CN1727471A - The purposes of alcohol soluble film in protein ground substance from source of plants - Google Patents
The purposes of alcohol soluble film in protein ground substance from source of plants Download PDFInfo
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- CN1727471A CN1727471A CN 200510079324 CN200510079324A CN1727471A CN 1727471 A CN1727471 A CN 1727471A CN 200510079324 CN200510079324 CN 200510079324 CN 200510079324 A CN200510079324 A CN 200510079324A CN 1727471 A CN1727471 A CN 1727471A
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Abstract
The present invention is that a kind of Phytogenous alcohol-soluble protein stroma of wheat, barley, naked barley, oat or corn that will comprise is used for the bio-medical material and the cytokine carrier of cell growth.This albumen substrate system is dissolved with alcohol by described Phytogenous alcohol-soluble protein, and further forms micropartical in alcoholic solution, perhaps with above-mentioned pure lysate, and dry onboard back film forming.Raw material sources of the present invention are wide, cheap, processing requirement is low, and a kind of new degradable is provided, reduced immunogenicity, and germ resistance has suitable physical strength biomaterial.
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 liquor-saturated molten albumen substrate film of plant-sourced composition, can be used as biomaterial.
Background technology
Prolamine is a storage protein in the cereal.This albumen and resin thereof have toughness, hydrophobicity, degradability, germ resistance etc.At present, zein 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 (EP 90559) 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) hair dye Morawsky et al (5518717), optical fiber Freeman (WO 95/01728) and textiles all many-sides such as (Cuq et al, 1999).
At present, the organic synthesis material is used for field of tissue engineering technology, but they or bad mechanical property; Or absorption rate is improper; Or be not easy degraded; 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; Tardy property inflammatory reaction [Bergsma et al., 1995] is also arranged behind PLA and the PGA implant into body; HA is difficult to be absorbed in vivo.And some natural biological material majorities do not have excellent mechanical intensity, can not provide good environment [Hubbell, 1995] for transplanted cells 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 [Grzesiaket 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 phase has not caused the immune response of body again 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 chromatic 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 purposes that the purpose of this invention is to provide a kind of alcohol soluble film in protein ground substance from source of plants, it is the biocompatibility that utilizes vitro culture technical evaluation Phytogenous alcohol-soluble protein, provide a kind of cell that is beneficial to attach the bio-medical material of the biocompatibility in the novel plant source of propagation and differentiation.
Alcohol soluble film in protein ground substance from source of plants of the present invention is a kind of degradable biological compatibility material of suitable cell growth needs.Bio-medical material and cytokine carrier as the cell growth.Described cytokine is the various types of cells factor that irritation cell is adherent, grow and break up.
Alcohol soluble film in protein ground substance from source of plants form of the present invention is the film that micropartical is unified into a slice.
Described micropartical is the micropartical of 50-2000nm.
Described Phytogenous alcohol-soluble protein is the protein,alcohol-soluble of rice, wheat, barley, naked barley, oat or corn.This is a zein.
Described alcohol is the alcohol of C1-4, recommends ethanol.
The alcoholic solution method for manufacturing thin film of Phytogenous alcohol-soluble protein of the present invention can adopt once to add alcohol or repeatedly add pure method and make respectively:
The aqueous solution of 20-60% alcohol will be added in the Phytogenous alcohol-soluble protein, the Phytogenous alcohol-soluble protein that contains 0.5-10mg in every ml soln contains the Phytogenous alcohol-soluble protein of 0.5-5mg in preferably every ml soln, this solution is on plate or sheet, drying forms circular micropartical.Perhaps Phytogenous alcohol-soluble protein is dissolved in the alcoholic solution of 40-100%, contains the Phytogenous alcohol-soluble protein of 0.5-20mg in every ml soln, add the alcohol of 1-100 times of 10-40% again, preferably add the alcohol of 2-10 times of 10-40%, form the 50-500nm micropartical.
Phytogenous alcohol-soluble protein is dissolved in the ethanol (0.5-100mg/ml) of 50-90%, adds the ethanol of 2-5 times of 50-90% again.The ultrasonic wave room temperature was handled 2-10 minute.Centrifugal 2-10 minute, dry formation micropartical was unified into the film of a slice, is a kind of film that is joined together by the micropartical of 50-2000nm.
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, as 24 well culture plates, and 50-300 μ l/ hole.
Description of drawings
Fig. 1, L-02 liver cell are in the different substrates of the present invention adherent rate of last 3 hour.
Fig. 2, L-02 liver cell vigor on different substrates of the present invention: (A) 3 days, (B) 6 days.
Fig. 3, L-02 liver cell vigor on different substrates of the present invention: (a) Tissue Culture Plate, (b) granule type matrix, (c) granule type membrane matrix.
Accompanying drawing 1 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 in 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 that contains different concns to form matrix in the accompanying drawing 2, the vigor of L-02 liver cell behind different substrates of the present invention last 3 day (3-1) and 6 days (3-2).Ordinate zou-relative reactivity wherein, the 200ul 0.2% (w/v) of the Phytogenous alcohol-soluble protein ethanolic soln of X-coordinate different concns-1.; 2. 200ul 0.4% (w/v); 3. 200ul 0.8% (w/v); 4. 200ul; 0. 1.6% (w/v).
Among attached Fig. 1 and 2, small-particle is represented granule type matrix, and macrobead is represented the granule type membrane matrix.
Phytogenous alcohol-soluble protein stroma of the present invention is a kind of degradable biological compatibility material of suitable cell growth needs.Specifically, utilize the biocompatibility of vitro culture technical evaluation Phytogenous alcohol-soluble protein, provide a kind of cell that is beneficial to attach the bio-medical 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.
Embodiment
To help to understand the present invention by following embodiment, but not limit content of the present invention.
Embodiment 1:
Zein film preparation and cell adhesion
Zein is dissolved in 80% the ethanol that compound concentration becomes 2,4,8,16mg/ml, the ethanol that adds 4 times of volumes 20%, room temperature ultrasonic was handled 5 minutes, added 24 well culture plates (the 200ul/ hole is used for cell cultures) or cover glass (the 100ul/ sheet is used for surface analysis) 300rpm centrifugal 5 minutes, drying at room temperature forms small-particle type matrix membrane.
Zein is dissolved in 70% the ethanol that compound concentration becomes 2,4,8,16mg/ml, the ethanol that adds triploid long-pending 70%, room temperature ultrasonic was handled 5 minutes, added 24 well culture plates (the 200ul/ hole is used for cell cultures) or cover glass (the 100ul/ sheet is used 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 100 μ g/ml penicillin and 100mg/ml Streptomycin sulphate at 37 ℃, is cultivated liver cell in the incubator of 5%CO2.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 * 105 cell/ml with RPMI-1640.The cell suspension inoculation in 1ml/ hole in 37 ℃, is cultivated in the incubator of 5%CO2 in 24 orifice plates that contain above-mentioned zein matrix membrane.Measure cell viability in 3 hours mtt assay, calculate adherence rate (the results are shown in Figure 1).
Embodiment 2
Maize alcohol-soluble protein film preparation and cell growth
Zein is dissolved in is mixed with 2,4,8 in 80% the ethanol, 16mg/ml, the ethanol that adds 4 times of volumes 20%, room temperature ultrasonic was handled 5 minutes, added 24 well culture plates (the 200ul/ hole is used for cell cultures) or cover glass (the 100ul/ sheet is used for surface analysis) 300rpm centrifugal 5 minutes, drying at room temperature forms small-particle type matrix membrane.
Zein is dissolved in is mixed with 2,4,8 in 70% the ethanol, 16mg/ml, the ethanol that adds triploid long-pending 70%, room temperature ultrasonic was handled 5 minutes, added 24 well culture plates (the 200ul/ hole is used for cell cultures) or cover glass (the 100ul/ sheet is used 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 100 μ g/ml penicillin and 100mg/ml Streptomycin sulphate at 37 ℃, is cultivated liver cell in the incubator of 5%CO2.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 * 105 cell/ml with RPMI-1640.The cell suspension inoculation in 1ml/ hole in 37 ℃, is cultivated in the incubator of 5%CO2 in 24 orifice plates that contain above-mentioned zein matrix membrane.Measured cell viability (the results are shown in Figure 2) in 3 days, 6 days with mtt assay.
Claims (7)
- The purposes of 1 one kinds of 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 bio-medical material and cytokine carrier as the cell growth.
- The purposes of 2 thin alcohol soluble film in protein ground substance from source of plants according to claim 1 is characterized in that described cytokine is the various types of cells factor that irritation cell is adherent, grow and break up.
- The purposes of 3 plant source protein matrix films according to claim 1 is characterized in that described plant source protein is the protein,alcohol-soluble of wheat, barley, naked barley, oat or corn.
- The purposes of 3 plant source protein matrix films according to claim 1 is characterized in that described Phytogenous alcohol-soluble protein film is molten with the described plant-derived alcohol of alcohol dissolving, and form micropartical film forming again in alcoholic solution; Perhaps with above-mentioned pure lysate, dry onboard back film forming.
- The purposes of 4 plant source protein matrix films according to claim 3 is characterized in that the alcoholic solution of above-mentioned dissolving Phytogenous alcohol-soluble protein with ultrasonication or centrifugation method.
- The purposes of 5 plant source protein matrix films according to claim 3 is characterized in that the following method that adds alcohol or repeatedly add alcohol:1) aqueous solution that will be in the Phytogenous alcohol-soluble protein adds 20-60% alcohol contains the Phytogenous alcohol-soluble protein of 0.5-100mg in every ml soln, this solution on plate or sheet, drying;2) or Phytogenous alcohol-soluble protein is dissolved in the alcoholic solution of 40-100%, contain the Phytogenous alcohol-soluble protein of 0.5-100mg in every ml soln, add the alcohol of 1-100 times of 10-40% again, form the circular micropartical of 50-500nm, drying; Perhaps;3) Phytogenous alcohol-soluble protein is dissolved in the ethanol (0.5-100mg/ml) of 50-90%, adds the ethanol of 2-5 times of 50-90% again, the ultrasonic wave room temperature was handled 2-10 minute, and centrifugal 2-10 minute, drying.
- The purposes of the 6 plant source protein matrix films of stating according to claim 5 is characterized in that described plate is the used culture plate of sheet glass, ceramic plate, stainless steel plate or biotechnology.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 true CN1727471A (en) | 2006-02-01 |
| CN1727471B CN1727471B (en) | 2013-03-06 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013078988A1 (en) * | 2011-11-29 | 2013-06-06 | 上海交通大学 | Micro-carrier based on grain protein and used for large-scale cell culture, preparation method therefor and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017058520A1 (en) | 2015-09-29 | 2017-04-06 | Biofunctions, Inc. | Dissolvable sample collection matrices and methods of using the same |
Family Cites Families (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 |
| ES2084712T3 (en) * | 1989-11-06 | 1996-05-16 | Alkermes Inc | METHOD FOR PRODUCING PROTEIN MICROSPHERES. |
| US5324351A (en) * | 1992-08-13 | 1994-06-28 | Euroceltique | Aqueous dispersions of zein and preparation thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013078988A1 (en) * | 2011-11-29 | 2013-06-06 | 上海交通大学 | Micro-carrier based on grain protein and used for large-scale cell culture, preparation method therefor and use thereof |
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| CN1727471B (en) | 2013-03-06 |
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Granted publication date: 20130306 Termination date: 20170920 |