Rapid hemostatic product for clinical operation wounds, as well as preparation method and application of rapid hemostatic product

A technique for surgical wounds and products, which is applied in the field of hemostatic compositions containing microbial transglutaminase and their preparation, and can solve problems such as insufficient activity and the like

Active Publication Date: 2014-05-28
李肯
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0035] Therefore, the idea of ​​the present invention is to overcome the shortcomings of the existing transglutaminase, which is insufficient in activity at normal temperature, or requires auxiliary agent activation (i.e., activating the enzyme), and provides a novel transglutaminase as the main active ingredient, Rapid hemostatic materials and composite hemostatic products for hemostasis in clinical surgical wounds

Method used

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  • Rapid hemostatic product for clinical operation wounds, as well as preparation method and application of rapid hemostatic product
  • Rapid hemostatic product for clinical operation wounds, as well as preparation method and application of rapid hemostatic product
  • Rapid hemostatic product for clinical operation wounds, as well as preparation method and application of rapid hemostatic product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Enzyme Activity Determination

[0083] 1) Preparation of reagents for measuring enzyme activity

[0084] Solution A (0.5L): Add 0.015mol (5.06g) of substrate Na-CBZ-Gln-Gly, 0.05mol (3.475g) of hydroxylamine hydrochloride, 0.005mol (1.536g) of reduced glutathione to 400ml of distilled water in a beaker After stirring with a magnetic stirrer for 20 minutes, add 0.1mol (12.11g) Tris, adjust the pH to 6.0 with 6mol / L (or 1mol / L) hydrochloric acid, move the solution to a 500ml volumetric flask, and wash the beaker with distilled water for 3 Pour it into a volumetric flask once, and set the volume to 500ml.

[0085] Liquid B: 3mol / L HCl, 12% trichloroacetic acid (W / V), 5% ferric chloride (W / V, dissolved in 0.1mol / L hydrochloric acid, then filtered) at a volume ratio of 1:1:1 mix.

[0086] 2) Determination of enzyme activity

[0087]The actinomycetes fermentation broth was filtered, and the filtrate was diluted 5 times. Experimental group: Take 0.2ml of diluted...

Embodiment 2

[0088] The mutagenesis of embodiment 2 wild strains

[0089] The wild-type mTG strain is Streptomyces mobaraensis (such as the strain ATCC No. 29032 of the American ATCC or the strain ATCC No. 27441 of the American ATCC, or the strains of the China Microorganism Conservation Center such as CGMCC No. 4.1719 and CGMCC No. 4.5591).

[0090] Medium configuration: Gaoshi No. 1 medium: soluble starch 20g / L, KNO 3 1g / L, MgSO 4 ·7H 2 O0.5g / L, K 2 HPO 4 ·3H 2 O0.5g / L, NaCl0.5g / L, FeSO 4 ·7H 2 O0.01g / L, agar 20g / L, pH7.2-7.4. Fermentation medium: glycerin 20g / L, yeast extract 6g / L, fish meal peptone 25g / L, MgSO 4 ·7H 2 O2g / L, K 2 HPO 4 ·3H 2 O2g / L, pH7.4.

[0091] Add 10ml of cold sterile water to Gao's No. 1 medium, use an inoculation needle to fully scrape the surface hyphae, break up the spores, and filter with sterile filter paper.

[0092] The operation is carried out under red light or dark conditions, and the ultraviolet light is turned on 0.5 hours in advance to st...

Embodiment 3

[0093] The acquisition of embodiment 3 mutant mTG gene

[0094] Genome extraction: Inoculate the fresh mycelium of the mutant strain with the highest enzyme activity in liquid culture in Example 2 into fresh medium, and cultivate for about 24 hours; collect 10 ml of thalline by centrifugation; fresh thalline in a mortar (-20 ℃ (pre-cooled), grind repeatedly with liquid nitrogen until fine powder, quickly and evenly distribute into two 1.5mL centrifuge tubes; add 550μL of TE buffer, add 30μL of 20% SDS solution preheated at 65℃, and vortex for 5 seconds , add 20 μL of 20 mg / mL proteinase K, mix gently, and incubate at 37°C for 1 hour; add an equal volume of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) for extraction, mix by inverting slightly, and centrifuge at 10,000 rpm for 10 minutes ; Carefully draw the supernatant into a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (24:1) for extraction, centrifuge at 10,000 rpm for 5 minutes; absorb...

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Abstract

The invention discloses a rapid hemostatic product for clinical operation wounds, as well as a preparation method and application of the rapid hemostatic product. The rapid hemostatic product comprises a matrix containing a microbial transglutaminase (mT6), wherein the amino acid sequence of the transglutaminase is the protein sequence coded by the gene shown in SEQ ID NO.: 3, or the protein sequence shown in SEQ ID NO.: 4. Specifically, the rapid hemostatic product is selected from a hemostatic, a hemostatic device, a hemostatic first-aid kit or a common hemostatic product; the matrix adopts a fluid or semisolid form, which can be absorbed by a human body, or a plastic gel or colloid form. The rapid hemostatic product contains the microbial transglutaminase and gelatin. The rapid hemostatic product has the advantages of rapid hemostatic property, convenience for use, prevention on organ adhesion, promotion on wound healing, low toxic and side effects, complete absorption and the like. The rapid hemostatic product can be used as a medical hemostatic material or product for clinical operation wounds, can stop bleeding rapidly, prevents organ adhesion, is easy to absorb, has little possibility of spreading diseases, and is low in cost.

Description

technical field [0001] The invention belongs to the field of rapid hemostatic products used for clinical surgical wounds, and in particular relates to a hemostatic composition containing microbial transglutaminase (mTG) and its preparation method and application. Background technique [0002] Controlling bleeding (or bleeding) is an important step in first aid and trauma care. During the operation, medical accidents that cause death due to excessive bleeding occur from time to time. In general, in clinical operations, hemostasis is often required for cutting tissues or blood vessels. Common hemostasis methods mainly include physical hemostasis and drug hemostasis. Among them, physical hemostasis mainly uses mechanical external force to clamp, ligate, suture and compress blood vessels to stop bleeding, while drug hemostasis mainly uses rapid hemostatic agents to stop bleeding. [0003] Clinically, rapid hemostatic agents are mainly divided into three types: [0004] 1. Sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54A61L15/38A61L15/44
CPCA61K38/00A61L15/44A61L29/048C12N9/1044C12Y203/02013
Inventor 易正芳丛晓楠吕方裴正培金明飞刘明耀
Owner 李肯
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