had killing effects on thyroid cancer cells. Or ergonovine maleate, moringa seed, and petracenin can increase the killing activity of γδT cells and then improve the killing effect of γδT cells on tumor cells.

The killing rate of experimental group 3 to experimental group 5 on lung cancer cells was significantly or extremely significantly higher than that of experimental group 1, indicating that moringa and petracenin have a killing effect on thyroid cancer cells, or that moringa and petracenin can kill thyroid cancer cells. Improve the killing activity of γδT cells and then improve the killing effect of γδT cells on tumor cells.

In addition, it can be seen from the comparison between group 5 and other groups that when the combination of ergonovine maleate, moringa seed and petracenin is applied to the γδ T cell preparation, it shows more significant tumor killing activity.

ANOVA results for kill rate

post hoc multiple comparison results

The above descriptions are only some embodiments of the present invention, and do not limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (6)

1. A gamma delta T cell preparation, containing gamma delta T cell liquid, is characterized in that, also contains 100-600 μ mol/L ergonovine maleate, 100-700 μ mol/L moringa seed, 100-1000 μ mol/L petogenin at least one of . 2 . The γδ T cell preparation according to claim 1 , wherein the cell concentration in the γδ T cell liquid is 2×10 ⁷ -10×10 ⁷ cells/ml. 3. The γδT cell preparation according to claim 1, characterized in that it contains at least one of 600 μmol/L ergonovine maleate, 600 μmol/L moringa seed, and 600 μmol/L petracenin. 4. The γδT cell preparation according to claim 1, characterized in that it contains γδT cell liquid, 200 μmol/L ergonovine maleate, 200 μmol/L moringa seed and 200 μmol/L petracenin. 5. Use of the γδT cell preparation according to any one of claims 1 to 4 in the preparation of antitumor drugs. 6. The application according to claim 4, wherein the tumor comprises lung cancer and thyroid cancer.

Images