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Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar

A targeted technology for hypertrophic scars, applied in drug combinations, peptide/protein components, medical preparations containing active ingredients, etc., can solve problems such as no effective solutions, reduce treatment costs, reduce drug dosage, The effect of reducing toxic side effects

Inactive Publication Date: 2011-07-06
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] But for hypertrophic scars, there is no effective solution

Method used

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  • Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar
  • Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Expression, purification and biological activity determination of CBD-IL-10

[0021] (1) Acquisition of CBD-IL-10 gene

[0022] 1. Extraction of total RNA

[0023] Mononuclear cells were separated from human peripheral blood by lymphocyte separation medium, washed twice with PBS, resuspended in 10% RPMI 1640 culture medium, added ConA with a final concentration of 10g / L, placed in a 5% CO2 incubator, and incubated at 37°C for 48h. Collect cells. According to the method of RNA extraction kit, total RNA was extracted and stored at -80°C for later use.

[0024] 2. Extraction of total RNA and amplification of IL-10 cDNA

[0025] Use the RNA extraction and inversion kit from Takara Company to extract total RNA according to the instructions, and perform RT-PCR reaction: total RNA 500ng, 5× reverse transcription Buffer 2μl, oligo dT 0.5μl, 6mer 0.5μl, AMV reverse transcriptase 0.5μl , DEPC water was added to 10 μl. Incubate at 37°C for 30 minutes, then incubate...

Embodiment 2

[0057]Example 2: CBD-IL-10 inhibits scar hyperplasia test

[0058] (1) Hypertrophic scar tissue

[0059] The hypertrophic scar tissue of the forearm (proven by pathology) 3 months after the burn was healed was red, hard and about 4-7 mm higher than the normal skin. Thirty BALA / c-nu male mice, aged 6-8 weeks and weighing 18-20 g, were used.

[0060] (2) Preparation of animal model of post-burn hypertrophic scar

[0061] The adipose tissue under the hypertrophic scar was removed, and the scar was cut into 6mm×5mm×3mm tissue pieces, placed in pre-cooled DMEM (containing 100 U / ml of penicillin and streptomycin double antibodies) under sterile conditions A small incision was made in the skin of the scapula of nude mice, and a small piece of scar tissue was transplanted under the skin without suturing. Complete 30 animal models within 3 hours. Observe whether the animal model has infection or rejection, and select a stable scar model as the treatment animal. The scar model was ...

Embodiment 3

[0070] Example 3: Collagen Specific Binding Identification

[0071] Identification by ELISA: after neutralization of the collagen soluble by sarcosine, add it to a 0.1 mg / well 96-well plate, overnight at 4°C, wash 3 times with PBST, add 200ul of 0.25% BSA to each well to block for 2 hours at room temperature. The same TBST was washed 3 times, and 0.157-10 uM CBD-IL-10 was added to the corresponding wells in 3 parallel wells. The control group was added with the same concentration of IL-10, incubated at 37°C for 1 hour, and washed 3 times with PBS. Add 50 ul mouse anti-IL-10 monoclonal antibody (diluted 1:1000) and incubate at room temperature for 1 hour, wash 3 times as above, add 100 ul alkaline phosphate-labeled goat anti-mouse antibody (diluted 1:10000), incubate at room temperature for 1 hour , wash 3 times as above. Add 100 ul of 2 mg / ml nitrobenzene phosphate ( p -NPP) at room temperature for 10 minutes, adding 100 ul of 0.2M NaOH to each well to terminate the reaction...

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Abstract

The invention belongs to the field of gene engineering and relates to a targeted hIL-10 recombinant protein called as CBD-IL-10 for short and the application of the CBD-IL-10. The recombinant protein is a recombinant protein expressed by a CBD polypeptide combined by human interlenkin10, hIL-10 and collagen specificity after the fusion in a gene level, and a new protein is generated after recombination. The protein has the anti-virus and anti-tumor activities as well as the immune function same as the hIL-10; and more importantly, the CBD-IL-10 can be combined with the collagen specificity in a targeted way during the wound healing process and can prevent and inhibit the formation of scars in the application of the drug for treating the hyperplastic scar and keloid.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the technical field of immune function regulation and gene recombinant expression in the field of immunology. The CBD polypeptide specifically combined with human interleukin 10 (hIL-10) and collagen at the gene level The recombinant protein expressed after fusion and the application of the recombinant protein. Background technique [0002] After the skin injury has healed, the scar continues to proliferate and develop into a hypertrophic scar. Hypertrophic scars protrude from the skin, irregular in shape, uneven, flushed and congested, and firm in texture. There is burning pain and itching, and the symptoms are aggravated when the ambient temperature increases, emotional, or eating spicy and irritating food. Hyperplasia often lasts for several months or years before gradually degenerating, manifested as a reduction in the height of the protrusions, dark...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K38/17A61P17/02
Inventor 石继红胡大海白晓智张战风朱雄翔韩军涛董茂龙汤朝武
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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