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Lentivirus, recombinant mesenchymal stem cells and construction method and application of recombinant mesenchymal stem cells

A technology of stromal stem cells and construction method, applied in the field of recombinant mesenchymal stem cells and their construction, and lentivirus, can solve the problems of difficulty in implementation, low transgenic efficiency, and high difficulty, and achieve improvement in allergy and immune regulation, low cytotoxicity, and difficulty. high effect

Inactive Publication Date: 2020-08-25
GUANGDONG AIE BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low efficiency and high difficulty of MSC transgenesis in the prior art, there are certain difficulties in realization, and it has not been realized in the field

Method used

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  • Lentivirus, recombinant mesenchymal stem cells and construction method and application of recombinant mesenchymal stem cells
  • Lentivirus, recombinant mesenchymal stem cells and construction method and application of recombinant mesenchymal stem cells
  • Lentivirus, recombinant mesenchymal stem cells and construction method and application of recombinant mesenchymal stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Preparation of lentivirus:

[0040]The lentiviral vector including the full-length sequence of the TGF-β1 gene and the enhanced green fluorescent protein gene, the packaging plasmid VSVG and the expression plasmid sPA were co-transfected into 293T cells at a mass ratio of 3:1:2, and collected and cultured 72 hours after transfection. Cleared, concentrated, ultrafiltration and centrifugation with an ultrafiltration tube with a molecular weight cut-off of 100kD from Millipore Company, so that the lentivirus stock solution was concentrated more than 50 times; the harvested lentivirus was suspended in PBS, and divided into sterile EP tubes, and stored in a -80°C refrigerator , to obtain a lentivirus; the sequence of the lentivirus vector is shown in SEQ ID NO.1.

[0041] Lentivirus titer detection:

[0042] The 293T cells were digested into a single cell suspension, and the cell density was counted by a cell counter. 293T cells were added to a 12-well plate for culture, 1...

Embodiment 2

[0044] 1) Obtaining mesenchymal stem cells: Centrifuge the isolated adipose tissue at 1500rpm for 5 minutes, discard the upper layer of fat and the lower bloody cell layer, take the middle fat layer, and wash the fat with PBS for 3-5 times until the cleaning solution is clear; finally Centrifuge the mixture obtained once at 1500rpm for 5min, absorb the fat, and record the fat volume; digest the obtained fat with digestive enzymes at a ratio of 1:1, and digest at 150-280rpm for 20-60min on a constant temperature shaker at 37°C; after digestion, centrifuge at 2000rpm for 5min, Discard the upper fat layer and the digested fat layer, leaving a layer of stem cell sediment and a small amount of digestion solution, and add an equal volume of FBS to terminate; after termination, the mixture is centrifuged at 1500rpm for 5min to collect fat stem cells, resuspended in PBS, and washed at 1500rpm for 2 The second time, the double antibody and FBS used in the previous stage were removed; th...

Embodiment 3

[0055] To obtain exosomes:

[0056] The recombinant mesenchymal stem cells obtained in Example 2 were centrifuged at 300, 2000 and 10000g to remove cells, cell debris and large vesicles, then the precipitate was collected at 110000g, and finally the precipitate was washed with phosphate buffered saline (PBS) to remove Soluble interfering proteins, exosomes were harvested.

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Abstract

The invention discloses a lentivirus, recombinant mesenchymal stem cells and a construction method and application of the recombinant mesenchymal stem cells. A lentivirus vector is a lentivirus vectorwith overexpression of TGF-beta1, can infect human mesenchymal stem cells and establish a stable expression cell line, and is high in infection efficiency; the recombinant mesenchymal stem cells areobtained by infecting mesenchymal stem cells with lentiviruses, the mesenchymal stem cells are infected with the lentivirus vector with overexpression of TGF-beta1, and a stable expression cell line can be established after infection; according to the application of the recombinant mesenchymal stem cells, the recombinant mesenchymal stem cells are cultured to obtain an exosome, and the exosome obtained in the invention has the effects of DNA damage prevention, cell proliferation promotion and damage repair, inhibits cell senescence, and has wide application in the fields of skin diseases and regeneration repair.

Description

technical field [0001] The invention relates to a lentivirus, a recombinant mesenchymal stem cell and a construction method and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Gene therapy is a novel method of recombinant protein delivery with distinct advantages, including stable, efficient and permanent gene expression, identical glycosylation pattern to the receptor, and non-immunogenicity to the receptor. Viral vectors can infect both dividing and non-dividing cells, and can make the target gene enter the target cell and be expressed stably for a long time. It can introduce multiple genes into the same cell, or transfect the same cell, which is not easy to cause The immune response of target cells is also high in biological safety. [0003] The accumulation of cellular damage over time is one of the key mechanisms that trigger aging and the onset of cancer. Cellular senescence may be driven by DNA, lipid or pr...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/867C12N5/10C12N15/12C12R1/91
CPCC07K14/495C12N7/00C12N15/86C12N2740/15021C12N2740/15043
Inventor 谢创丰吴曙霞李玉霞
Owner GUANGDONG AIE BIOSCIENCE CO LTD
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