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

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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 limit

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

Examples

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Example Embodiment

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

[0035] Among them, the umbilical cord mesenchymal stem cells are derived from isolated umbilical cord tissues, which are convenient to obtain materials, have a wide range of sources, and are not subject to ethical disputes and restrictions. At the same time, the materials are not traumatized to the donor, and are not affected by the age of the donor, and are isolated in vitro Simple cultivation, rapid amplification, low immunogenicity, no tumorigenicity, and 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. After continuous subculture and cryopreservation, they still have multi-directional differentiation potential and are ideal seed cells for cell transplantation therapy.

[0036] According to a prefer...

Example Embodiment

[0176] Example 1 Obtaining Lptn-CCL4 fusion gene and its recombinant plasmid

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

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

[0179] The above-mentioned human Lptn gene and human CCL4 gene were used to synthesize DNA molecules encoding human Lptn-CCL4 through Zhongmeitaihe Biotechnology (Beijing) Co., Ltd. The upstream and downstream contained BamHI and EcoRI restriction sites respectively. Among them, Lptn and CCL4 passed T2A sequence (the co...

Example Embodiment

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

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

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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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