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Application of quercitrin in preparation of human gamma delta T cell proliferator

A technology of cell proliferation, quercetin, applied in the field of medicine

Inactive Publication Date: 2014-06-18
NO 97 HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effects of quercetin on immune cells, especially on human γδT cells, have not been reported in the literature.

Method used

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  • Application of quercitrin in preparation of human gamma delta T cell proliferator
  • Application of quercitrin in preparation of human gamma delta T cell proliferator
  • Application of quercitrin in preparation of human gamma delta T cell proliferator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Effect of quercitrin on the proliferation of γδT cells

[0016] 1 Materials and methods

[0017] 1.1 Experimental materials

[0018] FITC-labeled anti-γδTCR, PE-labeled anti-perforin (anti-perforin), PE-labeled anti-granzyme B (anti-granzyme B), Fix&Perm membrane breaker were purchased from ebioscience; recombinant human interleukin-2 (rhIL-2), isopentenylpyrophosphate (IPP) were purchased from Xiamen Tebao Biological Engineering Co., Ltd.; RPMI-1640 medium, calf serum and trypsin were purchased from Gibco; lymphocyte separation fluid was purchased from Institute of Hematology, Chinese Academy of Sciences; CCK-8 kit, BCIP / NBT alkaline phosphatase chromogenic kit, and Western and IP cell lysate kits were purchased from Biyuntian Biotechnology Co., Ltd. Human AB serum was purchased from Xuzhou Blood Bank. Phosphoric acid solution was analytically pure, and water was redistilled water.

[0019] Quercitrin, from the Polygonum capitatum Buch.-Ham.ex D.Don in t...

Embodiment 2

[0031] Example 2: Effect of quercitrin on the expression of perforin and granzyme B on γδT cells

[0032] 1 Materials and methods

[0033] Among them, 1.1 Experimental materials; 1.2 γδT cell culture and identification; 1.3 Statistical processing are the same as in Example 1.

[0034] 1.4 Detection of expression of perforin and granzyme B on γδT cells by flow cytometry

[0035] The successfully cultured γδT cells were made into a cell suspension with a concentration of 1.0×105 / ml, inoculated in a 6-well culture plate, 3ml per well, and then added with final concentrations of 0μg / ml, 5μg / ml, 10μg / ml, and 20μg / ml, 40μg / ml, 80μg / ml quercitrin, each group had 3 replicate wells. At 37°C, 5% CO 2 Cultivate in the incubator for 48h. The cells were collected and washed twice with phosphate buffered saline (PBS), and 20 μl of anti-γδTCR-FITC was added to each tube, and incubated in the dark for 30 min. Add 100 μl of fixative and incubate at room temperature in the dark for 15 min...

Embodiment 3

[0038] Example 3: Effect of quercitrin on Bcl-2, p-ERK1 / 2 and p-Akt protein expression in γδT cells

[0039] 1 Materials and methods

[0040] Among them, 1.1 Experimental materials; 1.2 γδT cell culture and identification; 1.3 Statistical processing are the same as in Example 1.

[0041] 1.4 Western blot detection of Bcl-2, p-ERK1 / 2 and p-Akt protein expression

[0042] The successfully cultured γδT cells were prepared at a concentration of 1.0×10 5 / ml cell suspension, inoculated in 6-well culture plate, 3ml per well, then added quercetin with final concentrations of 0, 5, 10, 20, 40, 80 μg / ml respectively, at 37°C, 5% CO 2 Cultivate in the incubator for 48h. Cells were collected and washed once with PBS, fully lysed and extracted protein with 100 μL of Western and IP cell lysate, the protein concentration of the sample was measured by BCA method, separated by 10% SDS-PAGE, and then the protein was transferred to PVDF membrane, 5% delipidated The milk powder was blocked f...

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Abstract

The invention discloses an application of quercitrin in preparation of a human gamma delta T cell proliferator, and relates to the technical field of medicines. Quercitrin can remarkably promote proliferation of human gamma delta T cells and promote expression of perforin and granzyme B, so that the quercitrin is of certain dose dependency. The gamma delta T cell treated by the quercitrin can promote expression of p-ERK (Extracellular signal-Regulated Kinase)1 / 2 and p-Akt in active form and promote expression of an anti-apoptoasis protein Bcl-2, so that the gamma delta T cell is of certain dose dependency. The invention discovers the novel medical use of quercitrin and provides experimental references to development of tumor immunotherapy medicines.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to the application of quercitrin in the preparation of human γδT cell proliferation agent. Background technique [0002] In recent years, tumor biotherapy has become a research hotspot in the field of tumor treatment. Human γδT cells are mainly distributed in human mucosa and epithelial tissues, and have strong anti-tumor effects. They mainly recognize tumors in a non-restricted manner by major histocompatibility complex (MHC), and through granzyme B (granzyme B) and perforin (perforin) and other pathways kill tumor cells, and interact with other innate immune cells and adaptive immune cells to assist in the anti-tumor effect. Human γδT cells have become a new candidate effector cell for adoptive immunotherapy in cancer patients. It can be seen that it is very important and necessary to effectively expand human peripheral blood cells to meet the needs of γδT cell biological funct...

Claims

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Application Information

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IPC IPC(8): A61K31/7048A61P37/04A61P35/00
Inventor 刘军权杨阳陈永强郑璐周忠海陈复兴吕小婷
Owner NO 97 HOSPITAL OF PLA
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