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Application of sea elastase myroilysin in preparation of acellular dermal matrix

An elastase and dermal matrix technology, applied in the biological field, can solve the problems of complicated preparation method of ADM, low cell removal efficiency, low efficiency, etc., and achieve the effects of high speed, good biocompatibility and high efficiency

Active Publication Date: 2014-02-19
SHANDONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although enzymes such as Dispase, trypsin, and compound protease are currently used to remove cells in the preparation of ADM, the removal efficiency of these enzymes on cells is not high, and they often need to act for a long time or use multiple enzymes in combination, and then use them again. detergent treatment
Therefore, at present, the preparation method of ADM is cumbersome and inefficient due to the lack of efficient dermal decellularized protease

Method used

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  • Application of sea elastase myroilysin in preparation of acellular dermal matrix
  • Application of sea elastase myroilysin in preparation of acellular dermal matrix
  • Application of sea elastase myroilysin in preparation of acellular dermal matrix

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The preparation method of elastase myroilysin specifically comprises the steps:

[0045] Seed preparation

[0046] The components of the liquid seed culture medium are as follows, all in parts by weight:

[0047] 1 part of peptone, 0.5 part of yeast powder, 100 parts of artificial seawater, pH 7.5-8.5. It is obtained by mixing the above components, sterilizing and cooling.

[0048] The deep-sea psychrotroph Myroides profundi D25 was inoculated in the liquid seed medium, and cultured with shaking at 15°C for 2 days to activate the strain.

[0049] Preparation of Collagenase Preparation by Liquid Fermentation

[0050] The components of the liquid fermentation medium are as follows, all in parts by weight:

[0051] 2.0 parts of bean cake flour, 2.0 parts of corn flour, 1.0 parts of bran, KH 2 PO 4 0.03 parts, CaCl 2 0.1 part, 100 parts of water. The above components are mixed, sterilized and cooled to obtain a liquid fermentation medium.

[0052] Inoculate the bac...

Embodiment 2

[0055] Embodiment 2: the swelling effect of elastase myroilysin on collagen, specifically comprising the following steps:

[0056] (1) Accurately weigh 20 mg of bovine tendon type I insoluble collagen and place it in a test tube, add 5 ml of 0.1 mg / ml myroilysin, and incubate at 37°C for 1.5 hours with shaking to observe the swelling effect of elastase myroilysin on collagen.

[0057] (2) Comparative example 1: Accurately weigh 20mg of bovine tendon type I insoluble collagen and place it in a test tube, add 0.1% SDS, 0.25% trypsin and buffer for control respectively, and incubate at 37°C for 1.5h with shaking Then observe the swelling effect on collagen.

[0058] Result analysis

[0059] According to the method in the above example 2, after 20mg of bovine tendon type I insoluble collagen was treated with different reagents (0.1mg / ml myroilysin, 0.1wt% SDS, 0.25wt% trypsin and Buffer), the difference in swelling changes Obviously, myroilysin treatment has the best expansion ...

Embodiment 3

[0060] Embodiment 3: A method for efficiently preparing acellular dermal matrix by using elastase myroilysin, specifically comprising the following steps:

[0061] (1) Remove fat and epidermis from fresh pigskin, and process it into a size of 2.5×2×0.1cm.

[0062] (2) Use the elastase myroilysin prepared above to do the following treatment in a constant temperature shaker at 37°C: treat with 0.2 mg / ml lipase for 5 hours, and treat with 0.1 mg / ml elastase Myroilysin for 24 hours to obtain an acellular dermal matrix. During the treatment, 0.02% sodium azide or antibiotics were used to inhibit bacteria.

[0063] (3) The obtained acellular dermal matrix was observed by hematoxylin-eosin staining (HE staining) and Masson's trichrome staining to observe the effect of decellularization and deelastin.

[0064] Result analysis

[0065] According to the method in above-mentioned embodiment 3, pigskin is through myroilysin treatment 5h, and obvious expansion takes place, and its thick...

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Abstract

The invention relates to application of sea elastase myroilysin in the preparation of an acellular dermal matrix, and the preparation of the acellular dermal matrix comprises the following steps: (1) performing fermentation culture of an activated myroides profundi D25 strain, centrifugating, taking a supernatant, adding ammonium sulfate, collecting a precipitation, centrifugating after redissolving and dialysis, separating and purifying by ion exchange chromatography to obtain the elastase myroilysin; (2) dissolving the elastase myroilysin in a Tris-HCl buffer solution to prepare an enzyme solution; (3) dipping fatty layer and epidermis removed pigskin in the enzyme solution for processing, and taking out for washing to prepare the pigskin acellular dermal matrix. The sea-derived elastase myroilysin has high acellular and elastase removal efficiency, is fast in speed and small in use amount; and the prepared acellular dermal matrix is large in aperture and high in porosity, facilitates cell entry and proliferation in tissue engineering, and has wide uses in the tissue engineering.

Description

technical field [0001] The invention relates to the application of marine elastase myroilysin in preparing acellular dermal matrix, belonging to the technical field of biotechnology. Background technique [0002] Acellular dermal matrix (acellular dermal matrix, ADM) is a new type of dermal graft substitute material that has emerged in recent years. It is prepared from allogenic or heterogeneous skin after special treatment. Because ADM removes all the cell components and some soluble proteins in the skin, it has extremely low immunogenicity and excellent biocompatibility. It is currently used in skin burn treatment, abdominal wall defect repair, dura mater repair, soft and hard tissue filling and cosmetic plastic surgery. be widely used. [0003] There are many preparation methods of ADM, and enzymatic and chemical methods are commonly used. Commonly used chemical methods such as NaOH ablation method and NaCl-SDS method, etc. ADM prepared by chemical methods often have mo...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12R1/01
Inventor 张玉忠石梅陈秀兰刘德华刘畅解彬彬苏海楠张熙颖王昱凯周百成
Owner SHANDONG UNIV
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