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Preparation method and application of modified enhanced type targeting immune cell mass

A cell and interleukin technology, applied in the fields of cell biology, immunology, biology, and tumor treatment, can solve problems such as side effects and unsatisfactory treatment effects

Inactive Publication Date: 2017-06-13
深圳红石科创生物科技发展有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the treatment effect is mostly unsatisfactory, and often accompanied by serious side effects

Method used

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  • Preparation method and application of modified enhanced type targeting immune cell mass
  • Preparation method and application of modified enhanced type targeting immune cell mass
  • Preparation method and application of modified enhanced type targeting immune cell mass

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1. Preparation of lentivirus expressing human PD-1 fragment

[0059] 1. Construction of human PD-1 fragment lentivirus

[0060] Synthesize the double-stranded DNA molecule shown in SEQ ID NO: 4 of the sequence listing, link it into a commercial lentiviral vector, take the pWPXL vector system as an example, including but not limited to the pWPXL vector, and construct the recombinant plasmid pWPXL-PD -1- IRES-IL-2, identified by PCR, the results are as follows figure 1 As shown, lane 1 is the DL2000 Marker, lane 2 is the identification result of PD-1, and lane 3 is the identification result of IL-2.

[0061] Virus packaging is accomplished by the following routine:

[0062] a. The recombinant expression plasmid and the helper plasmid were co-transfected into HEK293 cells by the calcium phosphate method. Inoculate HEK293 cells in DMEM medium containing 10% fetal bovine serum, culture at 37ºC and 5% CO2 until logarithmic growth phase, collect cells, inoculat...

Embodiment 2

[0070] Example 2, Detection of PD-1 after recombinant virus infected CIK cells

[0071] In order to fully illustrate the beneficial effects of the present invention, the present invention also performs real-time PCR detection of PD-1 and IL-2 transcription levels in CIK cells infected with pWPXLd-PD-1-IRES-IL-2, the steps are as follows :

[0072] Total RNA was extracted from cells infected with pWPXLd–PD-1-IRES-IL-2 for 4h, 8h, and 12h respectively. At the same time, CIK cells infected with pWPXLd were used as controls, and uninfected CIK cells were used as blank controls. Then it was reverse-transcribed into a cDNA template, and the designed specific primers were used to detect whether the mRNAs of PD-1 and IL-2 were transcribed, and GAPDH was used as an internal reference. The primer sequences for GAPDH are:

[0073] Upstream primer: 5'-ACCACAGTCCATGCCATCAC-3'

[0074] Downstream primer: 5'-TCCACCACCCCTGTTGCTGTA-3';

[0075] The primer sequence of PD-1 is:

[0076] Ups...

Embodiment 3

[0083] Example 3, Preparation of modified DC-CIK (Triumph-DC-CIK) cells

[0084] 8. Preparation of PBMC from Peripheral Blood Mononuclear Cells

[0085] Take fresh anticoagulated whole blood, EDTA (sodium citrate or heparin) anticoagulant can be used. Dilute whole blood with an equal volume of PBS or 0.9% NaCl. Add a certain volume of separation liquid into the centrifuge tube, spread the diluted blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear. Separation solution, anticoagulated undiluted whole blood, PBS (or normal saline) at a volume of 1:1:1, centrifuged at 2000rpmx25min, separated to obtain PBMC, then washed twice with Hanks solution, counted the number of cells under a microscope, and finally used serum-free The culture medium VIVO-15 adjusted the cell density to make 5x106 / ml cell suspension;

[0086] 9. Non-adherent vs. Adherent Cell Isolation

[0087] Transfer the cell suspension into a 6...

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Abstract

The invention discloses a modified enhanced type DC-CIK cell targeting immune cell mass, and a preparation method and application thereof. A method for preparing a DC cell comprises the following steps: performing isolated culture on monocyte, thereby acquiring an immature DC cell; and performing tumor antigen load and induced differentiation culture on the immature DC cell, thereby acquiring a mature DC cell, wherein the monocyte is acquired by separating from a peripheral blood mononuclear cell of a sample. The method for preparing CIK comprises the following steps: performing in vitro differentiation culture on a CIK cell, thereby acquiring an immature CIK cell; preparing a modifying recombined slow virus for performing in vitro modification on the CIK cell; and co-culturing the modified CIK cell and the DC cell, thereby acquiring the mature modified CIK cell (Triumph-DC-CIK), so as to be used for achieving a better tumor immunotherapy effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a modified enhanced DC-CIK cell-targeted immune cell group and its preparation method and application, belonging to the fields of cell biology, immunology and tumor treatment. Background technique [0002] Malignant tumor is the main type of fatal disease in human beings and has become the number one killer threatening human health. According to the latest statistics from the Ministry of Health, according to the cancer-related report in 2012, there are about 3.37 million new cancer cases and 2.11 million deaths in my country every year. Cancer has become the leading cause of death in my country, accounting for a quarter of global cancer deaths. According to the 2012 World Cancer Report, there are approximately 14 million new cancer cases and 8 million deaths each year, which is a substantial increase compared with the 2008 statistics of 12.7 million. During t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/0784C12N5/10A61K39/00A61K35/17A61P35/00
Inventor 不公告发明人
Owner 深圳红石科创生物科技发展有限公司
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