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Trophoblast, preparation method thereof and application of trophoblast in efficient amplification of NK cells

A technology of trophoblasts and NK cells, which is applied in the fields of genetic engineering and cell biology, can solve the problems of unsatisfactory amplification multiples and low double-positive ratios, and achieve the effects of simplified workload, high amplification multiples, and high purity

Active Publication Date: 2020-06-19
中邦干细胞科技有限公司
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, this method also has an unsatisfactory amplification factor, and the expanded NK cell CD56 + CD16 + Defects such as low proportion of double positives

Method used

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  • Trophoblast, preparation method thereof and application of trophoblast in efficient amplification of NK cells
  • Trophoblast, preparation method thereof and application of trophoblast in efficient amplification of NK cells
  • Trophoblast, preparation method thereof and application of trophoblast in efficient amplification of NK cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Construction of pLenti-mbIL21-4.1BBL-CD16A-OX40L lentiviral plasmid vector

[0074] The OX40L cDNA (accession number NM_003326) sequence (as shown in SEQ ID NO: 1) was obtained by querying on NCBI and other websites, and the CD16 antibody heavy chain (heavy chain variable region VH) and light chain (light chain variable region VL) of antibody AFM13 were obtained , designed to form a membrane-bound CD16 single-chain antibody sequence (CD16A, as shown in SEQ ID NO: 2), and the membrane-bound part is the membrane-bound region sequence of CD8A;

[0075] Synthesize the gene of E2A-CD16A-P2A-OX40L-T2A-puro (as shown in SEQ ID NO: 3) by existing gene synthesis methods, and synthesize two restriction sites of BsmBI and SalI on both sides, coding region Avoid these two restriction sites;

[0076] The above-mentioned synthesized gene was double digested with two enzymes BsmBI and SalI, and then connected to the lentiviral vector pLenti-mbIL21-4.1BBL (as shown in SEQ ID NO: 4, pr...

Embodiment 2

[0080] Packaging preparation of recombinant lentivirus

[0081] 1. Add 4.5million 293FT cells and 9ml DMEM complete medium to each 10cm cell culture dish, mix well, and culture in a cell culture incubator (37°C, 5% (v / v) CO 2 );

[0082] 2. On the second day of culture, add the following reagents to each culture dish: 500 μL jetPRIME buffer, 6 μg lentiviral vector expressing IL21, 4.1BBL, CD16A, OX40L, 3 μg psPAX2 and 1.5 μg pMD2.G, mix well, and then Add jetPRIME to the system, 25μL / 10cm petri dish, mix again evenly, and let it stand at room temperature for 10min to obtain a mixture;

[0083] 3. Take out the 293FT cells used for packaging the virus from the incubator, add the mixed solution evenly to each culture dish, shake well, and put it into the incubator to continue culturing (37°C, 5% (v / v) CO 2 ). After culturing for 4 h, discard the old medium, add PBS to wash the cells, then add DMEM complete medium containing 10wt% FBS, and put them in an incubator for cultivati...

Embodiment 3

[0087] Preparation of K562-mbIL21-4.1BBL-CD16A-OX40L trophoblast cells

[0088] Put 1 million vigorously growing K562 cells into one well of a 24-well plate for culture, and the medium is 1 ml of 1640 containing 10wt% FBS;

[0089] Subsequently, 300 μL lentiviral particles concentrated by ultracentrifugation of the above-mentioned 30 ml virus supernatant were added to the wells, and cultured (37° C., 5% (v / v) CO 2 ) After 5 days, selective culture was carried out with 3 μg / ml of Puromycin;

[0090]The expression of these four genes IL21, 4.1BBL, CD16A, and OX40L in K562 cells was detected by flow cytometry, so that the expression of each gene was above 80% (see figure 2 and image 3 );

[0091] Then 100Gy of γ-rays were used to irradiate for 10 minutes, and after irradiation, branches were frozen in a -80°C refrigerator for future γδT cell culture experiments.

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Abstract

The invention relates to a trophoblast, a preparation method thereof and application of the trophoblast in efficient amplification of NK cells. The preparation method comprises the following steps: S1, constructing a plasmid vector: pLenti-mbIL21-4.1BBL-CD16A-OX40L; S2, packaging recombinant lentivirus; S3, preparing trophoblast: K562-mbIL21-4.1BBL-CD16A-OX40L; S4, preparing umbilical cord blood or peripheral blood leukocyte layer cells; and S5, performing in-vitro amplification of NK cells. The trophoblast provides a plurality of stimulating molecules such as IL21, 4.1BBL, CD16A and OX40L forthe NK cells to activate the NK cells, so that in-vitro mass proliferation of the NK cells is promoted; and the NK cells can be amplified by 10000 times or above after a 14-day proliferation period,and the purity of the prepared NK cells can reach 90% or above. Compared with traditional culture methods for stimulating NK by expressing IL21 and 4.1 BBL trophoblasts, the purity, quality and quantity of the NK cells obtained by the method disclosed by the invention are obviously improved.

Description

technical field [0001] The invention relates to the fields of genetic engineering and cell biology, in particular to a trophoblast capable of activating multiple signaling pathways, a preparation method thereof and an application in efficiently expanding NK cells. Background technique [0002] NK cells are a kind of innate immune cells, which are natural killers of virus-infected cells and tumor cells. Unlike T cells, NK cells can recognize abnormal cells through their inhibitory receptors KIR and NKG2A and activating receptors NKG2D, CD16, and NKp30. When target cells express low levels of inhibitory ligands and high levels of activating ligands When activated, NK cells are activated and release perforin and granzymes to kill target cells. [0003] In addition to the direct anti-tumor effect, NK cells can also participate in the acquired anti-tumor response and play the role of immune mediation. [0004] NK cells are mainly distributed in peripheral blood. In general, NK ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N5/0783
CPCC07K14/54C07K14/70535C07K14/47C07K14/70596C12N5/0646C12N15/86C12N5/0694C12N2502/30C12N2740/15043C12N2510/02Y02A50/30
Inventor 张云龙
Owner 中邦干细胞科技有限公司
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