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Recombinant mesenchymal stem cell, function-enhanced exosome as well as preparation method and application of function-enhanced exosome

A technology of stem cells and exosomes, applied in cell dissociation methods, extracellular fluid diseases, botanical equipment and methods, etc., to enhance function, improve hematopoietic microenvironment, and promote proliferation

Pending Publication Date: 2022-06-07
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the genetic modification of exosomes derived from mesenchymal stem cells to improve its repairing effect on radiation hematopoietic damage

Method used

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  • Recombinant mesenchymal stem cell, function-enhanced exosome as well as preparation method and application of function-enhanced exosome
  • Recombinant mesenchymal stem cell, function-enhanced exosome as well as preparation method and application of function-enhanced exosome
  • Recombinant mesenchymal stem cell, function-enhanced exosome as well as preparation method and application of function-enhanced exosome

Examples

Experimental program
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preparation example Construction

[0045] The present invention provides a method for preparing recombinant mesenchymal stem cells, comprising the following steps: infecting recipient mesenchymal stem cells with a recombinant vector expressing autocrine motor factor ATX to obtain recombinant mesenchymal stem cells.

[0046] In the present invention, the recombinant vector preferably includes recombinant adenovirus Ad-ATX. The recombinant adenovirus Ad-ATX described in the present invention and the examples is preferably purchased from Shandong Weizhen Biotechnology Co., Ltd., the article number is VH886323.

[0047] In the present invention, the number ratio of the recombinant vector to the recipient mesenchymal stem cells is preferably (50-150):1, more preferably (100-150):1, and more preferably 100:1; the The recipient mesenchymal stem cells preferably include P3 generation recipient mesenchymal stem cells, more preferably P3 generation dental pulp stem cells DPSC. Compared with bone marrow-derived, umbilica...

Embodiment 1

[0067] A recombinant mesenchymal stem cell consists of the following steps:

[0068] The P3 generation dental pulp stem cells (DPSC) were recovered, and the mesenchymal stem cell serum-free medium (purchased from Beijing Sanli Heze Biotechnology Co., Ltd., product number 120408) was used to expand, and after amplification, recombinant adenovirus Ad-ATX ( It was purchased from Shandong Weizhen Biotechnology Co., Ltd., the article number is VH886323) to infect DPSC (the ratio of Ad-ATX to DPSC is 100:1) to obtain recombinant mesenchymal stem cells;

[0069] The dental pulp stem cells (DPSC) were obtained from the normal intact third molars of healthy people; after the crown was cut, the dental pulp tissue was separated from the root and digested with collagenase and neutral protease for 40 min at 37°C; using Sodium chloride injection was washed once, and the liquid was removed after centrifugation at 1500 rpm for 5 min; the above tissue was placed in a 75 cm 2 The culture flask...

Embodiment 2

[0078] Preparation and identification of functionally enhanced exosomes

[0079] Resuscitate P3 generation DPSC, use mesenchymal stem cell serum-free medium for expansion, and infect DPSC with Ad-ATX after expansion (the specific method is the same as that in Example 1);

[0080] After 1 day of cell infection, the serum-free medium was replaced, and the culture supernatant was collected after 2 days; the culture supernatant was placed in a 50 mL centrifuge tube, 300 g, 4 °C, and centrifuged for 10 min; after centrifugation, the supernatant was directly poured into a new centrifuge tube, 2000 g , 4℃, centrifuge for 20min; after centrifugation, pour the supernatant directly into a new centrifuge tube, 10,000g, 4℃, centrifuge 30min; after centrifugation, pour the supernatant directly into a new centrifuge tube, 100,000g, 4℃ for 2 centrifuged for 70 min each time; after the centrifugation, discard the supernatant, rinse the side wall of the tube near the bottom with 200 μL of cold...

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Abstract

The invention relates to the technical field of hematopoietic injury, in particular to a recombinant mesenchymal stem cell, a function-enhanced exosome as well as a preparation method and application of the function-enhanced exosome. The preparation method comprises the following steps: infecting receptor mesenchymal stem cells with a recombinant vector expressing autotaxin ATX to obtain the recombinant mesenchymal stem cells. The recombinant mesenchymal stem cells prepared by the preparation method provided by the invention are subjected to gene modification by infecting a recombinant vector for expressing autotaxin ATX, so that the mesenchymal stem cells highly express ATX, the LPA expression of the ATX product in the secreted exosome is increased, the function of the exosome is further enhanced, and the expression rate of the exosome is increased. The exosome can promote proliferation of irradiated hematopoietic cells (irradiated by radiation), inhibit apoptosis of the irradiated hematopoietic cells, improve the hematopoietic microenvironment of irradiated mice and achieve the effect of treating hematopoietic injury.

Description

technical field [0001] The invention relates to the technical field of hematopoietic injury, in particular to a recombinant mesenchymal stem cell, a function-enhanced exosome and a preparation method and application thereof. Background technique [0002] With the increasing incidence of tumors and the widespread application of radiotherapy, local radioactive myelosuppression is one of the important complications and dose-limiting factors of radiotherapy for a variety of tumors. Complications of radiation hematopoietic injury also occur during preconditioning with total body irradiation for bone marrow transplantation. Acute radiation sickness is an important complication of nuclear radiation, and radiation hematopoietic injury is the most important type of tissue damage. Therefore, effective prevention of hematopoietic injury and early recovery of hematopoietic system function are the keys to successful treatment of radiation injury. [0003] Radiation hematopoietic injury...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/861C12N15/55C12N5/0775A61K35/28A61P7/00
CPCC12N5/0664C12N15/86C12N9/16C12Y301/04039A61K35/28A61P7/00C12N2510/00C12N2800/107C12N2710/10043C12N2509/10
Inventor 王华孔繁轩靳继德吴祖泽
Owner ACADEMY OF MILITARY MEDICAL SCI
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