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NK92 cell strain carrying TGF-betaRII and NKG2D genes, and preparation method and use thereof

A technology of TGF- and cell lines, applied in botany equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve problems such as increasing the risk of breast cancer

Active Publication Date: 2014-12-31
WUXI BEIRUIKANG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A study showed that low expression of TGF-βRII increased the risk of breast cancer, and also found that the TGF-βRII gene of cells isolated from human tumor lesions also mutated

Method used

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  • NK92 cell strain carrying TGF-betaRII and NKG2D genes, and preparation method and use thereof
  • NK92 cell strain carrying TGF-betaRII and NKG2D genes, and preparation method and use thereof
  • NK92 cell strain carrying TGF-betaRII and NKG2D genes, and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Construction of NK92 cell line carrying TN gene

[0021] (1) Construction of plasmid vector for recombinant fusion gene TN

[0022] 1.1 The preparation process of the fusion gene TN is as follows: extract the total RNA of human peripheral blood lymphocytes, obtain cDNA by RT-PCR, use the cDNA as a template, use primers P1 / P2 and P3 / P4 respectively, and PCR amplify TGF-βRⅡ extracellular The transmembrane region and the intracellular segment of NKG2D are two genes. The TGF-βRII and NKG2D genes were connected into a single-chain gene TN by overlapping extension PCR (splicing by overlapping extension PCR, SOE-PCR) with P1 and P4. The primers are shown in Table 1 below.

[0023] Table 1 Construction of primers for fusion gene TN

[0024]

[0025] The above PCR product was identified by 1% agarose gel electrophoresis, and there was an obvious band at the 750bp position, which was consistent with the expected molecular weight (720bp), which indicated that we h...

Embodiment 2

[0037] Example 2: The killing ability test of NK92-TN on liver cancer cells SMMC7721 under the action of TGF-β in vitro

[0038] Promega's Cytotox96 non-radioactive cytotoxicity detection kit was used to detect the killing effect of NK92-TN on SMMC7721. After 2ng of TGF-β was co-cultured with effector cells (NK92-TN and NK92-Venus) and target cells (SMMC7721) for 5 hours, the detection results Such as image 3 As shown, the killing effect of NK92-TN is stronger, which is significantly different from that of the control group, and the killing effect of NK92-TN is stronger than that without TGF-β, indicating that NK92-TN is effectively activated by (TGF-β).

Embodiment 3

[0039] Example 3: In vivo anti-tumor treatment test of NK92-TN

[0040] Establishment of liver cancer model in nude mice: subcutaneously inoculate 2×10 6 One SMMC7721 cell, a tumor can be seen at the inoculation site after one week.

[0041] Treatment test: The test was divided into 3 groups, and PBS, NK92-Venus and NK92-TN were respectively inoculated at the tumor site one week later. Tumor size was measured every three days. The result is as Figure 4 As shown, compared with the control group (inoculated with NK92-Venus), the tumor growth rate in the NK92-TN group was significantly reduced. This result indicated that NK92 cells carrying TN had a good anti-tumor effect.

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Abstract

The invention belongs to the tumor adoptive treatment field, and concretely relates to an NK92 cell strain carrying TGF-betaRII and NKG2D genes, and a preparation method and a use thereof. The NK92 cell strain is transfected with a fusion gene formed by a TGF-betaRII extracellular segment transmembrane domain and an NKG2D intracellular segment. The invention discloses the natural killer cell strain (NK92-TN) carrying the TGF-betaRII extracellular segment transmembrane domain and the NKG2D intracellular segment, and a preparation method and a use thereof. The method comprises the following steps: fusing the two genes of the TGF-betaRII extracellular segment transmembrane domain and the NKG2D intracellular segment, inserting the obtained fusion gene TN into the shuttle plasmid prepared through a lentivirus, transfectign a 293T cell with other package plasmids through a calcium phosphate process to obtain lentivirus particles, transducing the NK92 cell, and sorting YFP positive cells through a flow cell sorter. The cell stain is suitable for the clinic adoptive immune treatment of tumors, has a better tumor resistance capability than other targeting tumor antigen cell strains, can be effectively activated in the TGF-beta environment, and can effectively inhibit the transfer of the tumors.

Description

technical field [0001] The invention belongs to the field of tumor adoptive therapy, and specifically relates to a NK92 cell line carrying TGF-βRII and NKG2D genes, a preparation method and application. Background technique [0002] In the 1980s, TGF-β1, TGF-β2, and TGF-β3 were successively discovered. So far, TGF-β4 and TGF-β5 have been added to this family member. There are three types of receptors for members of this family: TGF-βRI, TGF-βRII (transforming growth factor βⅡ receptor) and TGF-βRⅢ. In the early stages of inflammation and tumorigenesis, TGF-β mainly inhibits the development of inflammation and tumors, but in the late stage of tumorigenesis, it mainly promotes the occurrence, differentiation, adhesion and migration of cells, thereby promoting tumor metastasis. Tumor cells secrete a large amount of TGF-β, resulting in a large amount of TGF-β in the local tumor microenvironment. In the early stage of tumorigenesis, some tumor cells can escape the induction of T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867A61K48/00A61P35/00
Inventor 刘海燕王仲娟吴艳
Owner WUXI BEIRUIKANG BIOTECH
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