Preparation method and application of trophoblast modified by genetic engineering

A trophoblast and genetic engineering technology, applied in genetically modified cells, genetic engineering, and cells modified by introducing foreign genetic material, etc., can solve the problems of unsatisfactory NK cell expansion efficiency, poor expansion fold repeatability, etc. The effect of improving the success rate of construction, reducing the difficulty of construction and shortening the working time

Pending Publication Date: 2021-12-24
中海峡福建细胞生物科技有限公司
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expansion efficiency of NK cells obtained by this method is still unsatisfactory, there is still a lot of room for improvement, and there are also shortcomings such as poor repeatability of expansion folds between PBMCs from different individuals

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method and application of trophoblast modified by genetic engineering
  • Preparation method and application of trophoblast modified by genetic engineering
  • Preparation method and application of trophoblast modified by genetic engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] A method for preparing trophoblasts modified by genetic engineering, comprising the following steps:

[0076] (1) Construction of pLV-mbIL-21-OX40L-CD48 lentiviral vector plasmid:

[0077] S1: Obtain the gene coding sequence of human IL-21, human OX40L, and human CD48; the gene coding sequence of human IL-21 is shown in SEQ ID NO.1, and the gene coding sequence of human OX40L is shown in SEQ ID NO.2 , the gene coding sequence of human CD48 is shown in SEQ ID NO.3. The gene coding sequence of the PGK promoter coding sequence is shown in SEQ ID NO.4; the gene coding sequence of the T2A self-cleaving peptide coding sequence is shown in SEQ ID NO.5.

[0078] S2: The coding sequence of the human IL-21 gene, the coding sequence of the PGK promoter, the coding sequence of the human OX40L gene, the coding sequence of the T2A self-cleaving peptide, and the coding sequence of the CD48 gene were sequentially concatenated to obtain mbIL-21-PGK-OX40L-T2A-CD48 Gene sequence; wherein ...

Embodiment 2

[0105] Get the NK cells prepared in Example 1 and carry out in vitro expansion culture, the specific steps are:

[0106] S6: Extract PBMC cells:

[0107] The specific steps are:

[0108] (1) Transfer 10ml of whole blood into a 50ml centrifuge tube, add 10ml of PBS solution to dilute, and mix gently;

[0109] (2) Take two 15ml centrifuge tubes, first add 5ml Ficoll solution. Then gently add the diluted blood to the Ficoll upper layer of the two centrifuge tubes, be sure to be gentle to avoid mixing the two solutions together, each centrifuge tube has 10ml of diluted blood;

[0110] (3) 2000rpm, 20min, note that the deceleration setting must be set to nobreak, or only 1-20% braking;

[0111] (4) The cell layer where PBMCs are located is white. At this time, you can use a pipette to absorb this layer of cells into another clean 15ml centrifuge tube; add fresh medium to 10-15ml, 1500rpm, centrifuge for 5min and remove the supernatant; add 5ml of medium to resuspend the cells, ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a preparation method and application of a trophoblast modified by genetic engineering, and aims to research whether K562 cells can stably express IL-21, OX40L and CD48 molecules at the same time and enhance NK cell proliferation and tumor killing ability or not. K562 gene engineering cells simultaneously expressed by the three molecules are used as trophoblasts to culture NK, so that the amplification multiple, purity and killing function of the NK are improved; according to the preparation method, K562 cells can be used for simultaneously and stably expressing membrane binding interleukin-21 (mbIL-21), OX40L molecules and CD48 molecules to prepare an engineering cell strain named as K562-ZHX trophoblast cells, then the engineering cell strain is irradiated with cobalt-60 to obtain trophoblast amplified NK cells, the activity of the NK cells prepared by the method is improved by 3 times, the cell amplification can be improved by more than 300 times, a large amount of time and cost can be saved, and a practical foundation can be laid for obtaining high-purity, high-amplification-multiple and high-killing-activity NK cells.

Description

technical field [0001] The invention relates to the technical field of NK cell expansion, in particular to a method for preparing trophoblasts modified by genetic engineering and its application. Background technique [0002] Cancer is still the number one killer that seriously threatens human health. From a global perspective, the incidence of cancer is increasing year by year. At present, traditional cancer therapy cannot achieve good results, and cannot meet the expectations of patients and their families for recovery. Cancer immunotherapy is currently the most promising tumor treatment method recognized by the medical community. Natural killer cell (NK) is an important innate immune cell of the body. Compared with T cells or other immune cells, it can rapidly release inflammatory cytokines without pre-stimulation, non-histocompatibility complex The major histocompatibility complex (MHC) restricts the killing of tumor cells. NK cells are used in tumor immunotherapy, an...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N15/64C12N15/24C12N15/12C12N5/10C12N5/0783
CPCC12N15/86C07K14/54C07K14/70575C07K14/70503C12N5/0694C12N5/0646C12N2740/15043C12N2740/15052C12N2800/107C12N2510/00C12N2502/99C12N2502/30C12N2501/2321C12N2501/599
Inventor 夏玉龙吴金芸叶华衍
Owner 中海峡福建细胞生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap