CN1727471B - Application of alcohol soluble film in protein ground substance from source of plants - Google Patents
Application of alcohol soluble film in protein ground substance from source of plants Download PDFInfo
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- CN1727471B CN1727471B CN 200510079324 CN200510079324A CN1727471B CN 1727471 B CN1727471 B CN 1727471B CN 200510079324 CN200510079324 CN 200510079324 CN 200510079324 A CN200510079324 A CN 200510079324A CN 1727471 B CN1727471 B CN 1727471B
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Abstract
An application of vegetative alcohol-soluble protein matrix prepared from wheat, barley, oats, corn, etc in preparing the biomedical material or cell factor carrier for the cell growth is disclosed. Said biomedical material is prepared through dissolving the vegetative alcohol-soluble protein matrix in alcohol, and generating particles in the alcohol solution or drying the alcohol solution on a plate to form a film.
Description
The present invention is that to be called " Phytogenous alcohol-soluble protein stroma, Preparation method and use ", the applying date be that September 20, application number in 2002 are 02137082.6 divide an application to name.
Technical field
The present invention relates to utilize the purposes of the alcohol soluble protein film of plant-sourced composition.Can be used as biomaterial.
Background technology
Prolamine is storage protein in the cereal.This albumen and resin thereof have toughness, hydrophobicity, degradability, germ resistance etc.At present, prolamine has obtained commercial applications.In medical treatment, the tamanori that has Coleman (2185110,2298548) to produce, binder; The odor mask of the oral pharmaceutical of Cuca (WO94/121578) and Meyer et al (5609909) invention; There is the makeup powder of Avalle (EP 882443) initiative the makeup aspect, on the food as the packaging material for food developed of Glasser (EP90559) and Wasa et al (WO 99/14076); Scientific research field has the successful microsphere of Mathiowitz et al design etc.In addition, the application of zein also relates to chewing gum Campbell et al (5164210), paint Reiners et al (3840515), marking ink Leckley (2570353), film Wang et al (EP 886177), degradability plastics (Lai et al, 1997) all many-sides such as hair dye Morawsky et al (5518717), optical fiber Freeman (WO 95/01728) and textiles (Cuq et al, 1999).
At present, the organic synthesis material has been used for field of tissue engineering technology, but their biocompatibilities own are undesirable; Or degradation speed is improper; Or after the degraded, its product causes that easily body produces untoward reaction.As: PLGA, its acid degradation thing have caused the inflammatory reaction [Hollinger et al., 1996] of body; Delayed onset inflammatory reaction [Bergsma et al., 1995] is also arranged behind PLA and the PGA implant into body; Hydroxyapatite is difficult to be absorbed in vivo.And some natural biological material majorities can not have good every biology performance simultaneously.Present this class material such as collagen (Nakahara et al.1989), scleroproein (Kawamura et al., 1988), gelatin and chitin etc. are as biomaterial, particularly reparation and the application aspect the regeneration at tissue is widely studied [Grzesiak et al., 1997; Sofia et al., 2001; Pamela et al., 2002; Coombes et al., 2002], but they self most bad mechanical strength, often must be modified or form mixture with other material, increase physical strength and biologically stable, but this can cause again the immune response of body simultaneously.As with the collagen behind the pentanedial decoration, in animal body, caused valvular calcification [Schoen et al., 1984].
Zein has fabulous physical strength, with 120 ℃ of pyroprocessing after 3 hours, gets its compositions through circle two-phase dispersion and electrophoretic analysis and does not change, and has proved that zein has good thermostability, can bear the complete processing of harshness.In addition, zein has stronger toughness [Padgett et al., 1998] through modifying, being processed to form degradable film than direct application.
Summary of the invention
The purpose of this invention is to provide a kind of Phytogenous alcohol-soluble protein stroma, this alcohol soluble protein Main Morphology is the micropartical of different-grain diameter, also can be the film that is linked to be by larger particle.
Purpose of the present invention also provides a kind of preparation method of above-mentioned Phytogenous alcohol-soluble protein stroma.
Another object of the present invention provides a kind of purposes of above-mentioned Phytogenous alcohol-soluble protein stroma.
The present invention is a kind of Phytogenous alcohol-soluble protein stroma, and the alcohol soluble protein Main Morphology is the micropartical of different-grain diameter, also can be the film that is linked to be by micropartical.
Described micropartical is the micropartical of 50-500nm, and described film is the film that the micropartical of 50-2000nm is unified into a slice.
Described Phytogenous alcohol-soluble protein is the protein,alcohol-soluble of wheat, barley, naked barley, oat or corn.
Described alcohol is C
1-4Fatty Alcohol(C12-C14 and C12-C18), recommend ethanol.
Phytogenous alcohol-soluble protein stroma preparation method of the present invention is to be dissolved with alcohol by described Phytogenous alcohol-soluble protein, and further forms micropartical in alcoholic solution, perhaps with above-mentioned pure lysate, becomes onboard micropartical or film after the drying.
Among the Phytogenous alcohol-soluble protein stroma preparation method of the present invention, preferably with the alcoholic solution ultrasonication of above-mentioned dissolving Phytogenous alcohol-soluble protein, can obtain the micropartical of even size particles, perhaps obtain to be unified into by even size particles the film of a slice.Also can adopt centrifugal method, remove larger particle in the alcoholic solution of above-mentioned dissolving Phytogenous alcohol-soluble protein, make the micropartical that obtains even size particles.
Phytogenous alcohol-soluble protein stroma preparation method of the present invention can adopt following once add alcohol or repeatedly add alcohol method obtain respectively:
The aqueous solution of 20-60% alcohol will be added in the Phytogenous alcohol-soluble protein, the Phytogenous alcohol-soluble protein that contains 0.5-100mg in every ml soln preferably contains the Phytogenous alcohol-soluble protein of 0.5-5mg in every ml soln, this solution is on plate or sheet, drying forms micropartical.Perhaps Phytogenous alcohol-soluble protein is dissolved in the alcoholic solution of 40-100%, the Phytogenous alcohol-soluble protein that contains 0.5-100mg in every ml soln, the alcohol that adds again 1-100 times of 10-40% preferably adds the alcohol of 2-10 times of 10-40%, forms the micropartical of 50-500nm.
Perhaps Phytogenous alcohol-soluble protein is dissolved in the ethanol (0.5-100mg/ml) of 50-90%, adds again the ethanol of 2-5 times of 50-90%.The ultrasonic wave room temperature was processed 2-10 minute.Centrifugal 2-10 minute, dry form the film that micropartical is unified into a slice, be the film that the micropartical of a kind of 50-2000nm joins together.
Described whizzer is 100-800rpm.
Described plate can be sheet glass, ceramic plate, stainless steel plate etc., also can be the used culture plate of biotechnology, such as 24 well culture plates, and 50-300 μ l/ hole.
Description of drawings
The granule type membrane matrix of Fig. 1, plant-derived zein of the present invention and granule type membrane matrix stereoscan photograph.
Fig. 2, L-02 liver cell are in the different substrates of the present invention adherent rate of upper 3 hour.
Fig. 3, L-02 liver cell vigor on different substrates of the present invention: (A) 3 days, (B) 6 days.
In the accompanying drawing 1, employing is carried out analytical results after containing the Phytogenous alcohol-soluble protein ethanolic soln formation matrix of different concns under 15000 times of scanning electronic microscope of amplification.Wherein 1-1～1-4 is granule type matrix stereoscan photograph, 1-5～1-8 is granule type membrane matrix stereoscan photograph, and their concentration is as follows respectively: 1-1:0.2% (w/v), 1-2:0.4% (w/v), 1-3:0.8% (w/v), 1-4:1.6％(w/v)，1-5：02％(w/v)，1-6：0.4％(w/v)，1-7：0.8％(w/v)，1-8：1.6％(w/v)。
In the accompanying drawing 2, expression L-02 liver cell on different substrates of the present invention to adherent rate, wherein; Ordinate zou-relative adherent rate, X-coordinate-0. health Buddhist nun Tissue Culture Plate, in the matrix set-up procedure, the concentration of Phytogenous alcohol-soluble protein ethanol is respectively: 1.200ul 0.2% (w/v); 2.200ul 0.4% (w/v); 3.200ul 0.8% (w/v); 4.200ul 1.6% (w/v).
After adopting the Phytogenous alcohol-soluble protein ethanolic soln contain different concns to form matrix in the accompanying drawing 3, the vigor of L-02 liver cell behind result on the different substrates of the present invention 3 days (3-1) and 6 days (3-2).Ordinate zou-relative reactivity wherein, the Phytogenous alcohol-soluble protein ethanolic soln-1.200ul 0.2% (w/v) of X-coordinate different concns; 2.200ul 0.4% (w/v); 3.200ul 0.8% (w/v); 4.200ul 0.1.6% (w/v).
In the accompanying drawing 2 and 3, small-particle represents granule type matrix, and macrobead represents the granule type membrane matrix.
The present invention not only preparation method is easy, and Phytogenous alcohol-soluble protein stroma of the present invention is a kind of degradable high mechanical strength biocompatible materials of suitable organizational project needs.Specifically, utilize the biocompatibility of Vitro Culture Techniques plant identification source property prolamine, provide a kind of cell that is beneficial to attach the tissue engineering material of the biocompatibility in the novel plant source of propagation and differentiation.The raw material sources of this material are wide, cheap, processing requirement is low, are fit to the used in tissue engineering material.
Embodiment
To help to understand the present invention by following embodiment, but not limit content of the present invention.
Embodiment 1:
Maize alcohol-soluble protein film preparation and cell adhesion
With the ethanol of zein dissolving 80% be mixed with 2,4,8,16mg/ml, the ethanol that adds 4 times of volumes 20%, room temperature ultrasonic was processed 5 minutes, added 24 well culture plates (the 200ul/ hole is for cell cultures) or cover glass (the 100ul/ sheet is for surface analysis) 300rpm centrifugal 5 minutes, drying at room temperature forms small-particle type matrix.
70% ethanolic soln preparation zein becomes 2,4,8,16mg/ml, the ethanol that adds triploid long-pending 70%, room temperature ultrasonic was processed 5 minutes, added 24 well culture plates (the 200ul/ hole is for cell cultures) or cover glass (the 100ul/ sheet is for surface analysis) 300rpm centrifugal 5 minutes, drying at room temperature forms macrobead type matrix membrane
The cover glass that is loaded with maize alcohol-soluble protein film places on the scanning electronic microscope sample analysis post, and metal spraying 3 minutes is under the 4-5KeV condition, amplify 15000 times and analyze that (small-particle type matrix is seen Fig. 1-1,1-2,1-3,1-4; Macrobead type matrix membrane is seen Fig. 1-5,1-6,1-7,1-8).
With containing 10%FBS, the RPMI-1640 substratum of 100g/ml penicillin and 100mg/ml Streptomycin sulphate, at 37 ℃, 5%CO
2Incubator in culture hepatocyte.With the cell that is cultured to full bottle with 0.25% tryptic digestion 4 minutes, collecting cell, viable cell dyeing and numeration: 1 cell suspension is mixed with 2 trypan blues, drip on the plate that counts, count after 2 minutes.Uncoloured is viable cell, is the blue dead cell that is.Transfer cell density to 2.5 * 10 with RPMI-1640
5Individual cell/ml.The cell suspension inoculation in 1ml/ hole in 24 orifice plates that contain above-mentioned zein, in 37 ℃, 5%CO
2Incubator in cultivate.Measure cell viability in 3 hours mtt assay, calculate adherence rate (the results are shown in Figure 2).
Maize alcohol-soluble protein film preparation and Growth of Cells
With the ethanol of zein dissolving 80% be mixed with 2,4,8,16mg/ml, the ethanol that adds 4 times of volumes 20%, room temperature ultrasonic was processed 5 minutes, added 24 well culture plates (the 200ul/ hole is for cell cultures) or cover glass (the 100ul/ sheet is for surface analysis) 300rpm centrifugal 5 minutes, drying at room temperature forms small-particle type matrix.
70% ethanolic soln preparation zein becomes 2,4,8,16mg/ml, the ethanol that adds triploid long-pending 70%, room temperature ultrasonic was processed 5 minutes, added 24 well culture plates (the 200ul/ hole is for cell cultures) or cover glass (the 100ul/ sheet is for surface analysis) 300rpm centrifugal 5 minutes, drying at room temperature forms macrobead type matrix membrane.
With containing 10%FBS, the RPMI-1640 substratum of 100g/ml penicillin and 100mg/ml Streptomycin sulphate, at 37 ℃, 5%CO
2Incubator in culture hepatocyte.With the cell that is cultured to full bottle with 0.25% tryptic digestion 4 minutes, collecting cell, viable cell dyeing and numeration: 1 cell suspension is mixed with 2 trypan blues, drip on the plate that counts, count after 2 minutes.Uncoloured is viable cell, is the blue dead cell that is.Transfer cell density to 2.5 * 10 with RPMI-1640
5Individual cell/ml.The cell suspension inoculation in 1ml/ hole in 24 orifice plates that contain above-mentioned zein, in 37 ℃, 5%CO
2Incubator in cultivate.Measured cell viability (the results are shown in Figure 3) in 3 days, 6 days with mtt assay.
Claims (2)
1. the purposes of an alcohol soluble film in protein ground substance from source of plants, this film is the film that the micropartical of 50-2000nm joins together, and it is characterized in that the bio-medical material as Growth of Cells, described film is standby in order to the below legal system:
Phytogenous alcohol-soluble protein is dissolved in 70% the ethanol, contains the Phytogenous alcohol-soluble protein of 2-16mg in every ml soln, add again 3 times 70% ethanol; The ultrasonic wave room temperature was processed 2-10 minute, and centrifugal 2-10 minute, the film that joins together of the dry micropartical that forms 50-2000nm onboard;
Described Phytogenous alcohol-soluble protein is zein.
2. the purposes of alcohol soluble film in protein ground substance from source of plants according to claim 1 is characterized in that described plate is the used culture plate of biotechnology.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 200510079324 CN1727471B (en) | 2002-09-20 | 2002-09-20 | Application of alcohol soluble film in protein ground substance from source of plants |
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| CN 200510079324 CN1727471B (en) | 2002-09-20 | 2002-09-20 | Application of alcohol soluble film in protein ground substance from source of plants |
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| CN 02137082 Division CN1401659B (en) | 2002-09-20 | 2002-09-20 | Phytogenous alcohol-soluble protein stroma preparing method |
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| CN1727471A CN1727471A (en) | 2006-02-01 |
| CN1727471B true CN1727471B (en) | 2013-03-06 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10228307B2 (en) | 2015-09-29 | 2019-03-12 | Biofunctions, Inc. | Dissolvable sample collection matrices and methods of using the same |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102516778A (en) * | 2011-11-29 | 2012-06-27 | 上海交通大学 | Cereal protein-based micro-carrier for large-scale culture of cells, and preparation method and application of micro-carrier |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991006286A1 (en) * | 1989-11-06 | 1991-05-16 | Enzytech, Inc. | Method for producing protein microspheres |
| EP0434760B1 (en) * | 1988-09-19 | 1994-01-12 | Opta Food Ingredients, Inc. | Hydrophobic protein microparticles and preparation thereof |
| EP0585688A2 (en) * | 1992-08-13 | 1994-03-09 | Euro-Celtique S.A. | Aqueous dispersions of zein and controlled release coatings derived therefrom |
-
2002
- 2002-09-20 CN CN 200510079324 patent/CN1727471B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0434760B1 (en) * | 1988-09-19 | 1994-01-12 | Opta Food Ingredients, Inc. | Hydrophobic protein microparticles and preparation thereof |
| WO1991006286A1 (en) * | 1989-11-06 | 1991-05-16 | Enzytech, Inc. | Method for producing protein microspheres |
| EP0585688A2 (en) * | 1992-08-13 | 1994-03-09 | Euro-Celtique S.A. | Aqueous dispersions of zein and controlled release coatings derived therefrom |
Non-Patent Citations (1)
| Title |
|---|
| T. PADGETT, et al..EFFECT OF LAURIC ACID ADDITION ON THE ANTIMICROBIAL EFFICACY AND WATER PERMEABILITY OF CORN ZEIN FILMS CONTAINING NISIN..Journal of Food Processing and Preservation.24.2001,24423-432. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10228307B2 (en) | 2015-09-29 | 2019-03-12 | Biofunctions, Inc. | Dissolvable sample collection matrices and methods of using the same |
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