Smooth muscle cell exosome for artificial blood vessel as well as preparation method and application of smooth muscle cell exosome

Abstract

The invention discloses a smooth muscle cell exosome for artificial blood vessels and a preparation method and application thereof, and relates to the technical field of biology, and the preparation method comprises the following steps: (1) obtaining smooth muscle cell precipitate; (2) taking out the smooth muscle cell precipitate, placing the smooth muscle cell precipitate in a complete culture medium containing fetal calf serum, continuously culturing until 90% of cells are fused, replacing a new complete culture medium containing fetal calf serum, adding sterilized fermentation liquor into the complete culture medium containing fetal calf serum, and continuously culturing; and (3) during continuous culture, firstly performing ultrasonic treatment, then performing continuous culture until 90% of cells are fused, and performing gradient centrifugation on the culture solution to obtain the product. The artificial blood vessel applying the exosome prepared by the invention has lower hemolysis rate, which indicates that hemolysis reaction is not caused, which is mainly characterized in that the blood compatibility of the artificial blood vessel is improved by adding the specific exosome, the influence of an allogenic material on red blood cells is reduced, and the exosome plays an important role in maintaining the integrity of the structure and the function of the red blood cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to smooth muscle cell exosomes for artificial blood vessels, a preparation method thereof, and an application thereof. Background Art

[0002] With the acceleration of the aging process of the population and the influence of unhealthy lifestyles, the morbidity and mortality rates of patients with cardiovascular diseases (CVD) continue to increase, and the cost of treating diseases is getting higher and higher, which makes the demand for clinical artificial blood vessel stents increasing.

[0003] However, the biocompatibility of the existing prepared artificial blood vessels is relatively poor, resulting in frequent problems of foreign material rejection and the occurrence of phenomena such as hemolysis of blood cells.

[0004] Based on this, a smooth muscle cell exosome for artificial blood vessels is provided to solve the existing technical problems. Summary of the Invention

[0005] The purpose of the present invention is to provide a smooth muscle cell exosome for artificial blood vessels and a preparation method thereof to make up for the deficiencies in the prior art.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A preparation method of smooth muscle cell exosomes for artificial blood vessels includes:

[0008] (1) First, obtain smooth muscle tissue, cut the smooth muscle tissue into pieces, culture the cut smooth muscle tissue pieces in a serum-free medium until 80% of the area adheres to the wall, filter to remove the smooth muscle cut tissue, then digest with 0.25% trypsin, and then centrifuge at a speed of 3000 r / min for 10 min, filter to obtain a smooth muscle cell precipitate;

[0009] Among them, the culture is carried out in an incubator at 37°C and 5% CO 2 ;

[0010] (2) Take out the obtained smooth muscle cell precipitate and place it in a complete medium containing fetal bovine serum and continue to culture until 90% cell confluence, replace the new complete medium containing fetal bovine serum, and add a sterilized fermentation broth to the complete medium containing fetal bovine serum, and continue to culture;

[0011] Among them, the addition amount of the sterilized fermentation broth accounts for 1-1.2% of the total mass of the complete medium;

[0012] (3) When continuing to culture, first perform ultrasonic treatment for 2-3 min, then continue to culture until 90% cell confluence, and perform gradient centrifugation on the culture solution to obtain smooth muscle cell exosomes for artificial blood vessels.

[0013] As a further technical solution, the complete medium is a complete medium containing fetal bovine serum;

[0014] Among them, the mass fraction of fetal bovine serum in the complete medium is 10%.

[0015] As a further technical solution, the serum-free medium is DMEM medium;

[0016] The specific preparation method is as follows:

[0017] Add 1 bag of DMEM medium powder directly to 1 L of ultrapure water. After mixing evenly, adjust the pH of the mixed system to 7.2, and then filter it through a 0.22 μm filter membrane to obtain the serum-free medium;

[0018] Among them, the pH is adjusted using sodium bicarbonate.

[0019] As a further technical solution, the preparation method of the sterilized fermentation broth is as follows:

[0020] Inoculate Thermus thermophilus HB27 into the fermentation medium and carry out activation culture at 67 °C for 24 hours to obtain the activated bacterial liquid;

[0021] Take 1 mL of the activated bacterial liquid and inoculate it into 1 L of the fermentation medium. Cultivate at 70 °C for 10 - 12 hours, then add soybean low molecular weight polypeptide to the medium, and then continue to cultivate for 30 - 32 hours to obtain the fermentation bacterial liquid;

[0022] Centrifuge the fermentation bacterial liquid at a speed of 4500 r / min for 20 min, then separate it, and take the supernatant;

[0023] Subject the supernatant to adsorption treatment to obtain the treated liquid;

[0024] Centrifuge the treated liquid again at a centrifugation speed of 6000 r / min, then separate it, retain the supernatant, and filter and sterilize the supernatant with a 0.22 μm filter membrane to obtain the fermentation broth.

[0025] As a further technical solution, the fermentation medium contains the following components per liter of the medium: 15 g of glucose, 3.5 g of yeast powder, 5 g of beef extract powder, 12 g of sodium nitrate, 0.3 g of sodium chloride, 10 g of peptone, 1.5 g of potassium dihydrogen phosphate, 0.8 g of dipotassium hydrogen phosphate, and the balance is sterile water.

[0026] As a further technical solution, the soybean low molecular weight polypeptide is added at 2.5 - 3 g per liter of the medium.

[0027] As a further technical solution: the probe frequency of the ultrasonic treatment is 0.3 - 0.4 MHz, the amplitude is 500 mV, the pulse repetition frequency is 120 Hz, and the pulse interval is 100 ms.

[0028] As a further technical solution, the gradient centrifugation is as follows:

[0029] Centrifuge the culture solution in an ultracentrifuge at 10,000×g and 4°C for 60 min, and take the supernatant once;

[0030] Centrifuge the supernatant in an ultracentrifuge at 50,000×g and 4°C for 90 min, and take the supernatant for the second time;

[0031] Centrifuge the supernatant for the second time in an ultracentrifuge at 150,000×g and 4°C for 60 min, and leave the precipitated exosomes.

[0032] The smooth muscle cell exosomes prepared by the described method are used for artificial blood vessels.

[0033] The smooth muscle cell exosomes are applied to artificial blood vessels.

[0034] Beneficial effects:

[0035] The artificial blood vessels applying the exosomes prepared by the present invention have a low hemolysis rate, indicating that they will not cause hemolytic reactions. This is mainly because the addition of specific exosomes improves the blood compatibility of artificial blood vessels, reduces the influence of foreign materials on red blood cells, and plays an important role in maintaining the integrity of the structure and function of red blood cells.

[0036] The smooth muscle cell exosomes prepared by the present invention can improve the re-endothelialization ability of artificial blood vessels, thereby improving the patency rate and long-term effect after artificial blood vessel transplantation, and greatly improving the biocompatibility of artificial blood vessels.

[0037] After the smooth muscle cell exosomes are applied to the surface of artificial blood vessels and the artificial blood vessels are implanted into the body, the inflammatory reaction can be reduced. This is mainly achieved by the anti-inflammatory factors carried by the smooth muscle cell exosomes or by regulating the functions of immune cells to alleviate this inflammatory reaction, thereby significantly reducing the occurrence of postoperative complications.

[0038] Since the smooth muscle cell exosomes prepared by the present invention have the ability to enhance tissue integration, they can promote the proliferation and differentiation of vascular wall cells such as fibroblasts and smooth muscle cells, thereby enhancing the integration ability between artificial blood vessels and tissues, and further improving the stability and durability of artificial blood vessels.

[0039] The exosomes prepared by the present invention have almost no cytotoxicity after being applied to artificial blood vessels and can support the adhesion and proliferation of venous endothelial cells. Description of the Drawings

[0040] Figure 1 For the column chart of hemolysis rate of each group in the experiment;

[0041] Figure 2 It is the electron microscopy image of the growth of venous endothelial cells on the surface of artificial blood vessels applied in Example 1. Specific implementation manners

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] 0.25% Trypsin: 0.25% Trypsin-EDTA Digest was purchased from Gibco, USA;

[0044] DMEM Medium and FBS Fetal Bovine Serum were purchased from Gibco, USA;

[0045] Example 1:

[0046] A method for preparing smooth muscle cell exosomes for artificial blood vessels, comprising:

[0047] (1) First, obtain smooth muscle tissue, cut the smooth muscle tissue into pieces, culture the cut smooth muscle tissue pieces in serum-free medium until 80% of the area adheres to the wall, filter to remove the smooth muscle cut tissue, then digest with 0.25% trypsin, and then centrifuge at a speed of 3000 r / min for 10 min, filter to obtain smooth muscle cell precipitate;

[0048] Among them, the culture is carried out in an incubator at 37°C and 5% CO 2 The complete medium is a complete medium containing fetal bovine serum;

[0049] Among them, the mass fraction of fetal bovine serum in the complete medium is 10%.

[0050] The serum-free medium is DMEM medium;

[0051] The specific preparation method is:

[0052] Directly add 1 bag of DMEM medium powder to 1 L of ultrapure water, mix evenly, adjust the pH of the mixed system to 7.2, and then filter through a 0.22 μm filter membrane to obtain serum-free medium;

[0053] Among them, the pH is adjusted using sodium bicarbonate.

[0054] (2) Take out the obtained smooth muscle cell precipitate and place it in a complete medium containing fetal bovine serum, and continue to culture until 90% cell confluence. Replace the fresh complete medium containing fetal bovine serum, add the sterilized fermentation broth to the complete medium containing fetal bovine serum, and continue to culture;

[0055] Among them, the addition amount of the sterilized fermentation broth accounts for 1% of the total mass of the complete medium; the preparation method of the sterilized fermentation broth is:

[0056] Inoculate Thermus thermophilus HB27 into the fermentation medium, and carry out activation culture at 67 °C for 24 hours to obtain an activated bacterial solution;

[0057] Take 1 mL of the activated bacterial solution and inoculate it into 1 L of the fermentation medium. Cultivate at 70 °C for 10 hours, then add soy low molecular weight polypeptide to the fermentation medium, and then continue to culture for 30 hours to obtain a fermented bacterial solution;

[0058] Centrifuge the fermented bacterial solution at a speed of 4500 r / min for 20 min, then carry out separation, and take the supernatant;

[0059] Subject the supernatant to adsorption treatment to obtain a treated solution;

[0060] Carry out secondary centrifugation treatment on the treated solution at a centrifugation speed of 6000 r / min, then carry out separation again, retain the supernatant, and filter and sterilize the supernatant with a 0.22 μm filter membrane to obtain a fermentation broth.

[0061] The fermentation medium contains the following components per liter of the medium: 15 g of glucose, 3.5 g of yeast powder, 5 g of beef extract powder, 12 g of sodium nitrate, 0.3 g of sodium chloride, 10 g of peptone, 1.5 g of potassium dihydrogen phosphate, 0.8 g of dipotassium hydrogen phosphate, and the balance is sterile water.

[0062] The soy low molecular weight polypeptide is added at 2.5 g per liter of the medium.

[0063] (3) When continuing to culture, first carry out ultrasonic treatment for 2 min, then continue to culture until 90% cell confluence, and carry out gradient centrifugation on the culture solution to obtain smooth muscle cell exosomes for artificial blood vessels; the probe frequency of the ultrasonic treatment is 0.3 MHz, the amplitude is 500 mV, the pulse repetition frequency is 120 Hz, and the pulse interval is 100 ms; the gradient centrifugation is:

[0064] Centrifuge the culture solution in an ultracentrifuge at 10000×g at 4 °C for 60 min, and take the first supernatant;

[0065] Centrifuge the supernatant in an ultracentrifuge at 50000×g at 4 °C for 90 min, and take the second supernatant;

[0066] Centrifuge the secondary supernatant at 150,000×g for 60 min at 4°C in an ultracentrifuge, and retain the precipitated exosomes.

[0067] Example 2

[0068] A method for preparing smooth muscle cell exosomes for artificial blood vessels, comprising:

[0069] (1) First, obtain smooth muscle tissue, cut the smooth muscle tissue into pieces, culture the cut smooth muscle tissue pieces in a serum-free medium until 80% of the area adheres to the wall, filter to remove the smooth muscle cut tissue, then digest with 0.25% trypsin, and then centrifuge at 3000 r / min for 10 min, filter to obtain smooth muscle cell precipitate;

[0070] Among them, the culture is carried out in an incubator at 37°C and 5% CO 2 ; The complete medium is a complete medium containing fetal bovine serum;

[0071] Among them, the mass fraction of fetal bovine serum in the complete medium is 10%.

[0072] The serum-free medium is DMEM medium;

[0073] The specific preparation method is:

[0074] Directly add 1 bag of DMEM medium powder to 1 L of ultrapure water, mix evenly, adjust the pH of the mixed system to 7.2, and then filter through a 0.22 μm filter membrane to obtain a serum-free medium;

[0075] Among them, the pH is adjusted using sodium bicarbonate.

[0076] (2) Take out the obtained smooth muscle cell precipitate and place it in a complete medium containing fetal bovine serum and continue to culture until 90% cell confluence, replace the new complete medium containing fetal bovine serum, and add a sterilized fermentation broth to the complete medium containing fetal bovine serum, and continue to culture;

[0077] Among them, the addition amount of the sterilized fermentation broth accounts for 1.1% of the total mass of the complete medium; The preparation method of the sterilized fermentation broth is:

[0078] Inoculate Thermus thermophilus HB27 into the fermentation medium and carry out activation culture at 67°C for 24 hours to obtain an activated bacterial liquid;

[0079] Take 1 mL of the activated bacterial liquid and inoculate it into 1 L of the fermentation medium, culture at 70°C for 11 hours, then add soy low molecular weight polypeptide to the medium, and then continue to culture for 31 hours to obtain a fermented bacterial liquid;

[0080] Centrifuge the fermented bacterial liquid at 4500 r / min for 20 min, then separate it and take the supernatant;

[0081] Subject the supernatant to adsorption treatment to obtain a treated liquid;

[0082] Centrifuge the treated liquid again at a centrifugation speed of 6000 r / min, then separate it again, retain the supernatant, and filter and sterilize the supernatant with a 0.22 μm filter membrane to obtain a fermentation broth.

[0083] The fermentation medium contains the following components per liter of the medium: 15 g of glucose, 3.5 g of yeast powder, 5 g of beef extract powder, 12 g of sodium nitrate, 0.3 g of sodium chloride, 10 g of peptone, 1.5 g of potassium dihydrogen phosphate, 0.8 g of dipotassium hydrogen phosphate, and the balance is sterile water.

[0084] Soybean low-molecular-weight polypeptide is added at 2.7 g per liter of the medium.

[0085] (3) When continuing the culture, first perform ultrasonic treatment for 2 - 3 min, then continue the culture until 90% cell fusion, and perform gradient centrifugation on the culture solution to obtain smooth muscle cell exosomes for artificial blood vessels; the probe frequency of the ultrasonic treatment is 0.35 MHz, the amplitude is 500 mV, the pulse repetition frequency is 120 Hz, and the pulse interval is 100 ms; the gradient centrifugation is as follows:

[0086] Centrifuge the culture solution in an ultracentrifuge at 10000×g and 4°C for 60 min, and take the first supernatant;

[0087] Centrifuge the supernatant in an ultracentrifuge at 50000×g and 4°C for 90 min, and take the second supernatant;

[0088] Centrifuge the second supernatant in an ultracentrifuge at 150000×g and 4°C for 60 min, and retain the precipitated exosomes.

[0089] Example 3

[0090] A method for preparing smooth muscle cell exosomes for artificial blood vessels, comprising:

[0091] (1) First, obtain smooth muscle tissue, cut the smooth muscle tissue into pieces, culture the cut smooth muscle tissue pieces in a serum-free medium until 80% of the area adheres to the wall, filter to remove the smooth muscle cut tissue, then digest it with 0.25% trypsin, and then centrifuge at 3000 r / min for 10 min and filter to obtain a smooth muscle cell precipitate;

[0092] Among them, the culture is carried out at 37°C and 5% CO 2Cultured in an incubator; the complete medium is a complete medium containing fetal bovine serum;

[0093] Among them, the mass fraction of fetal bovine serum in the complete medium is 10%.

[0094] The serum-free medium is DMEM medium;

[0095] The specific preparation method is as follows:

[0096] Add 1 bag of DMEM medium powder directly to 1 L of ultrapure water, mix evenly, adjust the pH of the mixed system to 7.2, and then filter through a 0.22 μm filter membrane to obtain the serum-free medium;

[0097] Among them, the pH is adjusted using sodium bicarbonate.

[0098] (2) Take out the obtained smooth muscle cell precipitate and place it in a complete medium containing fetal bovine serum and continue to culture until 90% cell confluence. Replace the fresh complete medium containing fetal bovine serum, and add the sterilized fermentation broth to the complete medium containing fetal bovine serum, and continue to culture;

[0099] Among them, the addition amount of the sterilized fermentation broth accounts for 1.2% of the total mass of the complete medium; the preparation method of the sterilized fermentation broth is as follows:

[0100] Inoculate Thermus thermophilus HB27 into the fermentation medium, and carry out activation culture at 67 °C for 24 hours to obtain the activated bacterial liquid;

[0101] Take 1 mL of the activated bacterial liquid and inoculate it into 1 L of the fermentation medium, culture at 70 °C for 10 hours, then add soy low molecular polypeptide to the medium, and then continue to culture for 31 hours to obtain the fermented bacterial liquid;

[0102] Centrifuge the fermented bacterial liquid at a speed of 4500 r / min for 20 min, then separate, and take the supernatant;

[0103] Subject the supernatant to adsorption treatment to obtain the treated liquid;

[0104] Carry out secondary centrifugation treatment on the treated liquid, the centrifugation speed is 6000 r / min, then separate again, keep the supernatant, and filter and sterilize the supernatant with a 0.22 μm filter membrane to obtain the fermentation broth.

[0105] The fermentation medium contains the following components per liter of medium: 15 g of glucose, 3.5 g of yeast powder, 5 g of beef extract powder, 12 g of sodium nitrate, 0.3 g of sodium chloride, 10 g of peptone, 1.5 g of potassium dihydrogen phosphate, 0.8 g of dipotassium hydrogen phosphate, and the balance is sterile water.

[0106] Soybean low-molecular-weight polypeptide was added at 2.8 g per liter of medium.

[0107] (3) When continuing the culture, first perform ultrasonic treatment for 2 min, and then continue the culture until 90% cell fusion. Perform gradient centrifugation on the culture medium to obtain smooth muscle cell exosomes for artificial blood vessels; the probe frequency of the ultrasonic treatment is 0.4 MHz, the amplitude is 500 mV, the pulse repetition frequency is 120 Hz, and the pulse interval is 100 ms; the gradient centrifugation is as follows:

[0108] Centrifuge the culture medium in an ultracentrifuge at 10,000×g for 60 min at 4°C, and take the first supernatant;

[0109] Centrifuge the supernatant in an ultracentrifuge at 50,000×g for 90 min at 4°C, and take the second supernatant;

[0110] Centrifuge the second supernatant in an ultracentrifuge at 150,000×g for 60 min at 4°C, and leave the precipitated exosomes.

[0111] Example 4

[0112] A method for preparing smooth muscle cell exosomes for artificial blood vessels, comprising:

[0113] (1) First, obtain smooth muscle tissue, cut the smooth muscle tissue into pieces, culture the cut smooth muscle tissue pieces in a serum-free medium until 80% of the area adheres to the wall, filter to remove the smooth muscle cut tissue, then digest with 0.25% trypsin, and then centrifuge at 3000 r / min for 10 min, filter to obtain smooth muscle cell precipitate;

[0114] Among them, the culture is carried out in an incubator at 37°C and 5% CO 2 The complete medium is a complete medium containing fetal bovine serum;

[0115] Among them, the mass fraction of fetal bovine serum in the complete medium is 10%.

[0116] The serum-free medium is DMEM medium;

[0117] The specific preparation method is as follows:

[0118] Add 1 bag of DMEM medium powder directly to 1 L of ultrapure water, mix evenly, adjust the pH of the mixed system to 7.2, and then filter through a 0.22 μm filter membrane to obtain a serum-free medium;

[0119] Among them, the pH is adjusted using sodium bicarbonate.

[0120] (2) Take out the obtained smooth muscle cell precipitate and place it in a complete medium containing fetal bovine serum for continued culture until 90% cell confluence. Replace the fresh complete medium containing fetal bovine serum, and add the sterilized fermentation broth to the complete medium containing fetal bovine serum, and continue the culture;

[0121] Among them, the addition amount of the sterilized fermentation broth accounts for 1.1% of the total mass of the complete medium; the preparation method of the sterilized fermentation broth is as follows:

[0122] Inoculate Thermus thermophilus HB27 into the fermentation medium and carry out activation culture at 67 °C for 24 hours to obtain an activated bacterial liquid;

[0123] Take 1 mL of the activated bacterial liquid and inoculate it into 1 L of the fermentation medium. Cultivate at 70 °C for 11 hours, then add soybean low molecular weight polypeptide to the medium, and then continue to cultivate for 32 hours to obtain a fermented bacterial liquid;

[0124] Centrifuge the fermented bacterial liquid at a speed of 4500 r / min for 20 min, then carry out separation, and take the supernatant;

[0125] Subject the supernatant to adsorption treatment to obtain a treated liquid;

[0126] Carry out secondary centrifugation treatment on the treated liquid at a centrifugation speed of 6000 r / min, then carry out separation again, retain the supernatant, and filter and sterilize the supernatant with a 0.22 μm filter membrane to obtain the fermentation broth.

[0127] The fermentation medium contains the following components per liter of the medium: 15 g of glucose, 3.5 g of yeast powder, 5 g of beef extract powder, 12 g of sodium nitrate, 0.3 g of sodium chloride, 10 g of peptone, 1.5 g of potassium dihydrogen phosphate, 0.8 g of dipotassium hydrogen phosphate, and the balance is sterile water.

[0128] The soybean low molecular weight polypeptide is added at 2.6 g per liter of the medium.

[0129] (3) During the continued culture, first perform ultrasonic treatment for 2.5 min, then continue the culture until 90% cell confluence, and perform gradient centrifugation on the culture solution to obtain smooth muscle cell exosomes for artificial blood vessels; the probe frequency of the ultrasonic treatment is 0.35 MHz, the amplitude is 500 mV, the pulse repetition frequency is 120 Hz, and the pulse interval is 100 ms; the gradient centrifugation is as follows:

[0130] Centrifuge the culture solution in an ultracentrifuge at 10000×g and 4 °C for 60 min, and take the first supernatant;

[0131] Centrifuge the supernatant in an ultracentrifuge at 50000×g and 4 °C for 90 min, and take the second supernatant;

[0132] Centrifuge the secondary supernatant at 150,000×g for 60 min at 4°C in an ultracentrifuge, and retain the precipitated exosomes.

[0133] Example 5

[0134] A method for preparing smooth muscle cell exosomes for artificial blood vessels, comprising:

[0135] (1) First, obtain smooth muscle tissue, cut the smooth muscle tissue into pieces, culture the cut smooth muscle tissue pieces in a serum-free medium until 80% of the area adheres to the wall, filter to remove the cut smooth muscle tissue, then digest with 0.25% trypsin, and then centrifuge at 3000 r / min for 10 min, filter to obtain a smooth muscle cell precipitate;

[0136] Among them, the culture is carried out in an incubator at 37°C and 5% CO 2 The complete medium is a complete medium containing fetal bovine serum;

[0137] Among them, the mass fraction of fetal bovine serum in the complete medium is 10%.

[0138] The serum-free medium is DMEM medium;

[0139] The specific preparation method is as follows:

[0140] Directly add 1 bag of DMEM medium powder to 1 L of ultrapure water, mix evenly, adjust the pH of the mixed system to 7.2, and then filter through a 0.22 μm filter membrane to obtain a serum-free medium;

[0141] Among them, the pH is adjusted using sodium bicarbonate.

[0142] (2) Take out the obtained smooth muscle cell precipitate and place it in a complete medium containing fetal bovine serum and continue to culture until 90% cell confluence, replace the new complete medium containing fetal bovine serum, and add a sterilized fermentation broth to the complete medium containing fetal bovine serum, and continue to culture;

[0143] Among them, the addition amount of the sterilized fermentation broth accounts for 1.2% of the total mass of the complete medium; the preparation method of the sterilized fermentation broth is as follows:

[0144] Inoculate Thermus thermophilus HB27 into a fermentation medium and carry out activation culture at 67°C for 24 hours to obtain an activated bacterial solution;

[0145] Take 1 mL of the activated bacterial solution and inoculate it into 1 L of the fermentation medium, carry out culture at 70°C for 12 hours, then add soybean low molecular polypeptide to the medium, and then continue to culture for 32 hours to obtain a fermentation bacterial solution;

[0146] Centrifuge the fermented bacterial liquid at 4500 r / min for 20 min, then separate it and take the supernatant;

[0147] Treat the supernatant through adsorption to obtain a treated liquid;

[0148] Centrifuge the treated liquid again at a centrifugal speed of 6000 r / min, then separate it again, keep the supernatant, and filter and sterilize the supernatant with a 0.22 μm filter membrane to obtain a fermentation broth.

[0149] The fermentation medium contains the following components per liter of the medium: 15 g of glucose, 3.5 g of yeast powder, 5 g of beef extract powder, 12 g of sodium nitrate, 0.3 g of sodium chloride, 10 g of peptone, 1.5 g of potassium dihydrogen phosphate, 0.8 g of dipotassium hydrogen phosphate, and the balance is sterile water.

[0150] Soybean low molecular weight polypeptide is added at 3 g per liter of the medium.

[0151] (3) When continuing the culture, first perform ultrasonic treatment for 3 min, then continue the culture until 90% cell fusion, and perform gradient centrifugation on the culture solution to obtain smooth muscle cell exosomes for artificial blood vessels; the probe frequency of the ultrasonic treatment is 0.4 MHz, the amplitude is 500 mV, the pulse repetition frequency is 120 Hz, and the pulse interval is 100 ms; the gradient centrifugation is as follows:

[0152] Centrifuge the culture solution in an ultracentrifuge at 10000×g at 4°C for 60 min, and take the first supernatant;

[0153] Centrifuge the supernatant in an ultracentrifuge at 50000×g at 4°C for 90 min, and take the second supernatant;

[0154] Centrifuge the second supernatant in an ultracentrifuge at 150000×g at 4°C for 60 min, and keep the precipitated exosomes.

[0155] Comparative Example 1:

[0156] On the basis of Example 1, the sterilized fermentation broth is not added, and the rest of the technical solutions remain unchanged.

[0157] Comparative Example 2:

[0158] On the basis of Example 1, the ultrasonic treatment is not performed, and the rest of the technical solutions remain unchanged.

[0159] Comparative Example 3:

[0160] On the basis of Example 1, the smooth muscle tissue is replaced with adipose tissue, and the rest of the technical solutions remain unchanged.

[0161] Experiment:

[0162] The surface of the artificial blood vessel was disinfected and sterilized with 75% alcohol, and then repeatedly rinsed with sterile PBS for 10 min;

[0163] The exosomes of the examples and the comparative examples were resuspended with PBS, and then a photo-polymerizable hydrogel-exosome suspension was prepared, where the mass concentration of the exosomes of the examples and the comparative examples was 100 μg / μL;

[0164] The artificial blood vessels were equally divided into 9 groups, with 10 in each group. The photo-polymerizable hydrogel-exosome suspension was respectively sprayed onto the surfaces of 8 groups of artificial blood vessels, and after photo-curing, the remaining group was sprayed with photo-polymerizable hydrogel on the surface and also underwent photo-curing;

[0165] The above artificial blood vessels were extracted in 37°C physiological saline for 12 hours for a hemolysis reaction test;

[0166] 35 mL of fresh rabbit blood was added with 3 mL of 2.8% potassium oxalate solution to prepare fresh anticoagulated rabbit blood for the test;

[0167] Another 10 mL of fresh anticoagulated rabbit blood was added with 15 mL of normal saline injection for dilution to obtain diluted anticoagulated rabbit blood;

[0168] Test procedure: The test tubes were placed in a 37 ± 1°C constant temperature water bath for 10 minutes. 0.5 mL of diluted anticoagulated rabbit blood was added to each test tube, and then the artificial blood vessels of the examples and the comparative examples were respectively added. After gently shaking and mixing, they were continued to be placed in the water bath for 60 minutes; the liquid in the tubes was aspirated, centrifuged at 1000 rpm for 10 min, the supernatant was aspirated and transferred into a cuvette, and the absorbance (OD) was measured at a wavelength of 545 nm with a 721 spectrophotometer and the results were recorded;

[0169] Calculate the hemolysis rate of the sample;

[0170] H(%) = (ODt - ODnc) / (ODpc - ODnc) × 100%;

[0171] In the formula, H% is the hemolysis rate;

[0172] ODt is the absorbance of the sample;

[0173] ODnc is the absorbance of the negative control sample;

[0174] ODpc is the absorbance of the positive control sample.

[0175] According to the standard of GB / T14233.2 - 1993, the in vitro hemolysis of the material was evaluated, and a hemolysis reaction greater than 5% was positive;

[0176] Table 1

[0177]

[0178]

[0179] As can be seen from Table 1, the artificial blood vessels prepared by applying the exosomes of the present invention have a low hemolysis rate, indicating that they will not cause hemolytic reactions. This is mainly because the addition of specific exosomes improves the blood compatibility of the artificial blood vessels, reduces the influence of foreign materials on red blood cells, and plays an important role in maintaining the integrity of the structure and function of red blood cells.

[0180] Inoculation and culture of venous endothelial cells on artificial blood vessels (poly(L-lactide-co-caprolactone) materials)

[0181] Inoculate venous endothelial cells into DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin, and culture them in a 37°C, 5% CO 2 cell incubator for 24 hours, then change the medium. When the venous endothelial cells reach 90% confluence, passage them. When passing the cells, treat them with 0.25% trypsin and digest them at 5% CO 2 , 37°C. Stop digestion when the cell tentacles disappear and the cells become round;

[0182] Sterilize the surface of the artificial blood vessels with 75% alcohol, and then rinse them repeatedly with sterile PBS for 10 min;

[0183] Resuspend the exosomes of the examples and comparative examples with PBS, and then prepare a photo-polymerizable hydrogel-exosome suspension, where the mass concentration of the exosomes of the examples and comparative examples is 100 μg / μL;

[0184] Divide the artificial blood vessels equally into 9 groups, with 10 in each group. Spray the photo-polymerizable hydrogel-exosome suspension onto the surfaces of 8 groups of artificial blood vessels, and after photo-curing, spray the photo-polymerizable hydrogel onto the surface of the remaining group, and also perform photo-curing;

[0185] Immerse the above artificial blood vessels in 37°C physiological saline for 12 hours, then transfer the treated artificial blood vessels to a 24-well plate, add 500 mL of DMEM and incubate for 24 hours. Suspend 4×10 4 / cm 2 venous endothelial cells onto the surface of the artificial blood vessels and place them in a 5% CO 2 , 37°C incubator for 4 hours. Change the medium after 24 hours:

[0186] After 7 days, the OD value can be measured with an enzyme-linked immunosorbent assay (ELISA) reader. The smaller the OD value, the stronger the cell proliferation ability and the stronger the cell activity, as shown in Table 2:

[0187] Table 2

[0188] OD value (540 nm) Example 1 0.38 Example 2 0.36 Example 3 0.35 Example 4 0.39 Example 5 0.40 Comparative Example 1 0.51 Comparative Example 2 0.46 Comparative Example 3 0.62 Blank control group 1.00

[0189] As can be seen from Table 2, under the action of the exosomes of the present invention, the activity of venous endothelial cells on the surface of artificial blood vessels can be improved;

[0190] Continue the above experiment and use the MTT method to detect the proliferation of venous endothelial cells on artificial blood vessels:

[0191] MTT detection: After culturing for 7 days, add 20 μL of MTT reagent (5 mg / mL) to each well, and continue to incubate in a cell culture incubator for 4 hours to allow MTT to enter living cells and be reduced by succinate dehydrogenase in mitochondria into insoluble blue-violet formazan crystals, and then observe and compare, as shown in Table 3:

[0192] Table 3

[0193]

[0194] As can be seen from Table 3, after the exosomes prepared by the present invention are applied to artificial blood vessels, there is almost no cytotoxicity and they can support the adhesion and proliferation of venous endothelial cells.

[0195] The above are only the preferred embodiments of the present invention, but the present invention is not limited to the shown scope of implementation. Any changes made according to the concept of the present invention, or equivalent embodiments modified into equivalent changes, still within the spirit covered by the specification, shall be within the protection scope of the present invention.

Claims

1. A method for preparing smooth muscle cell exosomes for artificial blood vessels, characterized in that: include: (1) First, smooth muscle tissue is obtained, the smooth muscle tissue is minced, and the minced smooth muscle tissue fragments are cultured in a serum-free medium until 80% of the area is attached to the wall, and the minced smooth muscle tissue is removed by filtration, and then digested with 0.25% trypsin, and then centrifuged at 3000 r / min for 10 minutes, filtered, and smooth muscle cell precipitation is obtained; Wherein, the culture is cultured in a 37°C, 5% CO2 incubator; (2) taking out the obtained smooth muscle cell pellet and placing it in a complete medium containing fetal bovine serum to continue culturing until the cells are 90% confluent, replacing it with a new complete medium containing fetal bovine serum, and adding sterilized fermentation liquid to the complete medium containing fetal bovine serum to continue culturing; The amount of sterilized fermentation broth added is 1-1.2% of the total mass of the complete culture medium; (3) When continuing to culture, the cells are first treated with ultrasound for 2-3 minutes, and then cultured until the cells are 90% confluent. The culture fluid is subjected to gradient centrifugation to obtain smooth muscle cell exosomes for artificial blood vessels.

2. The method for preparing smooth muscle cell exosomes for artificial blood vessels according to claim 1, characterized in that: The complete culture medium is a complete culture medium containing fetal bovine serum; The mass fraction of fetal bovine serum in the complete culture medium is 10%.

3. The method for preparing smooth muscle cell exosomes for artificial blood vessels according to claim 2, characterized in that: The serum-free culture medium is DMEM culture medium; The specific preparation method is: Add 1 bag of DMEM culture medium powder directly into 1L of ultrapure water, mix well, adjust the pH of the mixed system to 7.2, and then filter through a 0.22μm filter membrane to obtain a serum-free culture medium; The pH was adjusted using sodium bicarbonate.

4. The method for preparing smooth muscle cell exosomes for artificial blood vessels according to claim 1, characterized in that: The method for preparing the sterilized fermentation broth is as follows: Thermus thermophilus HB27 was inoculated into a fermentation medium, and activated and cultured at 67°C for 24 hours to obtain an activated bacterial solution; 1 mL of activated bacterial solution was inoculated into 1 L of fermentation medium, and cultured at 70° C. for 10-12 hours, and then soybean low molecular weight polypeptide was added to the fermentation medium, and then cultured for 30-32 hours to obtain fermentation bacterial solution; The fermented bacterial liquid was centrifuged at 4500 r / min for 20 min, and then separated, and the supernatant was taken; The supernatant is subjected to adsorption treatment to obtain a treated liquid; The treated liquid was centrifuged again at a centrifugal speed of 6000 r / min, and then separated, retaining the supernatant, which was filtered and sterilized using a 0.22 μm filter membrane to obtain a fermentation liquid.

5. The method for preparing smooth muscle cell exosomes for artificial blood vessels according to claim 4, characterized in that: The fermentation medium contains the following components per liter of medium: 15g of glucose, 3.5g of yeast powder, 5g of beef extract powder, 12g of sodium nitrate, 0.3g of sodium chloride, 10g of peptone, 1.5g of potassium dihydrogen phosphate, 0.8g of dipotassium hydrogen phosphate, and the balance is sterile water.

6. The method for preparing smooth muscle cell exosomes for artificial blood vessels according to claim 4, characterized in that: The soybean low molecular weight polypeptide is added at 2.5-3 g per L of culture medium.

7. The method for preparing smooth muscle cell exosomes for artificial blood vessels according to claim 1, characterized in that: The probe frequency of the ultrasonic treatment is 0.3-0.4 MHz, the amplitude is 500 mV, the pulse repetition frequency is 120 Hz, and the pulse interval is 100 ms.

8. The method for preparing smooth muscle cell exosomes for artificial blood vessels according to claim 1, characterized in that: The gradient centrifugation is: The culture solution was centrifuged at 10,000 × g and 4°C for 60 min in an ultracentrifuge and the supernatant was collected once; The supernatant was centrifuged at 50,000 × g and 4 °C for 90 min in an ultracentrifuge, and the secondary supernatant was taken; The secondary supernatant was centrifuged in an ultracentrifuge at 150,000 × g at 4°C for 60 min to precipitate the exosomes.

9. The method according to any one of claims 1 to 8 is used to prepare smooth muscle cell exosomes for artificial blood vessels.

10. The smooth muscle cell exosomes according to claim 9 are used in artificial blood vessels.

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