CN113274421A - Anti-inflammatory exosome preparation for knee joint treatment and preparation method and application thereof - Google Patents

Anti-inflammatory exosome preparation for knee joint treatment and preparation method and application thereof Download PDF

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CN113274421A
CN113274421A CN202110496557.9A CN202110496557A CN113274421A CN 113274421 A CN113274421 A CN 113274421A CN 202110496557 A CN202110496557 A CN 202110496557A CN 113274421 A CN113274421 A CN 113274421A
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exosome
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黄燕
王志伟
郑旭东
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Shenzhen Lailisai Biological Technology Co ltd
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Abstract

The invention provides an anti-inflammatory exosome preparation for treating knee joints, a preparation method and application thereof, wherein the exosome preparation comprises the following components: adipose-derived exosomes, miRNA-27b, dandelion tissue fluid extract, purslane extract and photoinduced imine cross-linked hydrogel; the preparation method comprises the following steps: s1: processing adipose tissues; s2: culturing adipose-derived mesenchymal stem cells; s3: preparing an adipose-derived mesenchymal stem cell exosome; s4: modifying an exosome; s5: preparing an anti-inflammatory exosome preparation; the prepared anti-inflammatory exosome preparation is applied to treatment of osteoarthritis. In a word, the invention has the advantages of advanced materials, perfect method, good inflammation treatment effect and the like.

Description

Anti-inflammatory exosome preparation for knee joint treatment and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medical preparations, and particularly relates to an anti-inflammatory exosome preparation for knee joint treatment, and a preparation method and application thereof.
Background
Gonarthritis is a disorder that is based on a degenerative change of pathology. The initial symptoms of most patients with gonarthritis are light, and the disease condition will gradually worsen if the patients are not treated. The main symptoms include knee pain, knee joint swelling, knee joint bounce, etc. Stiffness and coldness of knee joints are also one of symptoms of gonarthritis, and stiffness is the main symptom, fatigue, cold or slight trauma is aggravated, and severe people can have limited activity.
Exosomes are small membrane vesicles containing complex RNAs and proteins. All cultured cell types secrete exosomes and exosomes are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid and milk. Exosomes are found to have a wide variety of functions today with a large number of studies on their biological origin, their composition and transport of substances, the conduction of intercellular signals and distribution in body fluids. The function of exosomes depends on the cell type from which they are derived, and they can participate in aspects such as immune response, antigen presentation, cell migration, cell differentiation, tumor invasion, etc.
In recent years, the application of stem cell therapy in the field of regenerative medicine has become a research focus. It has been shown that mesenchymal stem cells have the potential to differentiate into various cells, promote their differentiation into chondrocytes under specific conditions, and maintain the function and stability of chondrocytes. Stem cell therapy has shown great potential in the field of regenerative medicine and was once considered the most promising therapy for repairing cartilage damage. With the progress of research, researchers have recognized that the therapeutic effects depend primarily on paracrine effects. The exosome which plays an important role in the paracrine mechanism process has the advantages of good stability, strong targeting property and low immune rejection, and can avoid the problems of teratogenicity, abnormal accumulation and the like of stem cell therapy to a certain extent. Not only stem cell-derived exosomes, but also other cell-derived exosomes can act on chondrocytes through bioactive molecules carried by the exosomes, so that the function of the chondrocytes is regulated, the proliferation of the chondrocytes is promoted, the apoptosis of the chondrocytes is inhibited, the effect of protecting the cartilage is further exerted, and the repair of the damaged cartilage of arthritis is promoted.
In the prior art, the preparation of the anti-inflammatory exosome for knee joint has no systematic preparation process, so the invention designs the anti-inflammatory exosome preparation for knee joint treatment and a preparation method and application thereof.
Disclosure of Invention
Aiming at the problems, the invention provides an anti-inflammatory exosome preparation for treating knee joints, a preparation method and application thereof.
The technical scheme of the invention is as follows: an anti-inflammatory exosome preparation for treating knee joints comprises the following components in parts by weight: 30-50 parts of adipose-derived stem cell exosomes, 3-8 parts of miRNA-27b, 8-10 parts of dandelion tissue fluid extract, 3-5 parts of purslane extract and 10-20 parts of photoinduced imine cross-linked hydrogel.
Further, the extraction process of the dandelion tissue fluid extract comprises the following steps: collecting and collecting milky white juice in dandelion stems, centrifugally separating the milky white juice on a centrifuge at the rotating speed of 5000-.
Further, the flow rate of the carbon dioxide in the supercritical carbon dioxide extraction circulation is 3-5m/s, the extraction temperature is 39-42 ℃, the extraction pressure is 13-15MPa, the extraction time is 120-160min, and the extract obtained by extraction has high effective components and high activity.
Further, the extraction process of the purslane extract comprises the following steps: cleaning and airing fresh purslane, cutting the purslane into small sections of 1-3cm, putting the small sections into a beater, adding a sodium chloride solution with the mass ratio of 2-3:1 to the purslane into the beater, starting the beater to beat the purslane into slurry, centrifugally separating the slurry in a centrifugal machine at the rotating speed of 6000-plus 9000r/min, taking supernatant after separation, distilling the supernatant with water vapor of 40-60 ℃ for 10-20min, and cooling to obtain the purslane extract which can inhibit the expression of inflammatory factors and has the effects of resisting inflammation and relieving pain.
Preferably, the mass concentration of the sodium chloride solution is 5-30%.
Furthermore, the miRNA-27b is obtained by amplifying and purifying the mesenchymal stem cell exosome, and the miRNA-27b can improve the proliferation capacity of chondrocytes under an inflammatory environment and inhibit the apoptosis of the chondrocytes.
The preparation method of the anti-inflammatory exosome preparation for treating knee joints comprises the following steps:
s1: adipose tissue treatment
Collecting a piece of adipose tissue, washing with PBS buffer solution for 3-5 times, placing in sterile operating dish, and shearing into 3-5mm with sterile scissors3Removing fibrous membranes and blood vessels in adipose tissues in the shearing process, adding PBS (phosphate buffer solution) which is over the adipose tissues into a sterile operating dish, soaking for 2-3d, and washing with PBS again to obtain the processed pure adipose tissues;
s2: adipose-derived mesenchymal stem cell culture
Transferring the pure adipose tissue obtained in the step S1 to a sterile culture dish, adding a digestion solution which is over the pure adipose tissue into the sterile culture dish, transferring the sterile culture dish to a constant-temperature water bath incubator, digesting for 15-18h at 40-45 ℃ to obtain a fat digestion solution, inoculating the fat digestion solution to a culture medium, culturing adipose mesenchymal stem cells in the constant-temperature incubator, and carrying out subculture when the fusion degree of the adipose mesenchymal stem cells reaches 80-90%;
s3: preparation of adipose-derived mesenchymal stem cell exosome
Selecting adipose-derived mesenchymal stem cells cultured to the 4 th-5 th generation to be placed in a centrifuge tube, precooling the temperature in an ultracentrifuge to 0-3 ℃, placing the centrifuge tube in an ultracentrifuge for centrifugation for 8-10min at 400g for removing the cells, placing supernatant in the centrifuge tube in a new centrifuge tube, centrifuging for 20-30min at 30000g for removing cell fragments, taking the supernatant in the centrifuge tube for filtration, removing the cell particles, placing the filtered supernatant in the new centrifuge tube, centrifuging for 90-100min at 120000g and 125000g for removing the supernatant in the centrifuge tube, and obtaining sediment at the bottom of the centrifuge tube as adipose-derived mesenchymal stem cell exosomes;
s4: exosome modification
Re-suspending the adipose-derived mesenchymal stem cell exosomes obtained in the step S3 in PBS buffer solution to prepare an exosome suspension with the mass concentration of 15-20mg/ml, adding miRNA-27b into the exosome suspension according to the mass part ratio, uniformly shaking to obtain a suspension, adding culture solution into the suspension, and then placing the suspension in an incubator to cultivate for 12-24 hours to obtain the miRNA-27b modified adipose-derived mesenchymal stem cell exosomes;
s5: preparation of anti-inflammatory exosome preparation
And (4) re-suspending the miRNA-27b modified adipose-derived mesenchymal stem cell exosomes obtained in the step (S4) in a PBS buffer solution to prepare a modified exosome suspension with the mass concentration of 35-40mg/ml, adding the photoinduced imine cross-linked hydrogel, the dandelion tissue fluid extract and the purslane extract into the modified exosome suspension according to the mass part ratio, and uniformly mixing in a water bath at the temperature of 35-38 ℃ to obtain the anti-inflammatory exosome preparation.
Preferably, the preparation steps of the PBS buffer solution are as follows: NaCl, KCl and Na with the mass ratio of 80:2:15:52HPO4、KH2PO4Fully dissolving the mixed solution in deionized water to obtain a mixed solution, adjusting the pH of the mixed solution to 7.1-7.5 by using HCl, and then continuously adding the deionized water into the mixed solution to fix the volume.
Preferably, the digestion solution is prepared from calcium chloride, magnesium sulfate, lipase and physiological saline in a mass ratio of 1:1:3: 5.
Further, the prepared anti-inflammatory type exosome preparation is applied to treatment of osteoarthritis.
The invention has the beneficial effects that: the invention provides an anti-inflammatory exosome preparation for treating knee joints, a preparation method and application, and is particularly suitable for repairing cartilage tissue cells in an inflammatory environment. In a word, the invention has the advantages of advanced materials, perfect method, good inflammation treatment effect and the like.
Drawings
FIG. 1 is an electron micrograph of an anti-inflammatory exosome formulation prepared in example 6 of the present invention.
Detailed Description
For the convenience of understanding the technical solution of the present invention, the following description is made with reference to fig. 1 and the specific embodiments, which are not intended to limit the scope of the present invention.
Example 1: an anti-inflammatory exosome preparation for treating knee joints comprises the following components in parts by weight: 30 parts of adipose mesenchymal stem cell-derived exosome, 3 parts of miRNA-27b obtained by amplifying and purifying mesenchymal stem cell exosome, 8 parts of dandelion tissue fluid extract, 3 parts of purslane extract and 10 parts of photoinduced imine crosslinked hydrogel,
the extraction process of the dandelion tissue fluid extract comprises the following steps: collecting and collecting milky white juice in the stem and stem of dandelion, centrifuging the milky white juice on a centrifuge at a rotating speed of 5000r/min, centrifuging, taking supernatant, adding sodium chloride solution with a mass ratio of 4:1 to the supernatant for leaching for 3h to obtain leaching liquor, wherein the mass concentration of the sodium chloride solution is 5-30%, performing supercritical carbon dioxide extraction circulation on the leaching liquor, the carbon dioxide flow rate of the supercritical carbon dioxide extraction circulation is 3m/s, the extraction temperature is 39 ℃, the extraction pressure is 13MPa, the extraction time is 120min, and obtaining dandelion tissue fluid extract after extraction is finished,
the extraction process of the purslane extract comprises the following steps: cleaning and air drying fresh purslane, cutting into 3cm small segments, placing into a pulping machine, adding a sodium chloride solution with the mass ratio of 2:1 to the purslane into the pulping machine, wherein the mass concentration of the sodium chloride solution is 5%, starting the pulping machine to pulp the purslane, centrifugally separating the pulp in a centrifugal machine at the rotating speed of 6000r/min, taking supernatant after separation, distilling the supernatant with water vapor at 40 ℃ for 10min, and cooling to obtain the purslane extract.
Example 2: an anti-inflammatory exosome preparation for treating knee joints comprises the following components in parts by weight: 40 parts of adipose mesenchymal stem cell-derived exosome, 5 parts of miRNA-27b obtained by amplifying and purifying mesenchymal stem cell exosome, 9 parts of dandelion tissue fluid extract, 4 parts of purslane extract and 15 parts of photoinduced imine crosslinked hydrogel,
the extraction process of the dandelion tissue fluid extract comprises the following steps: collecting and collecting milky white juice in the stem and stem of dandelion, centrifuging the milky white juice at 7000r/min on a centrifuge, centrifuging, collecting supernatant, adding sodium chloride solution with the mass ratio of 5:1 into the supernatant, leaching for 4h to obtain leaching solution with the mass concentration of 20%, performing supercritical carbon dioxide extraction circulation on the leaching solution, wherein the carbon dioxide flow rate of the supercritical carbon dioxide extraction circulation is 4/s, the extraction temperature is 40 ℃, the extraction pressure is 14MPa, the extraction time is 140min, and obtaining dandelion tissue fluid extract after extraction is finished,
the extraction process of the purslane extract comprises the following steps: cleaning and drying fresh purslane, cutting into 2cm small segments, putting into a pulping machine, adding a sodium chloride solution with the mass ratio of 3:1 to the purslane into the pulping machine, wherein the mass concentration of the sodium chloride solution is 20%, starting the pulping machine to pulp the purslane, centrifugally separating the pulp in a centrifugal machine at the rotating speed of 8000r/min, taking supernatant after separation, distilling the supernatant with water vapor at the temperature of 50 ℃ for 15min, and cooling to obtain the purslane extract.
Example 3: an anti-inflammatory exosome preparation for treating knee joints comprises the following components in parts by weight: 50 parts of adipose-derived exosome from mesenchymal stem cells, 8 parts of miRNA-27b obtained by amplifying and purifying the mesenchymal stem cell exosome, 10 parts of dandelion tissue fluid extract, 5 parts of purslane extract and 20 parts of photoinduced imine crosslinked hydrogel.
The extraction process of the dandelion tissue fluid extract comprises the following steps: collecting and collecting milky white juice in the stems of the dandelion, centrifugally separating the milky white juice on a centrifuge at the rotating speed of 8000r/min, taking supernatant after centrifugation, adding a sodium chloride solution with the mass ratio of 6:1 to the supernatant for leaching for 5h to obtain a leaching solution, wherein the mass concentration of the sodium chloride solution is 30%, performing supercritical carbon dioxide extraction circulation on the leaching solution, the carbon dioxide flow rate of the supercritical carbon dioxide extraction circulation is 5m/s, the extraction temperature is 42 ℃, the extraction pressure is 15MPa, the extraction time is 160min, and obtaining the dandelion tissue fluid extract after extraction.
The extraction process of the purslane extract comprises the following steps: cleaning and drying fresh purslane, cutting into 3cm small sections, putting into a pulping machine, adding a sodium chloride solution with the mass ratio of 3:1 to the purslane into the pulping machine, wherein the mass concentration of the sodium chloride solution is 30%, starting the pulping machine to pulp the purslane, centrifugally separating the pulp in a centrifugal machine at the rotating speed of 9000r/min, taking supernatant after separation, distilling the supernatant with 60 ℃ steam for 20min, and cooling to obtain the purslane extract.
Example 4: example 3 a method for preparing an anti-inflammatory exosome formulation for knee treatment, comprising the steps of:
s1: adipose tissue treatment
Taking a piece of adipose tissue, washing the adipose tissue with PBS buffer solution for 3 times, placing in a sterile operating dish, and shearing the adipose tissue into 5mm with a sterile scissors3Removing fibrous membranes and blood vessels in adipose tissues in the shearing process, adding PBS (phosphate buffer solution) which is over the adipose tissues into a sterile operating dish, soaking for 2d, and washing with the PBS buffer solution again to obtain the processed pure adipose tissues;
s2: adipose-derived mesenchymal stem cell culture
Transferring the pure adipose tissue obtained in the step S1 to a sterile culture dish, adding a digestion solution which is over the pure adipose tissue into the sterile culture dish, transferring the sterile culture dish to a constant-temperature water bath incubator, digesting for 15 hours at 40 ℃ to obtain a fat digestion solution, inoculating the fat digestion solution to a culture medium, culturing adipose-derived mesenchymal stem cells in the constant-temperature incubator, and performing subculture when the fusion degree of the adipose-derived mesenchymal stem cells reaches 80%;
s3: preparation of adipose-derived mesenchymal stem cell exosome
Selecting adipose-derived mesenchymal stem cells cultured to the 4 th generation in a centrifuge tube, precooling the temperature in an ultracentrifuge to 0 ℃, placing the centrifuge tube in the ultracentrifuge tube, centrifuging for 8min to remove the cells at 300g, taking supernatant in the centrifuge tube in a new centrifuge tube, centrifuging for 20min at 30000g to remove cell debris, taking supernatant in the centrifuge tube, filtering to remove cell particles, placing the filtered supernatant in the new centrifuge tube, centrifuging for 90min at 120000g, removing the supernatant in the centrifuge tube, and obtaining sediment at the bottom of the centrifuge tube, namely adipose-derived mesenchymal stem cell exosomes;
s4: exosome modification
Re-suspending the adipose-derived mesenchymal stem cell exosomes obtained in the step S3 in PBS buffer solution to prepare an exosome suspension with the mass concentration of 15mg/ml, adding miRNA-27b into the exosome suspension according to the mass part ratio, uniformly shaking to obtain a suspension, adding culture solution into the suspension, and then placing the suspension in an incubator to cultivate for 12 hours to obtain the adipose-derived mesenchymal stem cell exosomes modified by miRNA-27 b;
s5: preparation of anti-inflammatory exosome preparation
And (4) re-suspending the miRNA-27b modified adipose-derived mesenchymal stem cell exosomes obtained in the step (S4) in a PBS buffer solution to prepare a modified exosome suspension with the mass concentration of 35mg/ml, adding the photoinduced imine cross-linked hydrogel, the dandelion tissue fluid extract and the purslane extract into the modified exosome suspension according to the mass part ratio, and uniformly mixing the mixture in a water bath at the temperature of 35 ℃ to obtain the anti-inflammatory exosome preparation.
Example 5: example 3 a method for preparing an anti-inflammatory exosome formulation for knee treatment, comprising the steps of:
s1: adipose tissue treatment
Taking a piece of adipose tissue, washing the adipose tissue with PBS buffer solution for 4 times, placing the washed adipose tissue in a sterile operating dish, and shearing the adipose tissue into 4mm by using a sterile scissors3Removing fibrous membranes and blood vessels in adipose tissues in the shearing process, adding PBS (phosphate buffer solution) which is over the adipose tissues into a sterile operating dish, soaking for 3 days, and washing with the PBS buffer solution again to obtain the processed pure adipose tissues;
s2: adipose-derived mesenchymal stem cell culture
Transferring the pure adipose tissue obtained in the step S1 to a sterile culture dish, adding a digestion solution which is over the pure adipose tissue into the sterile culture dish, transferring the sterile culture dish to a constant-temperature water bath incubator, digesting for 17 hours at the temperature of 43 ℃ to obtain a fat digestion solution, inoculating the fat digestion solution to a culture medium, culturing adipose mesenchymal stem cells in the constant-temperature incubator, and carrying out subculture when the fusion degree of the adipose mesenchymal stem cells reaches 85%;
s3: preparation of adipose-derived mesenchymal stem cell exosome
Selecting adipose-derived mesenchymal stem cells cultured to the 5 th generation in a centrifuge tube, precooling the temperature in an ultracentrifuge to 2 ℃, placing the centrifuge tube in the ultracentrifuge tube, centrifuging 350g for 9min to remove the cells, taking supernatant in the centrifuge tube in a new centrifuge tube, centrifuging 32500g for 25min to remove cell debris, taking supernatant in the centrifuge tube, filtering, removing cell particles, placing the filtered supernatant in the new centrifuge tube, centrifuging 123000g for 95min, removing the supernatant in the centrifuge tube, and obtaining sediment at the bottom of the centrifuge tube, namely adipose-derived mesenchymal stem cell exosomes;
s4: exosome modification
Re-suspending the adipose-derived mesenchymal stem cell exosomes obtained in the step S3 in PBS buffer solution to prepare an exosome suspension with the mass concentration of 18mg/ml, adding miRNA-27b into the exosome suspension according to the mass part ratio, uniformly shaking to obtain a suspension, adding culture solution into the suspension, and then placing the suspension in an incubator to cultivate for 18 hours to obtain the adipose-derived mesenchymal stem cell exosomes modified by miRNA-27 b;
s5: preparation of anti-inflammatory exosome preparation
And (4) re-suspending the miRNA-27b modified adipose-derived mesenchymal stem cell exosomes obtained in the step (S4) in a PBS buffer solution to prepare a modified exosome suspension with the mass concentration of 38mg/ml, adding the photoinduced imine cross-linked hydrogel, the dandelion tissue fluid extract and the purslane extract into the modified exosome suspension according to the mass part ratio, and uniformly mixing in a water bath at 37 ℃ to obtain the anti-inflammatory exosome preparation.
Example 6: example 3 a method for preparing an anti-inflammatory exosome formulation for knee treatment, comprising the steps of:
s1: adipose tissue treatment
Taking a piece of adipose tissue, washing the adipose tissue with PBS buffer solution for 5 times, placing in a sterile operating dish, and shearing the adipose tissue into 3mm with a sterile scissors3Removing fibrous membranes and blood vessels in adipose tissues in the shearing process, adding PBS (phosphate buffer solution) which is over the adipose tissues into a sterile operating dish, soaking for 3 days, and washing with the PBS buffer solution again to obtain the processed pure adipose tissues;
s2: adipose-derived mesenchymal stem cell culture
Transferring the pure adipose tissue obtained in the step S1 to a sterile culture dish, adding a digestion solution which is over the pure adipose tissue into the sterile culture dish, transferring the sterile culture dish to a constant-temperature water bath incubator, digesting at 45 ℃ for 18h to obtain a fat digestion solution, inoculating the fat digestion solution to a culture medium, culturing adipose-derived mesenchymal stem cells in the constant-temperature incubator, and performing subculture when the fusion degree of the adipose-derived mesenchymal stem cells reaches 90%;
s3: preparation of adipose-derived mesenchymal stem cell exosome
Selecting adipose-derived mesenchymal stem cells cultured to the 4 th-5 th generation to be placed in a centrifugal tube, precooling the temperature in an ultracentrifuge to 3 ℃, placing the centrifugal tube in the ultracentrifuge for 400g and centrifuging for 10min to remove the cells, taking supernatant in the centrifugal tube to be placed in a new centrifugal tube, 35000g and centrifuging for 30min to remove cell debris, taking supernatant in the centrifugal tube and filtering, removing cell particles, placing the filtered supernatant in the new centrifugal tube, 125000g and centrifuging for 100min to remove the supernatant in the centrifugal tube, and taking sediment at the bottom of the centrifugal tube as adipose-derived mesenchymal stem cell exosomes;
s4: exosome modification
Re-suspending the adipose-derived mesenchymal stem cell exosomes obtained in the step S3 in PBS buffer solution to prepare an exosome suspension with the mass concentration of 20mg/ml, adding miRNA-27b into the exosome suspension according to the mass part ratio, uniformly shaking to obtain a suspension, adding culture solution into the suspension, and then placing the suspension in an incubator to cultivate for 24 hours to obtain the adipose-derived mesenchymal stem cell exosomes modified by miRNA-27 b;
s5: preparation of anti-inflammatory exosome preparation
And (2) suspending the miRNA-27b modified adipose-derived mesenchymal stem cell exosomes obtained in the step (S4) in PBS buffer solution to prepare a modified exosome suspension with the mass concentration of 40mg/ml, adding the photoinduced imine cross-linked hydrogel, the dandelion tissue fluid extract and the purslane extract into the modified exosome suspension according to the mass part ratio, and uniformly mixing the materials in a water bath at 38 ℃ to obtain the anti-inflammatory exosome preparation, wherein an electron microscope image of the anti-inflammatory exosome preparation is shown in figure 1.
In the above examples 4 to 6, the preparation of PBS buffer solution comprises the following steps: NaCl, KCl and Na with the mass ratio of 80:2:15:52HPO4、KH2PO4Fully dissolving the mixed solution in deionized water to obtain a mixed solution, adjusting the pH of the mixed solution to 7.2 by using HCl, and then continuously adding deionized water into the mixed solution to a constant volume;
the digestion solution is prepared from calcium chloride, magnesium sulfate, lipase and physiological saline in a mass ratio of 1:1:3: 5.
Experimental example 1: study on the Effect of the raw Material ratios on the anti-inflammatory exosome preparation
Experimental materials: the raw materials were taken in the ratios of the raw materials provided in examples 1 to 3, respectively, and the anti-inflammatory exosome formulations were prepared using the methods provided in example 6, respectively.
Subject: 10 rats, female and male, with 10 weeks of age, were injected with knee joint inflammation culture cell fluid provided by a hospital every other day, 1ml per injection, 1 week every time, 20 rats were randomly divided into 4 groups, numbered first, second, third, and fourth groups.
The experimental conditions are as follows: the anti-inflammatory exosome preparation prepared in example 1 was injected daily into the knee joint of the first group of rats in an amount of 1ml per injection for one week,
the anti-inflammatory exosome preparation prepared in example 2 was injected daily into the knee joint of the second group of rats in an amount of 1ml each time for one week,
the anti-inflammatory exosome preparation prepared in example 3 was injected daily into the knee joint of the third group of rats in an amount of 1ml per injection for one week,
the fourth group of rats was not treated.
The experimental results are as follows: the knee joints of rats and tissue fluid are taken for detection and analysis, and inflammatory cells at the knee joints of the rats of the first group, the second group and the third group are all obviously reduced, new cartilage tissues appear at the knee joints, the effect of the rat of the third group is most obvious, inflammatory cells at the knee joints of the rats of the fourth group slowly grow, and the cartilage cells are continuously apoptotic, so that the anti-inflammatory exosome preparation prepared from the raw materials provided by the embodiments 1-3 of the invention has the effect of treating the inflammation at the knee joints, and the embodiment 3 is the optimal raw material ratio.
Experimental example 2: research on the effect of the preparation method on the anti-inflammatory exosome preparation
Experimental materials: taking three parts of raw materials according to the raw material proportion provided in example 3, and preparing anti-inflammatory exosome preparations according to the methods provided in examples 4, 5 and 6 respectively;
subject: 15 rats, female and male, 10-year-old rats were injected with knee joint inflammation culture cell fluid provided by a hospital every other day, 1ml of the injection amount is injected every time, 1 week is injected, and 30 rats are randomly divided into 3 groups, and the numbers of the groups are fifth, sixth and seventh.
The experimental conditions are as follows: the anti-inflammatory exosome preparation prepared in example 4 was injected daily into the knee joint of the fifth group of rats in an amount of 1ml per injection for one week,
the anti-inflammatory exosome preparation prepared in example 5 was injected daily into the knee joint of the rats of the sixth group, 1ml per injection, continuously injected for one week,
the anti-inflammatory exosome preparation prepared in example 6 was injected daily into the knee joint of the seventh group of rats in an amount of 1ml per injection for one week continuously.
The experimental results are as follows: the knee joints of rats and tissue fluid are taken for detection and analysis, and inflammatory cells at the knee joints of the rats in the fifth, sixth and seventh groups are all obviously reduced, and newborn cartilage tissues appear at the knee joints, but the reduction rate of the inflammatory cells of the rats in the seventh group is more than two times that of the rats in the other two groups, and the size of the newborn cartilage tissues is more than two times that of the rats in the other two groups, so that the optimal preparation method of the anti-inflammatory exosome preparation is provided in example 6.

Claims (10)

1. The anti-inflammatory exosome preparation for treating the knee joint is characterized by comprising the following components in parts by weight: 30-50 parts of adipose-derived stem cell exosomes, 3-8 parts of miRNA-27b, 8-10 parts of dandelion tissue fluid extract, 3-5 parts of purslane extract and 10-20 parts of photoinduced imine cross-linked hydrogel.
2. The anti-inflammatory exosome preparation for knee joint treatment according to claim 1, wherein the extraction process of the dandelion tissue fluid extract comprises the following steps: collecting and collecting milky white juice in the dandelion stems, centrifugally separating the milky white juice on a centrifuge at the rotating speed of 5000-.
3. The preparation of an anti-inflammatory exosome for knee joint treatment according to claim 2, wherein the sodium chloride solution is at a mass concentration of 5-30%.
4. The anti-inflammatory exosome preparation for knee joint treatment according to claim 1, wherein the extraction process of the purslane extract comprises the following steps: cleaning and drying fresh purslane, cutting into small segments of 1-3cm, putting into a beater, adding a sodium chloride solution with the mass ratio of 2-3:1 to the purslane into the beater, starting the beater to beat the purslane into slurry, centrifugally separating the slurry in a centrifugal machine at the rotating speed of 6000-9000r/min, taking supernatant after separation, distilling the supernatant with water vapor at 40-60 ℃ for 10-20min, and cooling to obtain the purslane extract.
5. The preparation of an anti-inflammatory exosome for knee joint treatment according to claim 4, wherein the mass concentration of the sodium chloride solution is 5-30%.
6. The anti-inflammatory exosome preparation for knee joint treatment according to claim 1, wherein the miRNA-27b is obtained by amplifying and purifying mesenchymal stem cell exosomes.
7. The method for preparing an anti-inflammatory exosome formulation for knee joint treatment according to claim 1, comprising the steps of:
s1: adipose tissue treatment
Collecting a piece of adipose tissue, washing with PBS buffer solution for 3-5 times, placing in sterile operating dish, and shearing into 3-5mm with sterile scissors3Removing fibrous membranes and blood vessels in adipose tissues in the shearing process, adding PBS (phosphate buffer solution) which is over the adipose tissues into a sterile operating dish, soaking for 2-3d, and washing with PBS again to obtain the processed pure adipose tissues;
s2: adipose-derived mesenchymal stem cell culture
Transferring the pure adipose tissue obtained in the step S1 to a sterile culture dish, adding a digestion solution which is over the pure adipose tissue into the sterile culture dish, transferring the sterile culture dish to a constant-temperature water bath incubator, digesting for 15-18h at 40-45 ℃ to obtain a fat digestion solution, inoculating the fat digestion solution to a culture medium, culturing adipose mesenchymal stem cells in the constant-temperature incubator, and carrying out subculture when the fusion degree of the adipose mesenchymal stem cells reaches 80-90%;
s3: preparation of adipose-derived mesenchymal stem cell exosome
Selecting adipose-derived mesenchymal stem cells cultured to the 4 th-5 th generation to be placed in a centrifuge tube, precooling the temperature in an ultracentrifuge to 0-3 ℃, placing the centrifuge tube in an ultracentrifuge for centrifugation for 8-10min at 400g for removing the cells, placing supernatant in the centrifuge tube in a new centrifuge tube, centrifuging for 20-30min at 30000g for removing cell fragments, taking the supernatant in the centrifuge tube for filtration, removing the cell particles, placing the filtered supernatant in the new centrifuge tube, centrifuging for 90-100min at 120000g and 125000g for removing the supernatant in the centrifuge tube, and obtaining sediment at the bottom of the centrifuge tube as adipose-derived mesenchymal stem cell exosomes;
s4: exosome modification
Re-suspending the adipose-derived mesenchymal stem cell exosomes obtained in the step S3 in PBS buffer solution to prepare an exosome suspension with the mass concentration of 15-20mg/ml, adding miRNA-27b into the exosome suspension according to the mass part ratio, uniformly shaking to obtain a suspension, adding culture solution into the suspension, and then placing the suspension in an incubator to cultivate for 12-24 hours to obtain the miRNA-27b modified adipose-derived mesenchymal stem cell exosomes;
s5: preparation of anti-inflammatory exosome preparation
And (4) re-suspending the miRNA-27b modified adipose-derived mesenchymal stem cell exosomes obtained in the step (S4) in a PBS buffer solution to prepare a modified exosome suspension with the mass concentration of 35-40mg/ml, adding the photoinduced imine cross-linked hydrogel, the dandelion tissue fluid extract and the purslane extract into the modified exosome suspension according to the mass part ratio, and uniformly mixing in a water bath at the temperature of 35-38 ℃ to obtain the anti-inflammatory exosome preparation.
8. The method for preparing an anti-inflammatory exosome preparation for knee joint treatment according to claim 7, wherein the PBS buffer is prepared by the steps of: NaCl, KCl and Na with the mass ratio of 80:2:15:52HPO4、KH2PO4Fully dissolving in deionized water to obtainAnd (3) mixing the solution, adjusting the pH of the mixed solution to 7.1-7.5 by using HCl, and then continuously adding deionized water into the mixed solution to a constant volume.
9. The method for preparing an anti-inflammatory exosome preparation for treating knee joint according to claim 7, wherein the digestion solution is prepared from calcium chloride, magnesium sulfate, lipase and physiological saline in a mass ratio of 1:1:3: 5.
10. Use of an anti-inflammatory exosome formulation for knee treatment prepared according to the method of claim 7, characterised in that the anti-inflammatory exosome formulation is used in the treatment of osteoarthritis.
CN202110496557.9A 2021-05-07 2021-05-07 Anti-inflammatory exosome preparation for knee joint treatment and preparation method and application thereof Pending CN113274421A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112007049A (en) * 2020-09-21 2020-12-01 济南磐升生物技术有限公司 Stem cell exosome composition for treating knee osteoarthritis

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Title
生物探索: "Genomeweb：回顾基因产业十大热门新闻，NIPT假阳性赶超精准医", 《WWW.3GBIO.COM.CN/HTML/2016/NEWS_0104/1606.HTML》 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112007049A (en) * 2020-09-21 2020-12-01 济南磐升生物技术有限公司 Stem cell exosome composition for treating knee osteoarthritis

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