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Mesenchymal stem cell strain for overexpression of PD-L1 gene as well as a construction method and application of mesenchymal stem cell strain

A PD-L1, stem cell technology, applied in the field of biomedicine, can solve the problems of insignificant treatment effect, high disease recurrence rate, treatment of arthritis and other problems, achieve significant curative effect, inhibit inflammatory response, and reduce the degree of joint damage.

Pending Publication Date: 2020-11-20
樊克兴
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are no reports at home and abroad that combine MSC cells with PD-L1 to treat arthritis
At the present stage, the traditional means of treating arthritis-related diseases and traditional medicines can relieve the symptoms of rheumatoid arthritis, but the therapeutic effect is not obvious, and the recurrence rate of the disease is high, and the side effects are relatively large.

Method used

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  • Mesenchymal stem cell strain for overexpression of PD-L1 gene as well as a construction method and application of mesenchymal stem cell strain
  • Mesenchymal stem cell strain for overexpression of PD-L1 gene as well as a construction method and application of mesenchymal stem cell strain
  • Mesenchymal stem cell strain for overexpression of PD-L1 gene as well as a construction method and application of mesenchymal stem cell strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Preparation and Identification of Mesenchymal Stem Cells

[0034] 1. Mesenchymal stem cell culture

[0035] (1) Take 10-15ml of bone marrow (or 3-5ml of bone marrow from the ribs of normal people), add 100u heparin to each ml of bone marrow, mix thoroughly, store in a refrigerator at 4°C, and separate bone marrow mononuclear cells within 24 hours ( MNC). First add an equal amount of PBS or Hanks solution (or culture medium) to the bone marrow and mix well, then slowly add the diluted bone marrow to the surface of the ficoll liquid (density 1.077) at the ratio of 1 part of lymphocyte separation solution to 2 parts of diluted bone marrow, and horizontally centrifuge Centrifuge at 500g (2000r / min) for 25-30 minutes at 20°C. After the MNCs taken out from the interface were washed 2-3 times with PBS, an appropriate amount of culture medium was added for counting. Cell number up to 10 6 ~10 7 After / ml, the primary culture was carried out, and 5×10 6 / ml concentration i...

Embodiment 2

[0044] Construction of bone marrow mesenchymal stem cell line PD-L1-MSC overexpressing PD-L1 gene

[0045] 1. Design and amplification of PD-L1 gene PCR primers

[0046] The mouse PD-L1 gene sequence was queried in NCBI, the nucleotide sequence information is shown in SEQ ID NO.1, and the amino acid sequence information is shown in SEQ ID NO.8. PD-L1 primers were designed using Primer5.0 software.

[0047] Upstream primer PD-L1-F: tctagaATGAGGATATTTGCTGGCATT (SEQ ID NO: 2)

[0048] Downstream primer PD-L1-R: gaattcTTACGTCTCCTCGAATTGTG (SEQ ID NO: 3)

[0049] The mouse PD-L1 gene was amplified by PCR using the mouse lung cDNA as a template and using the primers designed above.

[0050] 2. Lentivirus packaging pCDH-PD-L1 plasmid

[0051] The PD-L1 sequence was constructed on the lentiviral vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro to obtain a lentiviral plasmid expressing pCDH-PD-L1.

[0052] Construction method: The above PCR product was connected to a T vector for sequencin...

Embodiment 3

[0056] Identification of PD-L1 gene expression in bone marrow mesenchymal stem cell lines transfected

[0057] 1. Fluorescence microscopy to observe the efficiency of lentivirus pCDH-PD-L1 transfection bone marrow stem cells

[0058] 24 hours after the lentivirus pCDH-PD-L1 transfected the bone marrow stem cells, the original medium was replaced with fresh complete medium, the culture was continued, and observed under a fluorescent inverted microscope 48 hours later. The result is as Figure 4 As shown, the transfection efficiency of PD-L1 was found to be greater than 90%.

[0059] 2. RT-PCR method to analyze the expression of PD-L1 gene

[0060] The RNA of bone marrow stem cells transfected with pCDH-PD-L1 was extracted by Trizol method, and reverse transcribed into cDNA. At the same time, the RNA of the stem cells transfected with the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro empty vector was extracted, reverse-transcribed into cDNA, and a control experiment was performed. Primer...

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Abstract

The embodiment of the invention discloses a mesenchymal stem cell strain for overexpression of a PD-L1 gene as well as a construction method and an application of the mesenchymal stem cell strain, andbelongs to the technical field of biomedicine. The method comprises the following steps: 1) constructing a recombinant slow virus carrying a PD-L1 gene; and 2) transfecting mesenchymal stem cells with the recombinant slow virus carrying the PD-L1 gene, and carrying out puromycin screening, so as to obtain a mesenchymal stem cell strain for over-expressing the PD-L1 gene. After the mesenchymal stem cell strain for over-expressing the PD-L1 gene is injected into a rheumatoid arthritis mouse model intravenously, the symptoms of rheumatoid arthritis can be remarkably relieved, the joint injury degree is reduced, the expression of inflammatory factors is regulated, inflammatory response is inhibited, and the mesenchymal stem cell strain has a remarkable curative effect on treatment of rheumatoid arthritis diseases.

Description

technical field [0001] The present invention relates to the field of biomedical technology, in particular to a genetically engineered mesenchymal stem cell strain overexpressing the programmed cell death ligand 1 (PD-L1) gene and a construction method thereof, and the overexpressing PD-L1 gene Application of mesenchymal stem cell lines in the treatment of rheumatoid arthritis. Background technique [0002] Rheumatoid arthritis (RA) is a systemic, inflammatory autoimmune disease characterized by articular cartilage lesions and synoviocyte hyperplasia. The diseased joint and its surrounding tissues show progressive destruction and lead to dysfunction of the damaged joint. Rheumatoid arthritis is a common and tormenting immune disease. The cause is that the body's immune system is disordered, and it attacks its own tissues as enemies, causing joint pain, swelling and destruction, and damage to the skin, eyes, and lungs and other organs. Rheumatoid arthritis has been called t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10A61K35/28A61P29/00A61P19/02
CPCC12N15/86C12N5/0663C07K14/70532A61K35/28A61P29/00A61P19/02C12N2740/15043C12N2510/00C12N2800/107
Inventor 樊克兴
Owner 樊克兴
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