CN1727471B - Application of alcohol soluble film in protein ground substance from source of plants - Google Patents

Abstract

The present invention is a biomedical material and a cytokine carrier using plant-derived prolamin matrix including wheat, barley, rye, oat or corn for cell growth. The protein matrix is obtained by dissolving the plant-derived prolamin with alcohol, and further forming microparticles in the alcohol solution, or drying the above alcohol solution on a plate to form a film. The invention has wide sources of raw materials, low price and low process requirements, and provides a new degradable biological material with low immunogenicity, antibacterial properties and proper mechanical strength.

Description

Uses of plant-derived gliadin matrix films

The present invention is a divisional application titled "plant-derived prolamin matrix, preparation method and application", filed on September 20, 2002, and numbered 02137082.6. the

technical field

The present invention relates to the use of gliadin matrix films utilizing ingredients of vegetable origin. Can be used as biological material. the

Background technique

Glamins are storage proteins in grains. The protein and its resin have toughness, hydrophobicity, degradability, antibacterial property and so on. At present, gliadin has been commercially applied. In medicine, there are adhesives and dressing tools produced by Coleman (2185110, 2298548); taste-masking agents for oral drugs invented by Cuca (WO94/121578) and Meyer et al (5609909); in cosmetics, there is Avalle (EP 882443) Cosmetic powder created, food packaging materials such as Glasser (EP90559) and Wasa et al (WO 99/14076) developed on food; in the field of scientific research, there are microspheres successfully designed by Mathiowitz et al. In addition, the application of zein also involves chewing gum Campbell et al (5164210), paint Reiners et al (3840515), printing ink Leckley (2570353), film Wang et al (EP 886177), degradable plastics (Lai et al , 1997) hair dye Morawsky et al (5518717), fiber optic Freeman (WO 95/01728) and textiles (Cuq et al, 1999) and many other aspects. the

At present, organic synthetic materials have been used in the field of tissue engineering, but their biocompatibility is not ideal; or the degradation rate is not suitable; or after degradation, their products are likely to cause adverse reactions in the body. Such as: PLGA, its acidic degradation product caused the body's inflammatory response [Hollinger et al., 1996]; PLA and PGA also have delayed inflammatory response after implantation in the human body [Bergsma et al., 1995]; hydroxyapatite in It is difficult to be absorbed in the body. Most of some natural biological materials cannot have good biological properties at the same time. At present, such materials as collagen (Nakahara et al. 1989), fibrin (Kawamura et al., 1988), gelatin and chitin have been widely studied as biomaterials, especially in the repair and regeneration of tissues [ Grzesiak et al., 1997; sofia et al., 2001; Pamela et al., 2002; Coombes et al., 2002], but most of them have poor mechanical strength and often have to be modified or form complexes with other materials to Increases mechanical strength and biological stability, but at the same time triggers an immune response in the body. For example, collagen modified with glutaraldehyde caused calcification of heart valves in animals [Schoen et al., 1984]. the

Zein has excellent mechanical strength. After being treated at 120°C for 3 hours, its composition has not changed through circular diphasic dispersion and electrophoresis analysis, which proves that zein has good thermal stability and can withstand Harsh processing technology. In addition, zein has been modified and processed to form a degradable film, which has stronger toughness than direct application [Padgett et al., 1998]. the

Contents of the invention

The purpose of the present invention is to provide a plant-derived gliadin matrix, the main form of the gliadin matrix is microparticles with different particle sizes, and it can also be a film formed by connecting larger particles. the

The object of the present invention is also to provide a preparation method of the above-mentioned plant-derived gliadin matrix. the

Another object of the present invention is to provide a use of the above-mentioned plant-derived gliadin matrix. the

The present invention is a plant-derived gliadin matrix. The main shape of the gliadin matrix is microparticles with different particle sizes, and it can also be a thin film formed by connecting microparticles. the

The microparticles are 50-500nm microparticles, and the thin film is a film in which the 50-2000nm microparticles are linked together. the

The plant-derived gliadin is the gliadin of wheat, barley, rye, oat or corn. the

The alcohol is a C _1-4 fatty alcohol, ethanol is recommended.

The preparation method of the plant-derived prolamin matrix of the present invention is to dissolve the plant-derived prolamin with alcohol, and further form microparticles in the alcohol solution, or dry the above-mentioned alcohol solution on a plate to form microparticles or membranes. the

In the preparation method of the plant-derived prolamin matrix of the present invention, it is preferable to treat the above-mentioned alcohol solution dissolving the plant-derived prolamin with ultrasonic waves to obtain fine particles of uniform size particles, or to obtain uniform-sized particles linked into a piece membrane. Centrifugation can also be used to remove larger particles in the alcohol solution in which the plant-derived gliadin is dissolved, so as to obtain fine particles of uniform size. the

The plant-derived gliadin matrix preparation method of the present invention can adopt the method for adding alcohol once or multiple times to obtain respectively:

Add 20-60% alcohol solution to the plant-derived prolamin, and each milliliter of solution contains 0.5-100 mg of plant-derived prolamin, preferably 0.5-5 mg of plant-derived prolamin per milliliter protein, the solution is dried on a plate or sheet to form microparticles. Or dissolve the plant-derived gliadin in 40-100% alcohol solution, containing 0.5-100mg of plant-derived gliadin per milliliter of solution, plus 1-100 times of 10-40% alcohol, the best Add 2-10 times of 10-40% alcohol to form 50-500nm microparticles. the

Alternatively, the plant-derived gliadin is dissolved in 50-90% ethanol (0.5-100 mg/ml), and 2-5 times of 50-90% ethanol is added. Ultrasonic treatment at room temperature for 2-10 minutes. Centrifuge for 2-10 minutes, and dry to form a film in which microparticles of 50-2000nm are connected in one piece. the

Described centrifuge is 100-800rpm. the

The plate can be a glass plate, a ceramic plate, a stainless steel plate, etc., or a culture plate used in biotechnology, such as a 24-well culture plate, 50-300 μl/well. the

Description of drawings

Fig. 1, the granular membrane matrix of the plant-derived zein of the present invention and the scanning electron micrograph of the granular membrane matrix. the

Fig. 2. The adherence rate of L-02 hepatocytes on different matrices of the present invention for 3 hours. the

Fig. 3. Viability of L-02 hepatocytes on different matrices of the present invention: (A) 3 days, (B) 6 days. the

In accompanying drawing 1, after using ethanol solutions containing different concentrations of plant-derived gliadins to form a matrix, the results are analyzed under a scanning electron microscope with a magnification of 15,000 times. Wherein 1-1～1-4 are scanning electron micrographs of granular matrix, 1-5～1-8 are scanning electron microscopic pictures of granular membrane matrix, and their concentrations are 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). the

In accompanying drawing 2, represent the adherence rate of L-02 hepatocyte on different substrates of the present invention, wherein; Ordinate-relative adherence rate, abscissa-0. Conny cell culture plate, in the substrate preparation process, The concentrations of plant-derived gliadin ethanol are: 1.200ul 0.2% (w/v); 2.200ul 0.4% (w/v); 3.200ul 0.8% (w/v); 4.200ul 1.6% (w/v ). the

In accompanying drawing 3, after adopting the ethanol solutions containing different concentrations of plant-derived gliadins to form substrates, L-02 hepatocytes produced results on different substrates of the present invention after 3 days (3-1) and 6 days (3-2) vitality. Among them, the ordinate-relative activity, the abscissa different concentrations of plant-derived gliadin ethanol solution-1.200ul 0.2% (w/v); 2.200ul 0.4% (w/v); 3.200ul 0.8% (w/v) ; 4.200ul 0.1.6% (w/v). the

In Figures 2 and 3, small particles represent granular substrates, and large particles represent granular membrane substrates. the

The preparation method of the invention is simple and convenient, and the plant-derived gliadin matrix of the invention is a degradable biocompatible material with high mechanical strength suitable for tissue engineering. Specifically, the biocompatibility of plant-derived gliadin is identified by in vitro culture technology, and a new plant-derived biocompatible tissue engineering material that is beneficial to cell attachment, proliferation and differentiation is provided. The material has wide sources of raw materials, low price and low process requirements, and is suitable for tissue engineering materials. the

Detailed ways

The following examples will help to understand the present invention, but do not limit the content of the present invention. the

Example 1:

Zein film preparation and cell adhesion

Dissolve zein in 80% ethanol to make 2, 4, 8, 16 mg/ml, add 4 times the volume of 20% ethanol, ultrasonicate at room temperature for 5 minutes, add to 24-well culture plate (200ul/well for cell culture) or The coverslips (100ul/sheet for surface analysis) were centrifuged at 300rpm for 5 minutes, and dried at room temperature to form a small particle matrix. the

Prepare zein into 2, 4, 8, 16 mg/ml with 70% ethanol solution, add three volumes of 70% ethanol, ultrasonicate at room temperature for 5 minutes, add to 24-well culture plate (200ul/well for cell culture) or cover Slides (100ul/sheet for surface analysis) centrifuged at 300rpm for 5 minutes, dried at room temperature to form large particle matrix membrane

The cover glass loaded with the zein film is placed on the scanning electron microscope sample analysis column, sprayed with gold for 3 minutes, and analyzed under the condition of 4-5KeV and magnified 15000 times (see Figure 1-1, 1 for the small particle matrix. -2, 1-3, 1-4; see Fig. 1-5, 1-6, 1-7, 1-8 for the large granular matrix membrane). the

Hepatocytes were cultured in RPMI-1640 medium containing 10% FBS, 100 g/ml penicillin and 100 mg/ml streptomycin in an incubator at 37 °C with 5% _CO2 . Digest the cells cultured to the full bottle with 0.25% trypsin for 4 minutes, collect the cells, stain and count live cells: mix 1 drop of cell suspension with 2 drops of trypan blue, drop on the counting plate, 2 minutes Post count. Live cells are unstained and dead cells are blue. Adjust the cell density to 2.5×10 ⁵ cells/ml with RPMI-1640. 1ml/well of the cell suspension was inoculated into a 24-well plate containing the above-mentioned zein, and cultured in an incubator at 37°C and 5% CO ₂ . The cell viability was measured by the MTT method at 3 hours, and the adhesion rate was calculated (results shown in Figure 2).

Example 2

Zein film preparation and cell growth

Dissolve zein in 80% ethanol to make 2, 4, 8, 16 mg/ml, add 4 times the volume of 20% ethanol, ultrasonicate at room temperature for 5 minutes, add to 24-well culture plate (200ul/well for cell culture) or The coverslips (100ul/sheet for surface analysis) were centrifuged at 300rpm for 5 minutes, and dried at room temperature to form a small particle matrix. the

Prepare zein into 2, 4, 8, 16 mg/ml with 70% ethanol solution, add three volumes of 70% ethanol, ultrasonicate at room temperature for 5 minutes, add to 24-well culture plate (200ul/well for cell culture) or cover Slides (100ul/sheet for surface analysis) were centrifuged at 300rpm for 5 minutes, and dried at room temperature to form a large particle matrix membrane. the

Hepatocytes were cultured in RPMI-1640 medium containing 10% FBS, 100 g/ml penicillin and 100 mg/ml streptomycin in an incubator at 37 °C with 5% _CO2 . Digest the cells cultured to the full bottle with 0.25% trypsin for 4 minutes, collect the cells, stain and count live cells: mix 1 drop of cell suspension with 2 drops of trypan blue, drop on the counting plate, 2 minutes Post count. Live cells are unstained and dead cells are blue. Adjust the cell density to 2.5×10 ⁵ cells/ml with RPMI-1640. 1ml/well of the cell suspension was inoculated into a 24-well plate containing the above-mentioned zein, and cultured in an incubator at 37°C and 5% CO ₂ . The cell viability was measured by MTT method on day 3 and day 6 (results are shown in Figure 3).

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.

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