Preparation method and application of modified enhanced DC-CIK targeting immune cell populations

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

Inactive Publication Date: 2016-05-11
哈尔滨新联合生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the treatment effect is mostly unsatisfac

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  • Preparation method and application of modified enhanced DC-CIK targeting immune cell populations
  • Preparation method and application of modified enhanced DC-CIK targeting immune cell populations
  • Preparation method and application of modified enhanced DC-CIK targeting immune cell populations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1. Preparation of lentivirus expressing human PD-1 fragment

[0054] 1. Construction of human PD-1 fragment lentivirus

[0055] Synthesize the double-stranded DNA molecule shown in SEQIDNO:1 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 , identified by PCR, the results are as follows figure 1 As shown, lane 1 is DL2000Marker, and lanes 2 and 3 are identification results. Virus packaging is accomplished by the following routine:

[0056] 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 at 37oC and 5% CO 2 Cultured to the logarithmic growth phase under certain conditions, the cells were collected and inoculated 5×10 cells in a cell culture dis...

Embodiment 2

[0065] Example 2. Detection of PD-1 after the recombinant virus infected CIK cells

[0066] In order to fully illustrate the beneficial effects of the present invention, the present invention also uses RT-PCR to perform real-time PCR detection of the PD-1 transcription level in CIK cells infected with pWPXLd-PD-1, and the steps are as follows:

[0067] Total RNA was extracted from the cells infected with pWPXLd-PD-1 for 24h, 48h and 72h 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 mRNA of PD-1 was transcribed, and GAPDH was used as an internal reference. The primer sequence of GAPDH is:

[0068] Upstream primer: 5'-ACCACAGTCCATGCCATCAC-3'

[0069] Downstream primer: 5'-TCCACCACCCCTGTTGCTGTA-3';

[0070] The primer sequence of PD-1 is:

[0071] Upstream primer: 5...

Embodiment 3

[0075] Example 3. Preparation of modified DC-CIK (Platinum-DC-CIK) cells

[0076] 1. Preparation of PBMC from Peripheral Blood Mononuclear Cells

[0077] 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 2000rpm for 25min, 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 5í10 6 / ml cell suspension;

[0078] 2. Non-adherent vs. Adherent Cell Isolation

[0079] Transfer the cell suspension into ...

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Abstract

The invention discloses a preparation method and application of modified enhanced DC-CIK targeting immune cell populations. A method for preparing DC cells includes subjecting monocyte to isolation culture to obtain immature DC cells, and subjecting the immature DC cells to tumor antigen loading and induction and differentiation culture so as to obtain mature DC cells, wherein the monocyte is obtained by separating single karyocyte of peripheral blood of samples. A method for preparing CIK includes subjecting CIK cells to in-vitro differentiation culture to obtain immature CIK cells, preparing recombinant lentivirus for modification to perform in-vitro modification on the CIK cells, and subjecting the modified CIK cells and the DC cells to co-culture so as to obtain mature modified CIK cells (Platinum-DC-CIK). The mature modified CIK cells (Platinum-DC-CIK) are used to improve tumor immunotherapy effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a modified 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 the same p...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/867C12N5/0784C12N5/0783A61K38/17A61P35/00
Inventor 不公告发明人
Owner 哈尔滨新联合生物科技有限公司
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