High-simulation multifunctional artificial simulation sputum and preparation method thereof

Abstract

The invention discloses a high-simulation multifunctional artificial simulated sputum and a preparation method thereof. The artificial simulated sputum is characterized by comprising the following components: 6-8% (w / v) of pig mucin; 0.4% (w / v) of low molecular weight salmon sperm DNA; 0.0059% (w / v) of diethylene triamine pentaacetic acid; 5% (w / v) of sodium chloride; 2.2% (w / v) of potassium chloride; 1.81% (w / v) of tris (hydroxymethyl) aminomethane; 0.1% (w / v) of cystine; 5 ml of egg yolk emulsion; nutrition and growth factors; a microbial simulation component; and a stability and protective agent; the simulated sputum prepared by the method is close to real sputum in component, physicochemical property and biological activity, and has good stability and repeatability.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically relates to a simulated sputum that combines multiple biological components and has high simulation and a preparation method thereof. The simulated sputum can be used in multiple fields such as scientific research, disease diagnosis, drug screening, and teaching and training. Background Art

[0002] Simulated sputum is an artificially synthesized substance used to simulate real sputum. It is often used in medical research, teaching, experiments, and testing of medical devices.

[0003] Simulated sputum can be made into different specifications and forms as needed. For example, some products exist in liquid form, while others may be provided in the form of culture medium. For example, artificial simulated synthetic sputum (BZ296) and artificial simulated synthetic sputum culture medium (ASM) BZ316 on the market are two different forms of simulated sputum products. These products usually have physical and chemical properties similar to real sputum in order to better simulate the real environment.

[0004] In terms of composition, simulated sputum may contain a variety of ingredients similar to real sputum, such as mucin, electrolytes, amino acids, etc., to simulate the viscosity, pH value, ionic strength and other characteristics of real sputum. In addition, some advanced simulated sputum may also add microbial components, such as bacteria and viruses, to more comprehensively simulate the biologically active components in real sputum. There are also many ways to make simulated sputum.

[0005] In general, simulated sputum, as an important experimental tool, plays an important role in medical research and teaching. By simulating the various characteristics of real sputum, it can help researchers better understand and study the physiological and pathological processes of sputum, and provide strong support for the diagnosis and treatment of related diseases. Traditional simulated sputum often contains only a few components, which makes it difficult to fully simulate the complexity and biological activity of real sputum. Summary of the invention

[0006] The purpose of the embodiments of the present application is to provide a highly simulated multifunctional artificial simulated sputum and a preparation method thereof and a preparation method thereof, so as to solve the problems existing in the related art.

[0007] To achieve the above object, the present invention provides a highly simulated multifunctional artificial simulated sputum and a preparation method thereof, characterized in that the artificial simulated sputum comprises the following components: porcine mucin: 6%-8% (w / v);

[0008] Low molecular weight salmon sperm DNA: 0.2-0.5% (w / v);

[0009] Diethylenetriaminepentaacetic acid: 0.002-0.008% (w / v);

[0010] Sodium chloride: 2-10% (w / v);

[0011] Potassium chloride: 1-5% (w / v);

[0012] Tris(hydroxymethyl)aminomethane: 1-3% (w / v);

[0013] Cystine: 0.05-0.2% (w / v);

[0014] Egg yolk emulsion: 2-8ml;

[0015] Nutrition and growth factors;

[0016] Microbial mimic ingredients;

[0017] and stabilizers and protectants.

[0018] Specifically, the artificial simulated sputum of the present invention includes the following components:

[0019] Porcine mucin: 6%-8% (w / v);

[0020] Low molecular weight salmon sperm DNA: 0.4% (w / v);

[0021] Diethylenetriaminepentaacetic acid: 0.0059% (w / v);

[0022] Sodium chloride: 5% (w / v);

[0023] Potassium chloride: 2.2% (w / v);

[0024] Tris(hydroxymethyl)aminomethane: 1.81% (w / v);

[0025] Cystine: 0.1% (w / v);

[0026] Egg yolk lotion: 5ml;

[0027] Nutrition and growth factors;

[0028] Microbial mimic ingredients;

[0029] and stabilizers and protectants;

[0030] Nutrition and growth factors include the following components:

[0031] Essential and non-essential L-amino acids: 0.25%;

[0032] L-cysteine: 0.25% (w / v), dissolved in 0.5 M potassium hydroxide solution;

[0033] L-Tyrosine: 0.25% (w / v), dissolved in sterile water.

[0034] Microbial simulation components include the following:

[0035] Cultured THP-1 cells: moderate amount, simulating immune cells in sputum

[0036] BCG-Pasteur bacteria: appropriate amount, simulating the bacterial components in sputum.

[0037] Stabilizers and protectants include the following components:

[0038] Glycerol: 1%-3% (v / v);

[0039] Catalytic enzyme (such as catalase): 0.01%-0.1% (w / v).

[0040] A pH adjustment was also performed by adjusting the pH to 7.0 using NaOH or HCl.

[0041] The highly simulated multifunctional artificial sputum used has the following preparation method:

[0042] 1) Mix 0.24 g 7H9 powder and 0.05 g Casitone in a 100 ml glass flask and add 0.2 g (0.4% w / v) salmon sperm DNA;

[0043] 2) Dissolve the above mixture in 49 ml of distilled water, then add 1 ml of glycerol and stir until completely dissolved;

[0044] 3) Use sodium hydroxide to adjust the pH to 7.0;

[0045] 4) Autoclave at 121°C for 20 minutes;

[0046] 5) After autoclaving, add 6 g of porcine mucin (Type II) and continue stirring until the mucin is completely dissolved;

[0047] 6) pasteurizing the resulting mixture in a preheated water bath at 80°C for 45 minutes;

[0048] 7) After cooling to room temperature, add 5 ml of egg yolk emulsion and appropriate amounts of cultured THP-1 cells and BCG-Pasteur bacteria;

[0049] 8) Mix gently to avoid disrupting the cells, and then store the final mixture at 4°C to maintain stability and extend shelf life.

[0050] The above technical solution has the following beneficial effects:

[0051] The mucin, DNA, amino acids and electrolytes from various literatures are combined to optimize the sputum simulation effect.

[0052] THP-1 cells and BCG-Pasteur bacilli were introduced to simulate the immune cells and bacteria in sputum, thus improving the biosimulation of simulated sputum.

[0053] By adding glycerol and catalytic enzyme, the stability and antioxidant capacity of the simulated sputum are improved, making it suitable for long-term storage and transportation.

[0054] The simulated sputum prepared by this method is close to real sputum in composition, physicochemical properties and biological activity, and has good stability and repeatability. DETAILED DESCRIPTION

[0056] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] Patent implementation method: Preparation and application of highly realistic simulated sputum.

[0058] To achieve the above object, the present invention adopts the following technical solutions:

[0059] Artificial simulated sputum includes the following components: porcine mucin: 6%-8% (w / v);

[0060] Low molecular weight salmon sperm DNA: 0.2-0.5% (w / v);

[0061] Diethylenetriaminepentaacetic acid: 0.002-0.008% (w / v);

[0062] Sodium chloride: 2-10% (w / v);

[0063] Potassium chloride: 1-5% (w / v);

[0064] Tris(hydroxymethyl)aminomethane: 1-3% (w / v);

[0065] Cystine: 0.05-0.2% (w / v);

[0066] Egg yolk emulsion: 2-8ml;

[0067] Nutrition and growth factors;

[0068] Microbial mimic ingredients;

[0069] and stabilizers and protectants.

[0070] Specifically, the artificial simulated sputum of the present invention includes the following components:

[0071] Recipe composition:

[0072] 1. Basic ingredients:

[0073] . Porcine Mucin (Type II): 6%-8% (w / v)

[0074] ο Low molecular weight salmon sperm DNA: 0.4% (w / v)

[0075] οDiethylenetriaminepentaacetic acid (DTPA): 0.0059% (w / v)

[0076] Sodium chloride (NaCl): 5% (w / v)

[0077] Potassium chloride (KCl): 2.2% (w / v)

[0078] Tris Base: 1.81% (w / v)

[0079] οCasitone: 0.1% (w / v)

[0080] οEgg yolk lotion: 5ml

[0081] 2. Nutrition and growth factors:

[0082] Essential and non-essential L-amino acids (except L-tyrosine and L-cysteine): 0.25% (w / v)

[0083] L-cysteine: 0.25% (w / v), dissolved in 0.5M potassium hydroxide solution

[0084] L-Tyrosine: 0.25% (w / v), dissolved in sterile water

[0085] 3. Microbial simulation ingredients:

[0086] Cultured THP-1 cells: moderate amount, simulating immune cells in sputum

[0087] BCG-Pasteur bacteria: appropriate amount, simulating the bacterial components in sputum

[0088] 4. Stability and protective agent:

[0089] Glycerol: 1%-3% (v / v), to prevent crystallization during freezing and maintain sample stability

[0090] Catalytic enzymes (such as catalase): 0.01%-0.1% (w / v), reduce oxidative stress

[0091] 5. pH adjustment:

[0092] Adjust pH to 7.0 using NaOH or HCl

[0093] Preparation method:

[0094] 1. Mix 0.24 g 7H9 powder and 0.05 g Casitone in a 100 ml glass flask and add 0.2 g (0.4% w / v) salmon sperm DNA.

[0095] 2. Dissolve the above mixture in 49 ml of distilled water, then add 1 ml of glycerol and stir until completely dissolved.

[0096] 3. Adjust pH to 7.0 using sodium hydroxide.

[0097] 4. Autoclave at 121°C for 20 minutes.

[0098] 5. After autoclaving, add 6 g of porcine mucin (Type II) and continue stirring until the mucin is completely dissolved.

[0099] 6. The resulting mixture was pasteurized in a preheated water bath at 80°C for 45 minutes.

[0100] 7. After cooling to room temperature, add 5 ml of egg yolk emulsion and appropriate amount of cultured THP-1 cells and BCG-Pasteur bacteria.

[0101] 8. Mix gently to avoid disrupting the cells, and then store the final mixture at 4°C to maintain stability and extend shelf life.

[0102] 1. BCG-Pasteur bacteria, also known as BCG Pasteur strain or BCG Pasteur.

[0103] 2. BCG-Pasteur bacteria, appropriate amount, the range is BCG-Pasteur bacteria use concentration is about 10^5 to 10^7 CFU / mL (colony forming units / mL). 3. Cultured THP-1 cells: appropriate amount, the range is 0.5×10^6 to 2×10^6 cells / mL.

[0104] The artificial sputum simulation of this scheme is explained in detail:

[0105] 1. Preparation of basic ingredients

[0106] Weigh 6-8 grams of porcine mucin (Type II) and place it in a clean container for later use.

[0107] Prepare 0.4 g of low molecular weight salmon sperm DNA, ensuring its purity and integrity.

[0108] Accurately weigh 0.0059 g of diethylenetriaminepentaacetic acid (DTPA).

[0109] Weigh 5 grams of sodium chloride and 2.2 grams of potassium chloride respectively.

[0110] Weigh 1.81 g of Tris Base.

[0111] Prepare 0.1 g of cystine.

[0112] 2. Nutrition and Growth Factor Addition

[0113] According to the ratio of essential and non-essential L-amino acids (except L-tyrosine and L-cysteine), a total amount of 0.25 g of the amino acid mixture was weighed.

[0114] Weigh 0.25 g of L-cysteine ​​and L-tyrosine respectively, and dissolve them in 0.5 M potassium hydroxide solution and sterile water respectively.

[0115] 3. Preparation of Microbial Simulation Components

[0116] An appropriate amount of THP-1 cells was cultured to simulate the immune cells in sputum.

[0117] Prepare an appropriate amount of BCG-Pasteur bacilli to simulate the bacterial components in sputum.

[0118] 4. Stability and protective agent addition

[0119] Measure 1-3 ml of glycerol as a stabilizer and protective agent.

[0120] Prepare 0.01-0.1 g of a catalytic enzyme (such as catalase) to reduce oxidative stress.

[0121] 5. Preparation of simulated sputum

[0122] Mix 0.24 g of 7H9 powder and 0.05 g of Casitone in a 100 ml glass flask, add 0.2 g of salmon sperm DNA, and add 49 ml of distilled water, stirring until completely dissolved.

[0123] Add 1 ml of glycerol and continue stirring until homogeneous.

[0124] Adjust pH to 7.0 using sodium hydroxide or hydrochloric acid.

[0125] The mixture was autoclaved at 121°C for 20 minutes.

[0126] After sterilization, add 6 g of porcine mucin and continue stirring until the mucin is completely dissolved.

[0127] The mixture was pasteurized in a water bath at 80°C for 45 minutes.

[0128] After cooling to room temperature, 5 ml of egg yolk emulsion and appropriate amounts of cultured THP-1 cells and BCG-Pasteur bacteria were added.

[0129] Mix gently to avoid disrupting the cell structure and store at 4°C.

[0130] The simulated sputum of the present invention can be used as a sputum substitute in scientific research, and can be used in multiple fields such as the establishment of disease models, drug screening, biomedical teaching and training, etc.

[0131] The simulated sputum of the present invention combines multiple biological components and physicochemical properties and has a high degree of simulation.

[0132] The introduction of THP-1 cells and BCG-Pasteur bacilli improved the biological activity of simulated sputum.

[0133] By adding glycerol and catalytic enzyme, the stability and antioxidant capacity of the simulated sputum are improved, making it suitable for long-term storage and transportation.

[0134] Those skilled in the art will readily appreciate other embodiments of the present application after considering the description and practicing the contents disclosed herein. The present application is intended to cover any modification, use or adaptation of the present application, which follows the general principles of the present application and includes common knowledge or customary techniques in the art that are not disclosed in the present application. The description and examples are intended to be exemplary only, and the true scope and spirit of the present application are indicated by the claims.

[0135] It should be understood that the present application is not limited to the precise structures that have been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present application is limited only by the appended claims.

Claims

1. Highly simulated multifunctional artificial sputum and preparation method thereof, characterized in that: Artificial sputum includes the following components: Porcine mucin: 6%-8% (w / v); Low molecular weight salmon sperm DNA: 0.2-0.5% (w / v); Diethylenetriaminepentaacetic acid: 0.002-0.008% (w / v); Sodium chloride: 2-10% (w / v); Potassium chloride: 1-5% (w / v); Tris(hydroxymethyl)aminomethane: 1-3% (w / v); Cystine: 0.05-0.2% (w / v); Egg yolk emulsion: 2-8ml; Nutrition and growth factors; Microbial mimic ingredients; and stabilizers and protectants.

2. The highly emulational multifunctional artificial simulated sputum and the preparation method thereof according to claim 1, characterized in that: Artificial simulated sputum includes the following components: porcine mucin: 6%-8% (w / v); Low molecular weight salmon sperm DNA: 0.4% (w / v); Diethylenetriaminepentaacetic acid: 0.0059% (w / v); Sodium chloride: 5% (w / v); Potassium chloride: 2.2% (w / v); Tris(hydroxymethyl)aminomethane: 1.81% (w / v); Cystine: 0.1% (w / v); Egg yolk lotion: 5ml; Nutrition and growth factors; Microbial mimic ingredients; and stabilizers and protectants.

3. The highly emulational multifunctional artificial simulated sputum and the preparation method thereof according to claim 1 or 2, characterized in that: Nutrition and growth factors include the following components: Essential and non-essential L-amino acids: 0.25%; L-cysteine: 0.25% (w / v), dissolved in 0.5 M potassium hydroxide solution; L-Tyrosine: 0.25% (w / v), dissolved in sterile water.

4. The highly emulational multifunctional artificial simulated sputum and the preparation method thereof according to claim 1 or 2, characterized in that: Microbial simulation components include the following: Cultured THP-1 cells: moderate amount, simulating immune cells in sputum BCG-Pasteur bacteria: appropriate amount, simulating the bacterial components in sputum.

5. The highly emulational multifunctional artificial simulated sputum and the preparation method thereof according to claim 1 or 2, Features: Stabilizers and protectants include the following components: Glycerol: 1%-3% (v / v); Catalytic enzyme: 0.01%-0.1% (w / v).

6. The highly realistic multifunctional artificial simulated sputum and the preparation method thereof according to claim 1 or 2, characterized in that: pH adjustment was also performed by adjusting the pH to 7.0 using NaOH or HCl.

7. A method for preparing a highly realistic multifunctional artificial simulated sputum, characterized in that: The highly simulating multifunctional artificial simulated sputum according to any one of claims 1 to 5 is prepared by: 1) Mix 0.24 g 7H9 powder and 0.05 g cystine in a 100 ml glass flask and add 0.2 g (0.4% w / v) salmon sperm DNA; 2) Dissolve the above mixture in 49 ml of distilled water, then add 1 ml of glycerol and stir until completely dissolved; 3) Adjust the pH to 7.0 using sodium hydroxide; 4) Autoclave at 121°C for 20 minutes; 5) After autoclaving, add 6 g of porcine mucin and continue stirring until the mucin is completely dissolved; 6) pasteurizing the resulting mixture in a preheated water bath at 80°C for 45 minutes; 7) After cooling to room temperature, add 5 ml of egg yolk emulsion and appropriate amounts of cultured THP-1 cells and BCG-Pasteur bacteria; 8) Mix gently to avoid damaging the cells, and then store the final mixture at 4°C.