CN103589680B - A kind of ocean elastoser myroilysin is in the application prepared in cell-less corium ground substance - Google Patents
A kind of ocean elastoser myroilysin is in the application prepared in cell-less corium ground substance Download PDFInfo
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- CN103589680B CN103589680B CN201310528391.XA CN201310528391A CN103589680B CN 103589680 B CN103589680 B CN 103589680B CN 201310528391 A CN201310528391 A CN 201310528391A CN 103589680 B CN103589680 B CN 103589680B
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Abstract
The present invention relates to a kind of ocean elastoser myroilysin in the application preparing in cell-less corium ground substance, comprise the steps: that (1) is by after the deep-sea bacterium D25 strain fermentation of activation is cultivated, centrifugal, get supernatant liquor, add ammonium sulfate, collecting precipitation, centrifugal after heavy molten, dialysis, again through ion exchange chromatography separation and purification, obtained elastoser myroilysin; (2) elastoser myroilysin is dissolved in Tris-HCl damping fluid, it is made into enzyme liquid; (3) pigskin removing lipid layer and epidermis is immersed in enzyme liquid, take out after process, washing, obtained pigskin cell-less corium ground substance. The efficiency height of the de-cell of marine source elastoser myroilysin of the present invention and removal elastin, speed is fast, and consumption is little; The aperture of the cell-less corium ground substance prepared is big, voidage height, is conducive to entering and breeding of cell in organizational project, and in organizational project, tool has been widely used.
Description
Technical field
The present invention relates to a kind of ocean elastoser myroilysin in the application preparing in cell-less corium ground substance, belong to technical field of biotechnology.
Background technology
Cell-less corium ground substance (acellulardermalmatrix, ADM) is the novel dermal transplantation substitute material in recent years risen, and is prepared from through special processing by allosome or xenogenesis skin. The whole cellular constituent in skin and part soluble proteins is eliminated due to ADM, immunogenicity is extremely low, biocompatibility is extremely good, is used widely at present in skin burn treatment, abdominal-wall defect repairing, endocranium reparation, hard and soft tissue filling and beauty and shaping.
The preparation method of ADM is a lot, and conventional is enzyme method and chemical process. Conventional chemical process NaOH ablation method and NaCl-SDS method etc., ADM prepared by chemical process has often remained more antigenic component in matrix, and biocompatibility is not good enough. Therefore, mostly adopt enzyme method to prepare ADM at present. Enzyme class used is mainly Dispase, trypsinase or compound protease etc. Can not thoroughly remove cell and other antigenic components due to these enzyme classes, enzyme method is general all with the use of stain remover. Such as: Dispase-Triton method, first digest skin sheet with Dispase, then with Triton-100 process washing, remove the cell in tissue and antigenic component; Trypsinase-SDS method, first with tryptic digestion skin sheet, then processes wash-out cell with SDS liquid. Collagen protein in ADM may be had sex change destruction by the stain remover used in these enzyme methods.
Although removing cell in having the enzyme classes such as Dispase, trypsinase and compound protease to prepare for ADM at present, but these enzyme classes are not high to the removal efficiency of cell, often need effect long period or multiple enzyme conbined usage, also to be used detergent-treatment again. Therefore, at present owing to lacking the de-leukoprotease of efficient corium so that the preparation method of ADM is loaded down with trivial details, and efficiency is low.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that the elastoser myroilysin of a kind of marine source is in the application prepared in cell-less corium ground substance.
Technical solution of the present invention is as follows:
The elastoser myroilysin of a kind of marine source is in the application prepared in cell-less corium ground substance.
Technique scheme, comprises the steps:
(1) by through deep-sea bacterium (Myroidesprofundi) the D25 inoculation of activation in liquid fermentation medium after fermentation culture, culture is centrifugal, get supernatant liquor, adding ammonium sulfate to ammonium sulfate saturation ratio in supernatant liquor is 55%, collecting precipitation, after heavy molten, the dialysis of Tris-HCl centrifugal, then through ion exchange chromatography separation and purification, obtained elastoser myroilysin;
(2) the elastoser myroilysin that step (1) is obtained is dissolved in Tris-HCl damping fluid, it is made into the enzyme liquid that enzyme concn is 0.1��0.2mg/mL;
(3) pigskin removing lipid layer and epidermis is immersed in the obtained enzyme liquid of step (2), process and take out after 12��48 hours, be placed in distilled water washing 24 hours, obtained pigskin cell-less corium ground substance.
Preferred according to the present invention, in described step (1), activate as when 13��18 DEG C, cultivating 2��3 days in liquid seed culture medium.
Preferred further according to the present invention, aforesaid liquid seed culture medium component is as follows, is weight part:
Peptone 1 part, yeast powder 0.5 part, artificial seawater 100 parts, pH is 7.5��8.5.
Preferred according to the present invention, in described step (1), liquid fermentation medium component is as follows, is weight part:
Soybean cake powder 2.0 parts, Semen Maydis powder 2.0 parts, 1.0 parts, wheat bran, KH2PO40.03 part, CaCl20.1 part, 100 parts, water.
Preferred according to the present invention, in described step (1), bacterial load is the 1% of liquid fermentation medium volume.
Preferred according to the present invention, in described step (1), fermentation culture conditions is: 13��18 DEG C, 200r/min when shaking culture 2��3 days;
Preferred further according to the present invention, in described step (1), culture temperature is 15 DEG C.
Preferred further according to the present invention, in described step (1), incubation time is 72 hours.
Preferred according to the present invention, in described step (1), ion exchange chromatography is by the gradient elution separation purifying of the NaCl of 0��0.8M with DEAE anion column.
Preferred according to the present invention, in described step (2), Tris-HCl buffer concentration is 50mM, pH9.0.
Preferred according to the present invention, in described step (2), the enzyme concn of proteolytic enzyme myroilysin is 0.1mg/ml.
Preferred according to the present invention, in described step (3), the treatment time is 24 hours.
The excellent results of the present invention:
1, various animal skin can be carried out de-cell process by marine source elastoser myroilysin of the present invention, and this elastoser has and expands but the characteristic of not degrade collagen albumen; The efficiency height of de-cell and removal elastin, speed is fast, and consumption is little; The aperture of the cell-less corium ground substance prepared is big, voidage height, is conducive to entering and breeding of cell in organizational project, and in organizational project, tool has been widely used;
2, ocean elastoser myroilysin of the present invention processes animal skin, owing to not using chemical reagent, the cell-less corium ground substance toxicological harmless of preparation, good biocompatibility, primary formation is not destroyed, and obtained cell-less corium ground substance can be used for the fields such as skin burn treatment, abdominal-wall defect repairing, endocranium reparation, hard and soft tissue filling and beauty and shaping.
Accompanying drawing illustrates:
The electrophoresis result photo of the proteolytic enzyme myroilysin of Fig. 1, SDS-PAGE analytical separation purifying;
Wherein: M:marker, A: proteolytic enzyme myroilysin;
Fig. 2, proteolytic enzyme myroilysin, trypsinase, the photo of SDS and buffer after 37 DEG C of process collagen protein 1.5h;
Wherein: A:0.1mg/ml proteolytic enzyme myroilysin, B:0.25wt% trypsinase, C:0.1wt%SDS, D:Buffer;
Fig. 3, employing proteolytic enzyme myroilysin process the expansion effect figure that cell-less corium ground substance prepared by pigskin;
Wherein: A: pigskin in the proteolytic enzyme myroilysin of 0.1mg/ml, process 5h in 37 DEG C after photo, at B:37 DEG C, 0.1mg/ml proteolytic enzyme myroilysin processes the histogram of pigskin variation in thickness after pigskin different time;
The photo that the hematoxylin-eosin staining method (hematoxylin-eosinstaining, HE dye) of cell of going Fig. 4, proteolytic enzyme myroilysin dyes;
Wherein: A: comparison, B:0.1mg/ml proteolytic enzyme myroilysin processes pigskin 5h, C:0.1mg/ml trypsin treatment pigskin 5h;
Fig. 5, proteolytic enzyme myroilysin remove the masson trichrome stain photo of elastin;
Wherein: A: comparison, B:0.1mg/ml proteolytic enzyme myroilysin processes pigskin 5h, C:0.1mg/ml trypsin treatment pigskin 5h;
Fig. 6, proteolytic enzyme myroilysin are to the scanning electron microscope (SEM) photograph of pigskin modified effect;
Wherein: A: comparison, B:0.1mg/ml proteolytic enzyme myroilysin processes the pigskin after 16h.
Embodiment
Below in conjunction with embodiment, the technical scheme of the present invention is described further, but institute of the present invention protection domain is not limited to this.
Biological material source:
Deep sea cold-adaptive microbe bacterium strain described in embodiment (Myroidesprofundi) D25, purchased from China typical culture collection center, culture presevation CCTCCM2012534.
Embodiment 1:
The preparation method of elastoser myroilysin, specifically comprises the steps:
The preparation of seed
Liquid seed culture medium component is as follows, is weight part:
Peptone 1 part, yeast powder 0.5 part, artificial seawater 100 parts, pH is 7.5��8.5. By sterilizing after said components mixing, cool and get final product.
Being inoculated in liquid seed culture medium by deep sea cold-adaptive microbe bacterium strain MyroidesprofundiD25, under 15 DEG C of conditions, concussion is cultivated 2 days, activated spawn.
Liquid fermenting prepares collagen protein zymin
Liquid fermentation medium component is as follows, is weight part:
Soybean cake powder 2.0 parts, Semen Maydis powder 2.0 parts, 1.0 parts, wheat bran, KH2PO40.03 part, CaCl20.1 part, 100 parts, water. By sterilizing after said components mixing, cooling, obtained liquid fermentation medium.
By above-mentioned steps activate bacterial classification by 1%(v/v) inoculum size be inoculated in liquid fermentation medium, at 15 DEG C, when 200r/min, concussion cultivate 72 hours, obtain fermented liquid.
The separation and purification of elastoser myroilysin
By above-mentioned obtained fermented liquid at 4 DEG C, centrifugal 15min when 10000r/min, collects supernatant liquor, and slowly adding ammonium sulfate to most saturation ratio is 55%, precipitation of spending the night. 4 DEG C will be deposited in, centrifugal 10min when 8500r/min, with 50mM, the heavy molten precipitation of the Tris-HCl of pH9.0, again with centrifugal after the Tris-HCl dialysis of 50mM, pH9.0, supernatant DEAE anion column is by the gradient elution separation purifying of NaCl of 0��0.8M, the proteolytic enzyme myroilysin that wash-out obtains is by the SDS-PAGE electrophoretic analysis purity of 12.5%, and result is as shown in Figure 1.
Embodiment 2: elastoser myroilysin, to the expansion of collagen protein, specifically comprises the steps:
(1) accurately take the 20mg ox tendon soluble collagen protein of I type and it is placed in test tube, add the myroilysin of 5ml0.1mg/ml, after 37 DEG C of concussions hatch 1.5h hour, observe elastoser myroilysin to the expansion of collagen protein.
(2) comparative example 1: accurately take the 20mg ox tendon soluble collagen protein of I type and be placed in test tube, add the SDS of 0.1% respectively, the Buffer of the trypsinase of 0.25% and comparison, observes the expansion to collagen protein after 37 DEG C of concussions hatch 1.5h hour.
Result is analyzed
According to the method in above-described embodiment 2, the 20mg ox tendon soluble collagen protein of I type is through the different reagent (myroilysin of 0.1mg/ml, the SDS of 0.1wt%, the trypsinase of 0.25wt% and Buffer) process after, expand change obvious difference, the expansion effect of myroilysin process is best, the trypsin treatment of 0.25% also have some to expand, but not as myroilysin successful, 0.1% SDS process with control group Buffer process be more or less the same, substantially can't see its expansion effect. In above-mentioned process, the concentration of trypsinase is much larger than myroilysin, but collagen protein expansion effect is far not as myroilysin. These results show, the ability of proteolytic enzyme myroilysin expansion collagen protein very outstanding (as shown in Figure 2).
Embodiment 3: utilize elastoser myroilysin efficiently to prepare the method for cell-less corium ground substance, specifically comprise the steps:
(1) fresh porcine skin is removed fat and epidermal area, it is processed into the specification of 2.5 �� 2 �� 0.1cm.
(2) in 37 DEG C of constant-temperature tables, following process is done with above-mentioned obtained elastoser myroilysin: with 0.2mg/ml lipase treatment 5h, the elastoser Myroilysin of 0.1mg/ml processes the obtained cell-less corium ground substance of 24h, antibacterial with 0.02% sodiumazide or microbiotic in whole treating processes.
(3) cell-less corium ground substance obtained is gone cell by hematoxylin-eosin staining method (hematoxylin-eosinstaining, HE dye) and the observation of Masson trichrome stain and is gone elastin effect.
Result is analyzed
According to the method in above-described embodiment 3, pigskin processes 5h through myroilysin, occurs significantly to expand, and its thickness has obvious increase (as Suo Shi Fig. 3 A, B). Shown by HE dyeing observation, after myroilysin processes 5h, cell in pig dermis is removed totally substantially, and also retains a lot of cell (as shown in Figure 4) in the corium of trypsin treatment, and this shows that myroilysin goes the ability of dermal cell obviously higher than trypsinase conventional at present. Being observed by masson trichrome stain and show, myroilysin does not substantially observe elastin, and also retains some elastins (as shown in Figure 5) in the corium of trypsin treatment after processing 5h in pig dermis. This shows that myroilysin goes the ability of corium elastase obviously higher than trypsinase. Scanning electron microscope sem observe it may be seen that through myroilysin process 5h, it is possible to remove the cell in pig dermis efficiently, and by expansion collagen protein, aperture increased, generation vesicular structure (as shown in Figure 6).
Claims (9)
1. the elastoser myroilysin of a marine source is in the application prepared in cell-less corium ground substance, it is characterised in that, comprise the steps:
(1) by through deep-sea bacterium (Myroidesprofundi) the D25 inoculation of China typical culture collection center preserving number CCTCCM2012534 of activation in liquid fermentation medium after fermentation culture, culture is centrifugal, get supernatant liquor, adding ammonium sulfate to ammonium sulfate saturation ratio in supernatant liquor is 55%, collecting precipitation, after heavy molten, the dialysis of Tris-HCl centrifugal, then through ion exchange chromatography separation and purification, obtained elastoser myroilysin;
(2) the elastoser myroilysin that step (1) is obtained is dissolved in Tris-HCl damping fluid, it is made into the enzyme liquid that enzyme concn is 0.1mg/mL;
(3) pigskin removing lipid layer and epidermis is immersed in the obtained enzyme liquid of step (2), process and take out after 24 hours, be placed in distilled water washing 24 hours, obtained pigskin cell-less corium ground substance.
2. apply as claimed in claim 1, it is characterised in that, in described step (1), activate as when 13��18 DEG C, cultivating 2��3 days in liquid seed culture medium.
3. apply as claimed in claim 2, it is characterised in that, aforesaid liquid seed culture medium component is as follows, is weight part:
Peptone 1 part, yeast powder 0.5 part and artificial seawater 100 parts, pH is 7.5��8.5.
4. apply as claimed in claim 1, it is characterised in that, in described step (1), liquid fermentation medium component is as follows, is weight part:
Soybean cake powder 2.0 parts, Semen Maydis powder 2.0 parts, 1.0 parts, wheat bran, KH2PO40.03 part, CaCl20.1 part and 100 parts, water.
5. apply as claimed in claim 1, it is characterised in that, in described step (1), bacterial load is the 1% of liquid fermentation medium volume.
6. apply as claimed in claim 1, it is characterised in that, in described step (1), fermentation culture conditions is: 13��18 DEG C, 200r/min when shaking culture 2��3 days.
7. apply as claimed in claim 6, it is characterised in that, in described step (1), culture temperature is 15 DEG C; Incubation time is 72 hours.
8. apply as claimed in claim 1, it is characterised in that, in described step (1), ion exchange chromatography is by the gradient elution separation purifying of the NaCl of 0��0.8M with DEAE anion column.
9. apply as claimed in claim 1, it is characterised in that, in described step (2), Tris-HCl buffer concentration is 50mM, pH9.0.
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| CN107213515B (en) * | 2017-06-28 | 2020-05-19 | 山东大学 | Method for efficiently preparing corneal acellular matrix tissue engineering scaffold by enzyme method |
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| CN102218162A (en) * | 2011-05-24 | 2011-10-19 | 山西奥瑞生物材料有限公司 | Preparation method of homologous acellular dermal matrix |
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| CN102218162A (en) * | 2011-05-24 | 2011-10-19 | 山西奥瑞生物材料有限公司 | Preparation method of homologous acellular dermal matrix |
Non-Patent Citations (2)
| Title |
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| Ecological Function of Myroilysin, a Novel Bacterial M12 Metalloprotease with Elastinolytic Activity and a Synergistic Role in Collagen Hydrolysis,in Biodegradation of Deep-Sea High-Molecular-Weight Organic Nitrogen;Xiu-Lan Chen等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20090430;第75卷(第7期);1838-1844 * |
| 低浓度胰蛋白酶消化加反复冻融法制备猪脱细胞真皮基质;谭谦等;《中华烧伤杂志》;20041231;第20卷(第6期);354-356 * |
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