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Biologically active peptide and method for proliferating CIK cell in vitro

A bioactive peptide and in vitro amplification technology, applied in the field of bioactive peptides and in vitro expansion of CIK cells, to achieve the effects of low cost, high purity, and high survival rate

Active Publication Date: 2017-05-17
杭州鑫生源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, no research has proved that cicada monkey bioactive peptides can improve the proliferation efficiency and cytotoxic activity of CIK cells in vitro

Method used

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  • Biologically active peptide and method for proliferating CIK cell in vitro
  • Biologically active peptide and method for proliferating CIK cell in vitro
  • Biologically active peptide and method for proliferating CIK cell in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 bioactive peptide

[0030] 1. Experimental materials

[0031] 1. Instrument reagents

[0032] The ultrafiltration cup and ultrafiltration membrane were purchased from Shanghai Mosu Scientific Equipment Co., Ltd.;

[0033] Trypsin (1:250U / mg) was purchased from Guangzhou Qiyun Biotechnology Co., Ltd.

[0034] 2. Experimental samples

[0035] Fresh cicada monkeys just unearthed from trees were collected, stored in water, and frozen at -20°C. Thaw naturally at room temperature before use.

[0036] 2. Experimental methods and results

[0037] 1. Enzymatic hydrolysis method

[0038] Wash the cicada monkeys and twist them into minced meat. Take 10g of the minced meat and put it in 50mL of ultra-pure water. Add trypsin for enzymolysis. The amount of trypsin added is 0.25% of the weight of the substrate minced meat. The enzymatic hydrolysis conditions are: pH value , 8.0; enzymatic hydrolysis temperature, 45°C; enzymatic hydrolysis time, 1...

Embodiment 2

[0044] Example 2 In vitro culture, expansion, survival rate determination and immunophenotype detection of CIK cells

[0045] 1. Experimental method

[0046] 1. In vitro culture, expansion and survival rate determination of CIK cells

[0047] A total of 150 mL of peripheral venous blood was extracted from 30 healthy people, and peripheral blood mononuclear cells were extracted with lymphocyte separation medium. After washing with PBS, the cell suspension was made with KBM551 serum-free medium, and the cell concentration was adjusted to 1×10 5 / mL, were equally divided into 6 parts, and were cultured with the bioactive peptide induction culture method of the present invention and the traditional induction culture method respectively, and each method operated 3 parts in parallel.

[0048] Bioactive peptide induction culture method: Before culturing for 24 hours, put 250 mL of ice-cold PBS containing 20 μg / mL bioactive peptide and 5 μg / mL anti-human CD3 monoclonal antibody in a ...

Embodiment 3

[0062] Example 3 Determination of in vitro tumor killing rate of CIK cells

[0063] 1. Experimental method

[0064] 1. Tumor cell culture

[0065] Human lung cancer cell line A549 was incubated with RPMI-1640 medium containing 10% calf serum at 37°C and 5% CO 2 Under normal conditions, cultured and subcultured. Digest with 0.25% trypsin for 3 minutes during subculture, pipette with culture medium to make cell suspension, and inoculate according to the required concentration. When mixed with CIK cells, as target cells.

[0066] 2. Determination of tumor killing rate of CIK cells in vitro by MTT method (%)

[0067] Human lung cancer cell line A549 in the logarithmic growth phase was selected, and the cell concentration was adjusted to 8×10 4 / mL, add 100 μL to each well of a 96-well plate, 37°C, 5% CO 2 Incubate for 4 h in the incubator. The CIK cells on the 15th day of culture were taken as effector cells, and CIK cells were added according to the effect-to-target ratio ...

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Abstract

The invention discloses a biologically active peptide and a method for proliferating a CIK cell in vitro. The method comprises the following steps: drawing peripheral venous blood, separating a single karyocyte, culturing the single karyocyte in a culture flask pre-coated with the biologically active peptide and an anti-CD3 monoclonal antibody by virtue of a serum-free culture medium, only adding IL-2 and the serum-free culture medium in a culture system afterwards, and performing culturing for 14 to 21 days under the conditions of 37 DEG C and 5% CO2, wherein the biologically active peptide is obtained by performing enzymatic hydrolysis on a raw material cicada with trypsin, entrapping a component of a molecular weight of 3,000 to 1,000u by virtue of an ultrafiltration membrane and performing freeze-drying. The biologically active peptide can be used for efficiently inducing in-vitro proliferation of the CIK cell, high survival rate and high purity are achieved, and the CIK cell obtained by the method for proliferating the CIK cell in vitro has good in-vitro tumor cytotoxicity effects and in-vivo tumor suppression effects; in addition, the biologically active peptide which is low in cost and readily available is used according to the method instead of recombinant human IFN-gamma and IL-1alpha in a conventional culturing method, so that the cost is low.

Description

technical field [0001] The invention belongs to the field of medicine and relates to the preparation and application of polypeptide medicines, in particular to a biologically active peptide and a method for expanding CIK cells in vitro. Background technique [0002] Lung cancer is the most common malignant tumor in the world, and its mortality rate ranks first among malignant tumors. The incidence of lung cancer in my country has been increasing year by year, with an average annual increase of nearly 2%. Lung cancer is divided into multiple tissue types according to different pathological characteristics, and the treatment measures are also different for different types of lung cancer tissue. According to the 2004 edition of the WHO classification, common histopathological types of lung cancer are divided into non-small cell carcinoma (NSCLC) and small cell carcinoma (SCLC). Non-small cell carcinomas are subdivided into squamous cell carcinoma (SCC), adenocarcinoma (AC) an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12P21/06C07K1/34A61K35/17A61P35/00
CPCA61K35/17C07K1/34C12N5/0037C12N5/0646C12N2500/90C12N2501/2302C12N2501/515C12N2501/998C12P21/06
Inventor 郭守河其他发明人请求不公开姓名
Owner 杭州鑫生源生物科技有限公司
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