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Cultivation method for improving CIK cell tumor killing activity

A culture method and cell technology, applied to cell culture active agents, tissue culture, animal cells, etc., can solve the problems of complex steps, reliance on repeated column chromatography, and inapplicability to large-scale preparation

Inactive Publication Date: 2018-10-12
王欢
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no prior art that epiprocurcumenol, curcurenol, picrolactone A, picrolactone F, and vichakulide A can be deposited into the expression of granzyme B in CIK cells
[0006] In addition, the current methods for preparing epiprocurcumenol, curcumenol, picrolactone A, picrolactone F, and wechakucurin A are complicated in steps, rely on repeated column chromatography, and are not suitable for large-scale production.

Method used

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  • Cultivation method for improving CIK cell tumor killing activity
  • Cultivation method for improving CIK cell tumor killing activity
  • Cultivation method for improving CIK cell tumor killing activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Effects of Epiprocurcumenol, Curcumenol, Kukulide A, Kukulide F and Wichakucurin A on the Expression Level of Granzyme B in CIK Cells (Granzyme B Activator)

[0085] 1. Experimental materials

[0086] Lymphocyte separation medium was purchased from Jiahe Biotechnology.

[0087] Fetal bovine serum and GT-T551 medium were purchased from Gibco, USA.

[0088] CD3 mAb was purchased from Beijing Tongliyuan Biotechnology Co., Ltd., and IL-2 was purchased from Jiangsu Jinsili Pharmaceutical Co., Ltd.

[0089] 2. Experimental method

[0090] 1. Isolation and induction culture of CIK cells

[0091] Take 100mL of peripheral anticoagulant blood from healthy volunteers, add lymphocyte separation medium, centrifuge at 1500rpm for 15min, absorb the mononuclear cell layer, wash with normal saline three times, and centrifuge at 1500rpm for 10min each time; 5×10 6 cells / mL plus GT-T551 medium containing 500 μg / L CD3 mAb, 1000 U / mL IL-2 and 10% FBS to induce and culture in...

Embodiment 2

[0113] Embodiment 2: the culture method of CIK cell and tumor killing activity assay

[0114] 1. Experimental materials

[0115] Lymphocyte separation medium was purchased from Jiahe Biotechnology.

[0116] Fetal bovine serum and GT-T551 medium were purchased from Gibco, USA.

[0117] CD3 mAb was purchased from Beijing Tongliyuan Biotechnology Co., Ltd., and IL-2 was purchased from Jiangsu Jinsili Pharmaceutical Co., Ltd.

[0118] 2. Experimental method

[0119] 1. Culture of CIK cells

[0120] Take 100mL of peripheral anticoagulant blood from healthy volunteers, add lymphocyte separation medium, centrifuge at 1500rpm for 15min, absorb the mononuclear cell layer, wash with normal saline three times, and centrifuge at 1500rpm for 10min each time; 5×10 6 cells / mL plus GT-T551 medium containing 500 μg / L CD3 mAb, 1000 U / mL IL-2 and 10% FBS to induce and culture into CIK cells, half of the medium was changed every 2-3 days.

[0121] After 7 days of induction culture, adjust t...

Embodiment 3

[0135] Embodiment 3: Preparation of granzyme B activator epigenal curcumenol, curcumenol

[0136] 1. Instruments and materials

[0137] TBE-300A high-speed countercurrent chromatography was purchased from Shanghai Tongtian Biochemical Technology Co., Ltd., China.

[0138] ADS-17 macroporous adsorption resin was purchased from Anhui Sanxing Resin Technology Co., Ltd.

[0139] 2. Separation method and results

[0140] 1. Preparation of epiprotocurcumenol and crude curcumenol

[0141] After crushing the curcuma herb, pass it through a 40-mesh sieve, take 800g and use 4L ethanol solution with a concentration of 75% by volume to extract by microwave for 20min at 400W power, extract twice, combine the filtrate and concentrate it under reduced pressure until it has no alcohol smell, and put it on the sample On the ADS-17 macroporous adsorption resin column (75cm × 3.5cm, resin 450mL), first eluted with 45% ethanol of 5 times of column bed volume, and then eluted with 75% ethanol o...

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Abstract

The invention discloses a cultivation method for improving the CIK cell tumor killing activity. Epi-procurcumenol, curcumenol, physagulin A, physagulin F and withangulatin A can activate experssion ofgranzyme B in CIK cells and are adopted as effective granzyme B activating agents; the granzyme B activating agents of epi-procurcumenol, curcumenol, physagulin A, physagulin F and withangulatin A can remarkably improve killability of CIK cells for tumor cells. The invention further provides a method for preparing Epi-procurcumenol, curcumenol, physagulin A, physagulin F and withangulatin A. Themethod does not depend on repeated column chromatography, and is applicable to industrial large-scale preparation. In the prior, no technical scheme of the method is disclosed.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to the cultivation of CIK cells and the preparation method of inducing compounds. Background technique [0002] Malignant tumors seriously endanger human health. Currently, conventional surgery, radiotherapy and chemotherapy cannot completely eliminate tumor cells in the body. Adoptive immunotherapy has become an important adjuvant therapy for tumors due to its advantages of conforming to physiology, low toxicity and high efficiency. Among them, cytokine-induced killer cells (cytokine-induced killer cells, CIK cells) are a very promising adoptive immune cell discovered in recent years, with the advantages of rapid proliferation, broad-spectrum and high-efficiency tumoricidal activity, and minimal toxic and side effects. , has become a new option in the field of tumor immunotherapy. CIK is a heterogeneous cell population dominated by CD3+CD56+ T cells, which has the characteristic...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2501/2302C12N2501/515C12N2501/734
Inventor 王欢
Owner 王欢
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