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Recombinant mesenchymal stem cell, and preparation method and application thereof

A technology of stem cells and recombinant plasmids, applied in the field of genetic engineering, can solve the problems such as the inability to expand the scope of immunotherapy applications, the inability to efficiently achieve "cold" and "hot" tumor transformation, and the inability to efficiently change the immune microenvironment. To achieve the effect of changing the immune microenvironment, improving the efficiency, and increasing the content

Active Publication Date: 2020-05-12
北京华奥玄德生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, in the prior art, the immune microenvironment cannot be efficiently changed, that is, the transformation of "cold" and "hot" tumors cannot be efficiently realized, which limits the application of PD1 antibodies in the treatment of solid tumors, and at the same time cannot expand the scope of application of immunotherapy and Improve the efficiency of immunotherapy

Method used

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  • Recombinant mesenchymal stem cell, and preparation method and application thereof
  • Recombinant mesenchymal stem cell, and preparation method and application thereof
  • Recombinant mesenchymal stem cell, and preparation method and application thereof

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Embodiment approach

[0034] According to a preferred embodiment of the present invention, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0035] Wherein, the umbilical cord mesenchymal stem cells are derived from the isolated umbilical cord tissue, which is convenient to obtain, has a wide range of sources, is not subject to ethical debate and restrictions, and is not traumatic to the donor, and is not affected by the age of the donor. The culture is simple, the expansion is rapid, the immunogenicity is low, and there is no tumorigenicity, and it can be used as a cell carrier for gene therapy. Under specific induction conditions in vivo or in vitro, umbilical cord mesenchymal stem cells can differentiate into a variety of tissue cells, and they still have multidirectional differentiation potential after continuous subculture and cryopreservation. They are ideal seed cells for cell transplantation therapy.

[0036] According to a preferred embodiment of the present invention...

Embodiment 1

[0176] Embodiment 1 obtains Lptn-CCL4 fusion gene and recombinant plasmid thereof

[0177] (1) According to the human Lptn gene sequence in Genebank, the human Lptn gene was synthesized from the whole gene of Zhongmei Taihe Biotechnology (Beijing) Co., Ltd., its coding nucleotide sequence is the sequence shown in SEQ ID NO.5, and its amino acid The sequence is the sequence shown in SEQ ID NO.6.

[0178] According to the human CCL4 gene sequence in Genebank, the human CCL4 gene was synthesized from the whole gene of Zhongmei Taihe Biotechnology (Beijing) Co., Ltd., its coding nucleotide sequence is the sequence shown in SEQ ID NO.7, and its amino acid sequence is SEQ ID NO.7 The sequence shown in ID NO.8.

[0179] The above-mentioned human Lptn gene and human CCL4 gene were synthesized by Zhongmei Taihe Biotechnology (Beijing) Co., Ltd. into a DNA molecule encoding human Lptn-CCL4. The T2A sequence (the coding nucleotide sequence is the sequence shown in SEQ ID NO.3, and th...

Embodiment 2

[0182] Example 2 Preparation of recombinant lentivirus carrying Lptn-CCL4

[0183] (1) Take out a tube of frozen 293T cells (purchased from ATCC) from liquid nitrogen and quickly put it in a 37°C water bath until the ice cubes disappear, add dropwise to a 15ml centrifuge tube containing 5ml preheated medium, and centrifuge at 1200rpm for 3min , Discard the supernatant, resuspend the cells in 293T medium (10% FBS + 1mM sodium pyruvate + 2mM glutamine + 1% non-essential amino acids + DMEM) and inoculate them in a 150mm culture dish at 37°C, 5% CO 2 Cultivate in saturated humidity. During the culture process, when the confluence of the cells reaches more than 90%, carry out subculture, discard the old medium, add 5ml sterilized PBS solution, shake gently, discard the PBS solution after washing the cells, and add 2ml 0.25% Trypsin- EDTA digestion solution, digest for 1-2min until the cells are completely digested; add serum-containing medium to stop the digestion, and centrifug...

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Abstract

The invention discloses a recombinant mesenchymal stem cell, and a preparation method and application thereof. The recombinant mesenchymal stem cell is obtained by modifying mesenchymal stem cells bychemokine genes, can convert non-immunogenic tumors into immunogenic tumors, and can be combined with a PD1 antibody to inhibit proliferation of solid tumors, and the content of infiltrating lymphocytes in the tumors is obviously increased. The preparation method of the recombinant mesenchymal stem cells has the advantages of simple operation, easy control of conditions and low cost, and is suitable for large-scale application.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, specifically relates to the technical field of stem cells, in particular to a recombinant mesenchymal stem cell and its preparation method and application. Background technique [0002] Immunotherapy has become an important progress in the field of tumor treatment after targeted therapy. Especially for patients who have not targeted driver gene mutations and therapeutic targets, the emergence of immunotherapy has brought them new hope. At the same time, clinical studies have shown that once tumor patients respond to immunotherapy, they can often bring long-term benefits, even complete remission of the disease. [0003] Currently, several PD-1 / PD-L1 inhibitors (such as nivolumab and atezolizumab) have been approved for 12 solid tumors including non-small cell lung cancer and urothelial carcinoma And the second / third-line or even first-line treatment of all microsatellite instability ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/28A61P35/00
CPCA61K35/28A61P35/00C07K14/521C07K14/523C12N5/0665C12N15/86C12N2510/00C12N2740/15043
Inventor 段海峰薛冰华于婷婷肖秀孝解晶庞如梦张群伟弓景波
Owner 北京华奥玄德生物医药科技有限公司
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