Preparation method for genetically recombinant stem cell preparation and application of same to skin injury repair and scar inhibition

A technology of stem cell preparation and gene recombination, which is applied in the field of preparation of medicines for repairing skin damage and/or inhibiting scar generation, can solve the problems of short half-life of HGF, maintaining HGF concentration, affecting protein activity, etc., to reduce scar generation and maintain effective concentration , the effect of effective treatment

Inactive Publication Date: 2017-03-15
ZHEJIANG SHENGCHUANG PRECISION MEDICAL TECH CO LTD
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AI Technical Summary

Problems solved by technology

[0003] However, HGF is not easy to collect and obtain in large quantities. At present, the most widely used production method in industrial production is the prokaryotic protein expression system. This system mainly uses E. coli to express proteins. E. coli has a high expression level of HGF, but it is also easy to form inclusion bodies and affect the protein. active
However, the eukaryotic expression system for transient transfection avoids the formation of inclusion bodies, but the production cost is extremely high due to the low expression level
In addition, the existing injections or paints that use HGF as an active ingredient are not targeted for the repair of skin scars, and there is a short half-life of HGF in the treatment methods that directly use HGF protein administration. Injection, it is also difficult to maintain a high level of HGF concentration in the body, resulting in huge HGF dosage and economic costs
On the other hand, limited by the level of protein preparation purification technology, some existing HGF preparations are prone to adverse reactions during treatment, which will bring negative effects to the treatment.

Method used

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  • Preparation method for genetically recombinant stem cell preparation and application of same to skin injury repair and scar inhibition
  • Preparation method for genetically recombinant stem cell preparation and application of same to skin injury repair and scar inhibition
  • Preparation method for genetically recombinant stem cell preparation and application of same to skin injury repair and scar inhibition

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preparation example Construction

[0017] The present disclosure provides a method for preparing a genetically recombined stem cell preparation for wound repair and scar suppression, the stem cell preparation includes independently stored cell injections and spraying agents; the preparation method includes:

[0018] (1) Pack the lentiviral vector pLVX-EF1α-hHGF shown in SEQ ID NO.1 into lentiviral infection particles, infect uterine blood stem cells, and screen to obtain a cell line stably expressing hepatocyte growth factor HGF; the uterine blood Stem cells are CD3-positive and CD28-positive uterine blood stem cells;

[0019] (2) The cell line stably expressing HGF is subjected to expansion culture for 4-6 generations, then cultured in serum-free medium for 48-72 hours, and then the cell components and culture supernatant are separated;

[0020] (3) The cell component is prepared as a cell injection; the culture supernatant is prepared as a spraying agent.

[0021] Wherein, the promoter of the lentiviral vect...

Embodiment 1

[0059] 1) Preparation of lentiviral infection particles: Synthesize the lentiviral vector pLVX-EF1α-hHGF shown in SEQ ID NO.1. The lentiviral vector pLVX-EF1α-hHGF was mixed with liposome transfection reagent to transfect 293T cells to obtain transfected packaging cells; the transfected packaging cells were cultured and lentiviral infection particles were collected.

[0060] 2) Acquisition of uterine blood stem cells: collect menstrual products, separate the uterine blood from the endometrial tissue in the menstrual products; use Ficoll density gradient centrifugation to separate the mononuclear cells in the uterine blood, and resuspend the mononuclear cells in normal saline Impurities such as red blood cells, platelets and cell debris are removed by centrifugation. Then use GIBCO MSC SFM medium was used to culture the cells adherently to obtain adherent cells; the endometrial tissue was digested with trypsin and separated by sieving, the mononuclear cells were resuspended i...

Embodiment 2

[0069] The stem cell preparation prepared in Example 1 was tested on Swiss nude mice for wound repair.

[0070] Eighteen nude mice weighing 18-22g were anesthetized by intraperitoneal injection of ketamine-xylazine (80mg / kg-12mg / kg; Ref.K-113, Sigma, France), and the animals were randomly divided into 3 experiments Group and 3 control groups, 3 mice in each group, then percutaneous injury (length 1 cm, depth about 1 to 2 mm) was performed on the right flank of each mouse with a 0.5 / 10 razor blade.

[0071] (1) Experimental group 1 and control group 1

[0072] 10 minutes after the injury in the control group 1, 200 μL of normal saline was applied locally on the wound of the animal; and 1 ml of normal saline was injected through the tail vein;

[0073] Experimental group 1 topically applied 200 μL of the spray agent prepared in Example 1 to the animal wound 10 minutes after injury; and injected 1 ml of the cell injection prepared in Example 1 through the tail vein.

[0074] Th...

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Abstract

The invention discloses a genetically recombinant stem cell preparation used for wound repair and scar inhibition and a preparation method thereof. The genetically recombinant stem cell preparation comprises a cell injection and a spraying agent which are separately stored. The preparation method comprises the following steps: subjecting a cell line stably expressing HGF to multiplication culture and separating out a cell component and a cultural supernatant; preparing the cell injection from the cell component; and preparing the spraying agent from the cultural supernatant. The invention also provides application of intrauterine blood stem cells stably expressing HGF to preparation of drugs used for repairing skin injuries and / or inhibiting scar formation. The genetically recombinant stem cell preparation can realize safe and efficient repair of wounds and inhibit scar formation.

Description

technical field [0001] The present disclosure relates to the technical field of drug preparation and cell culture, in particular, to a preparation method and stem cell preparation for wound repair and scar suppression, and the use of uterine blood stem cells stably expressing HGF in the preparation of repairing skin damage and / or Or use in medicines for inhibiting scar production. Background technique [0002] Hepatocyte growth factor (hepatocyte growth factor, HGF) is an important anti-fibrosis factor. Existing studies have shown that it can promote effective, high-quality and rapid healing of skin wounds, reduce scar formation, and have an effect on bone remodeling. It has an important role and is a protein therapeutic drug with broad application prospects. [0003] However, HGF is not easy to collect and obtain in large quantities. At present, the most widely used production method in industrial production is the prokaryotic protein expression system. This system mainly ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/0789A61K38/18A61K35/14A61P17/02
CPCC12N15/86A61K9/0014A61K9/0019A61K9/12A61K35/14A61K38/1833C12N5/0634C12N2509/00C12N2740/15043
Inventor 李程郭垚示项春生
Owner ZHEJIANG SHENGCHUANG PRECISION MEDICAL TECH CO LTD
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