CN117100785A - Composition containing stem cell exosomes and application of composition in preparation of drugs with liver injury treatment effect - Google Patents
Composition containing stem cell exosomes and application of composition in preparation of drugs with liver injury treatment effect Download PDFInfo
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- CN117100785A CN117100785A CN202311106377.0A CN202311106377A CN117100785A CN 117100785 A CN117100785 A CN 117100785A CN 202311106377 A CN202311106377 A CN 202311106377A CN 117100785 A CN117100785 A CN 117100785A
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Classifications
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Abstract
The invention discloses a composition containing stem cell exosomes and application thereof in preparing a medicament with a liver injury treatment effect, wherein the composition containing stem cell exosomes comprises the following components in parts by weight: 0.1-0.5 part of umbilical mesenchymal stem cell exosome, 3-6 parts of kudzu root extract, 0.2-1 part of honey, 0.06-0.32 part of compound vitamin, 0.3-0.7 part of olive oil, 0.08-0.2 part of starch, 0.03-0.15 part of glutamic acid, 0.03-0.15 part of cysteine, 0.03-0.15 part of glycine, 0.05-0.35 part of glucuronolactone, 0.05-0.18 part of sodium carboxymethyl cellulose and 22-37 parts of deionized water. The stem cell exosome can regulate apoptosis, growth, proliferation and differentiation pathways through the transcriptome and proteome of the receptor cells, so that the occurrence and development of liver diseases are delayed, the pueraria extract is rich in various flavonoid and isoflavone substances, can promote liver cell regeneration, and can provide needed nutrition for the liver in cooperation with other components, thereby effectively treating liver injury.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a composition containing stem cell exosomes and application thereof in preparing a medicine with a liver injury treatment effect.
Background
The liver is one of five important organs of the human body and plays a key role in metabolism of the human body. Viral infection, drug intoxication, lipid deposition and autoimmune response various liver toxicity causes liver injury, and sustained liver injury causes excessive accumulation of extracellular matrix, thereby losing normal structure and progressing to liver fibrosis.
The stem cell exosome is a membrane-derived nanoscale vesicle, has important biological functions of reducing apoptosis, reducing inflammatory reaction, improving tissue repair and the like, and can regulate apoptosis, growth, proliferation and differentiation pathways by changing extracellular matrix, transcriptome and proteome of receptor cells, thereby delaying occurrence and development of liver diseases.
Research shows that the exosome has better safety and better treatment effect than cell transplantation treatment, and based on the safety, how to prepare a medicine containing a stem cell exosome composition becomes one direction of current research.
Disclosure of Invention
The invention firstly provides a composition containing stem cell exosomes and application thereof in preparing a medicament with a liver injury treatment effect, wherein the composition can synergistically provide needed nutrition for the liver, promote liver cell regeneration and effectively treat liver injury.
The invention solves the technical problems by adopting the following technical scheme:
the invention provides a composition containing stem cell exosomes, which is characterized by comprising the following components in parts by weight: 0.1-0.5 part of umbilical mesenchymal stem cell exosome, 3-6 parts of kudzu root extract, 0.2-1 part of honey, 0.06-0.32 part of compound vitamin, 0.3-0.7 part of olive oil, 0.08-0.2 part of starch, 0.03-0.15 part of glutamic acid, 0.03-0.15 part of cysteine, 0.03-0.15 part of glycine, 0.05-0.35 part of glucuronolactone, 0.05-0.18 part of sodium carboxymethyl cellulose and 22-37 parts of deionized water.
Preferably, the composition containing stem cell exosomes comprises, in parts by weight: 0.2-0.4 part of umbilical mesenchymal stem cell exosome, 4-5 parts of kudzu root extract, 0.4-0.8 part of honey, 0.18-21 parts of compound vitamin, 0.4-0.6 part of olive oil, 0.12-0.16 part of starch, 0.07-0.11 part of glutamic acid, 0.07-0.11 part of cysteine, 0.07-0.11 part of glycine, 0.17-0.23 part of glucuronolactone, 0.09-0.14 part of sodium carboxymethyl cellulose and 27-32 parts of deionized water.
Preferably, the composition containing stem cell exosomes comprises, in parts by weight: 0.3 part of umbilical mesenchymal stem cell exosome, 4 parts of radix puerariae extract, 0.6 part of honey, 0.2 part of compound vitamin, 0.5 part of olive oil, 0.14 part of starch, 0.09 part of glutamic acid, 0.09 part of cysteine, 0.09 part of glycine, 0.21 part of glucuronolactone, 0.12 part of sodium carboxymethyl cellulose and 29 parts of deionized water.
Preferably, the preparation method of the umbilical mesenchymal stem cell exosome comprises the following steps:
s1, taking umbilical cord tissue, placing the umbilical cord tissue into PBS buffer solution for cleaning for 3 times, each time for 8-10min, removing the artery and vein to obtain Walton gum, shearing the Walton gum into tissue blocks with the size of 4-6mm, flushing the tissue blocks with deionized water for 15-30min, and then cleaning the tissue blocks with the PBS buffer solution for 5 times, each time for 5-8min to obtain the tissue blocks;
s2, mixing the tissue blocks and collagenase I solution according to a weight ratio of 1: (0.2-0.28) and reacting for 15-30min to obtain a mixture;
s3, centrifuging the mixture at 800-1200r/min for 20-30min, passing through a 0.4 mu m sterile filter membrane to obtain cell sediment, and washing with PBS buffer solution for 3 times;
s4, washing the cell sediment for 3 times by using a balanced salt buffer solution, re-suspending the cells by using a balanced salt buffer solution with the volume of 2 times, and purifying the cell suspension by using a density gradient centrifugation method to obtain umbilical cord mesenchymal primordial stem cells;
s5, inoculating umbilical cord mesenchymal primordial stem cells into a culture medium at a density of 1X 10/ml, placing the culture medium in an incubator for culturing for 2-4d, controlling the passage times within P10, and collecting culture solution supernatant, namely first supernatant, when the cell fusion rate reaches 80-90%;
s6, centrifuging the first clear liquid for 10-30min at the temperature of 4 ℃ and the speed of 1800r/min, collecting the supernatant, ultrafiltering, concentrating to remove small molecules, and filtering and sterilizing by a sterile filter membrane with the thickness of 0.3 mu m to obtain umbilical mesenchymal cell sediment;
s7, washing umbilical cord mesenchymal cell sediment 3 times by using PBS buffer solution, inoculating the umbilical cord mesenchymal cell sediment into a serum-free culture medium at the density of 1X 10/ml, placing the umbilical cord mesenchymal cell sediment into an incubator for culturing for 42-48 hours, and collecting culture solution supernatant, namely second supernatant;
s8, transferring the second clear liquid to a sucrose heavy water density pad with the mass concentration of 30%, centrifuging for 120min at 4 ℃ by a density gradient of 80000-160000g, and collecting a bottom buffer pad;
s9, washing the bottom buffer cushion for 3 times by using PBS solution, performing ultrafiltration concentration to remove small molecules, and performing filter sterilization by using a sterile filter membrane with the diameter of 0.25 mu m to obtain umbilical mesenchymal stem cell exosomes.
As a preferred embodiment, the PBS buffer contains 1-3wt% penicillin and 1.2-3.6wt% streptomycin sulfate; the culture medium is DMEM culture medium containing 8-12wt% FBS; the culture conditions of the incubator are 36-38 ℃ and 4-6% CO 2 Saturated humidity;
preferably, the preparation method of the kudzuvine root extract comprises the following steps:
s21, cleaning the kudzuvine root with deionized water, carrying out ultrasonic treatment at 100W and 20 ℃ for 15min, drying, and crushing to 20-40 meshes to obtain kudzuvine root powder;
s22, mixing radix puerariae powder and 65-85% ethanol solution according to a weight ratio of 1: (5-8) mixing, leaching at 40-50deg.C for 30-90min, centrifuging, and filtering to obtain first extractive solution;
s23, mixing the first extracting solution and activated carbon according to the weight ratio of 1: (0.006-0.018), centrifuging, and filtering to obtain second extractive solution;
s24, mixing the second extracting solution with 1.5% chitosan solution according to the weight ratio of 1: (0.002-0.01), mixing, centrifuging, and filtering to obtain a third extractive solution;
s25, introducing the third extracting solution into an adsorption column filled with macroporous resin, eluting at the speed of 2-3BV/h, flushing the adsorption column with deionized water with the volume of 3 times of the resin column at the flow rate of 1.5-2.5V/h and ethanol solution with the volume of 2 times of 50wt% of the resin column at the flow rate of 1-3BV/h, and collecting the ethanol solution to obtain the radix puerariae extract.
As a preferable scheme, the centrifugation condition is 25 ℃,12000 r/min and the centrifugation time is 20min; the pore diameter of the filter membrane is 0.3 mu m; the macroporous adsorption resin is HPD-100.
Preferably, the compound vitamin consists of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 according to the weight ratio of 1.5:0.5:1:2:1.
Preferably, the preparation method of the composition containing stem cell exosomes comprises the following steps:
s31, dissolving honey, compound vitamin, olive oil, starch, glutamic acid, cysteine, glycine, glucuronolactone and sodium carboxymethyl cellulose in deionized water, and stirring at 45 ℃ and 240-360rmp for 30-45min to completely dissolve the mixture to obtain a mixture A;
s32, adding umbilical mesenchymal stem cell exosomes and radix puerariae extract into the mixture A, and stirring at 40 ℃ and 120-180rmp for 15-30min to obtain a mixture B;
s33, refluxing the mixture B at 37 ℃ for 1-2h, filtering, concentrating to a relative density of 1.5, and freeze-drying to obtain the composition containing stem cell exosomes.
The invention also provides application of the composition containing stem cell exosomes in preparing a medicament with a liver injury treatment effect.
The invention has the beneficial effects that:
the composition containing the stem cell exosome comprises the components of compound vitamins, amino acids and the like, the stem cell exosome regulates cell growth and proliferation by changing the transcriptome and proteome of receptor cells, and the radix puerariae extract synergistically promotes liver cell regeneration to provide substances and energy required by normal physiological activities for the liver, so that liver injury is effectively treated.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below in connection with specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1.
The composition containing the stem cell exosomes of the embodiment comprises the following components in parts by weight: 0.1 part of umbilical mesenchymal stem cell exosome, 3 parts of radix puerariae extract, 0.2 part of honey, 0.06 part of compound vitamin, 0.3 part of olive oil, 0.08 part of starch, 0.03 part of glutamic acid, 0.03 part of cysteine, 0.03 part of glycine, 0.05 part of glucuronolactone, 0.05 part of sodium carboxymethyl cellulose and 22 parts of deionized water.
The preparation method of the umbilical cord mesenchymal stem cell exosome of the embodiment comprises the following steps:
s1, taking umbilical cord tissue, putting the umbilical cord tissue into PBS buffer solution for cleaning for 3 times for 8min each time, removing artery and vein to obtain Walton gum, shearing the Walton gum into tissue blocks with the size of 4mm, flushing the tissue blocks with deionized water for 15min, and then cleaning the tissue blocks with the PBS buffer solution for 5 times for 5min each time to obtain tissue blocks;
s2, mixing the tissue blocks and collagenase I solution according to a weight ratio of 1:0.2, and reacting for 15min to obtain a mixture;
s3, centrifuging the mixture at 800r/min for 20min, passing through a 0.4 mu m sterile filter membrane to obtain cell sediment, and washing with PBS buffer solution for 3 times;
s4, washing the cell sediment for 3 times by using a balanced salt buffer solution, re-suspending the cells by using a balanced salt buffer solution with the volume of 2 times, and purifying the cell suspension by using a density gradient centrifugation method to obtain umbilical cord mesenchymal primordial stem cells;
s5, inoculating umbilical cord mesenchymal primordial stem cells into a culture medium at a density of 1X 10/ml, placing into an incubator for culturing at 36 ℃ and 4% CO 2 Culturing under saturated humidity for 2d, and controlling passage times within P10 for cell fusionWhen the rate reaches 80%, collecting culture solution supernatant, namely first clear solution;
s6, centrifuging the first clear liquid for 10min at the temperature of 4 ℃ and the speed of 1800r/min, collecting the supernatant, ultrafiltering, concentrating to remove small molecules, and filtering and sterilizing by a sterile filter membrane with the thickness of 0.3 mu m to obtain umbilical mesenchymal cell sediment;
s7, washing umbilical cord mesenchymal cell sediment with PBS buffer solution for 3 times, inoculating into serum-free culture medium at a density of 1×10/ml, placing into an incubator for culturing, and culturing at 36 ℃ with 4% CO 2 Culturing for 42h under saturated humidity, and collecting culture solution supernatant, namely second supernatant;
s8, transferring the second clear liquid onto a sucrose heavy water density pad with the mass concentration of 30%, centrifuging for 120min at 4 ℃ by a density gradient of 80000g, and collecting a bottom buffer pad;
s9, washing the bottom buffer cushion for 3 times by using PBS solution, performing ultrafiltration concentration to remove small molecules, and performing filter sterilization by using a sterile filter membrane with the diameter of 0.25 mu m to obtain umbilical mesenchymal stem cell exosomes.
The PBS buffer of this example contained 1wt% penicillin and 1.2wt% streptomycin sulfate; the medium of this example was DMEM medium containing 8wt% fbs.
The preparation method of the kudzuvine root extract of the embodiment comprises the following steps:
s21, cleaning the kudzuvine root with deionized water, carrying out ultrasonic treatment at 100W and 20 ℃ for 15min, drying, and crushing to 20 meshes to obtain kudzuvine root powder;
s22, mixing radix puerariae powder and 65% ethanol solution according to a weight ratio of 1:5 mixing, leaching at 40deg.C for 30min, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain first extractive solution;
s23, mixing the first extracting solution and activated carbon according to the weight ratio of 1: mixing at 0.006, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain second extractive solution;
s24, mixing the second extracting solution with 1.5% chitosan solution according to the weight ratio of 1:0.002, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain third extractive solution;
s25, introducing the third extracting solution into an adsorption column filled with macroporous resin, eluting at a speed of 2BV/h, flushing the adsorption column with deionized water with a volume which is 3 times that of the resin column at a flow speed of 1.5BV/h and an ethanol solution with a volume which is 2 times that of 50wt% of the resin column at a flow speed of 1BV/h, and collecting the ethanol solution to obtain the radix puerariae extract.
The macroporous adsorbent resin of this example was HPD-100.
The compound vitamin of the embodiment consists of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 according to the weight ratio of 1.5:0.5:1:2:1.
The preparation method of the composition containing stem cell exosomes of the present embodiment comprises the following steps:
s31, dissolving honey, compound vitamins, olive oil, starch, glutamic acid, cysteine, glycine, glucuronolactone and sodium carboxymethyl cellulose in deionized water, and stirring at 45 ℃ and 240rmp for 30min to completely dissolve the mixture to obtain a mixture A;
s32, adding umbilical mesenchymal stem cell exosomes and radix puerariae extract into the mixture A, and stirring at 40 ℃ and 120rmp for 15min to obtain a mixture B;
s33, refluxing the mixture B at 37 ℃ for 1h, filtering, concentrating to a relative density of 1.5, and freeze-drying to obtain the composition containing the stem cell exosomes.
Example 2.
The composition containing the stem cell exosomes of the embodiment comprises the following components in parts by weight: 0.5 part of umbilical mesenchymal stem cell exosome, 6 parts of radix puerariae extract, 1 part of honey, 0.32 part of compound vitamin, 0.7 part of olive oil, 0.2 part of starch, 0.15 part of glutamic acid, 0.15 part of cysteine, 0.15 part of glycine, 0.35 part of glucuronolactone, 0.18 part of sodium carboxymethyl cellulose and 37 parts of deionized water.
The preparation method of the umbilical cord mesenchymal stem cell exosome of the embodiment comprises the following steps:
s1, taking umbilical cord tissue, putting the umbilical cord tissue into PBS buffer solution for cleaning for 3 times for 10min each time, removing artery and vein to obtain Walton gum, shearing the Walton gum into tissue blocks with the size of 6mm, flushing the tissue blocks with deionized water for 30min, and then cleaning the tissue blocks with the PBS buffer solution for 5 times for 8min each time to obtain tissue blocks;
s2, mixing the tissue blocks and collagenase I solution according to a weight ratio of 1:0.28, and reacting for 30min to obtain a mixture;
s3, centrifuging the mixture for 30min under the condition of 1200r/min, passing through a 0.4 mu m sterile filter membrane to obtain cell sediment, and washing 3 times with PBS buffer solution;
s4, washing the cell sediment for 3 times by using a balanced salt buffer solution, re-suspending the cells by using a balanced salt buffer solution with the volume of 2 times, and purifying the cell suspension by using a density gradient centrifugation method to obtain umbilical cord mesenchymal primordial stem cells;
s5, inoculating umbilical cord mesenchymal primordial stem cells into a culture medium at a density of 1X 10/ml, placing into an incubator for culturing, and culturing at 38 ℃ with 6% CO 2 Culturing for 4d under saturated humidity, controlling the passage times within P10, and collecting culture solution supernatant, namely first supernatant, when the cell fusion rate reaches 90%;
s6, centrifuging the first clear liquid for 30min at the temperature of 4 ℃ and the speed of 1800r/min, collecting the supernatant, ultrafiltering, concentrating to remove small molecules, and filtering and sterilizing by a sterile filter membrane with the thickness of 0.3 mu m to obtain umbilical mesenchymal cell sediment;
s7, washing umbilical cord mesenchymal cell sediment with PBS buffer solution for 3 times, inoculating into serum-free culture medium at a density of 1×10/ml, placing into an incubator for culturing, culturing at 38deg.C under 6% CO 2 Culturing for 48h under saturated humidity, and collecting culture solution supernatant, namely second supernatant;
s8, transferring the second clear liquid onto a sucrose heavy water density pad with the mass concentration of 30%, centrifuging at 4 ℃ with a density gradient of 160000g for 120min, and collecting a bottom buffer pad;
s9, washing the bottom buffer cushion for 3 times by using PBS solution, performing ultrafiltration concentration to remove small molecules, and performing filter sterilization by using a sterile filter membrane with the diameter of 0.25 mu m to obtain umbilical mesenchymal stem cell exosomes.
The PBS buffer of this example contained 3wt% penicillin and 3.6wt% streptomycin sulfate; the medium of this example was DMEM medium containing 12wt% fbs.
The preparation method of the kudzuvine root extract of the embodiment comprises the following steps:
s21, cleaning the kudzuvine root with deionized water, carrying out ultrasonic treatment at 100W and 20 ℃ for 15min, drying, and crushing to 40 meshes to obtain kudzuvine root powder;
s22, mixing radix puerariae powder and 85% ethanol solution according to a weight ratio of 1:8, mixing, leaching at 50deg.C for 90min, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with a filter membrane with pore diameter of 0.3 μm to obtain a first extractive solution;
s23, mixing the first extracting solution and activated carbon according to the weight ratio of 1:0.018, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain second extractive solution;
s24, mixing the second extracting solution with 1.5% chitosan solution according to the weight ratio of 1:0.01, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain third extractive solution;
s25, introducing the third extracting solution into an adsorption column filled with macroporous resin, eluting at a speed of 3BV/h, flushing the adsorption column with deionized water with a volume of 3 times of the resin column at a flow speed of 2.5BV/h and an ethanol solution with a volume of 2 times of 50wt% of the resin column at a flow speed of 3BV/h, and collecting the ethanol solution to obtain the radix puerariae extract.
The macroporous adsorbent resin of this example was HPD-100.
The compound vitamin of the embodiment consists of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 according to the weight ratio of 1.5:0.5:1:2:1.
The preparation method of the composition containing stem cell exosomes of the present embodiment comprises the following steps:
s31, dissolving honey, compound vitamins, olive oil, starch, glutamic acid, cysteine, glycine, glucuronolactone and sodium carboxymethyl cellulose in deionized water, and stirring at 45 ℃ and 360rmp for 45min to completely dissolve the mixture to obtain a mixture A;
s32, adding umbilical mesenchymal stem cell exosomes and radix puerariae extract into the mixture A, and stirring at 40 ℃ and 180rmp for 30min to obtain a mixture B;
s33, refluxing the mixture B for 2 hours at 37 ℃, filtering, concentrating to a relative density of 1.5, and freeze-drying to obtain the composition containing the stem cell exosomes.
Example 3.
The composition containing the stem cell exosomes of the embodiment comprises the following components in parts by weight: 0.3 part of umbilical mesenchymal stem cell exosome, 4 parts of radix puerariae extract, 0.6 part of honey, 0.2 part of compound vitamin, 0.5 part of olive oil, 0.14 part of starch, 0.09 part of glutamic acid, 0.09 part of cysteine, 0.09 part of glycine, 0.21 part of glucuronolactone, 0.12 part of sodium carboxymethyl cellulose and 29 parts of deionized water.
The preparation method of the umbilical cord mesenchymal stem cell exosome of the embodiment comprises the following steps:
s1, taking umbilical cord tissue, putting the umbilical cord tissue into PBS buffer solution for cleaning for 3 times for 9min each time, removing artery and vein to obtain Walton gum, shearing the Walton gum into tissue blocks with the size of 5mm, flushing the tissue blocks with deionized water for 22min, and then cleaning the tissue blocks with the PBS buffer solution for 5 times for 8min each time to obtain tissue blocks;
s2, mixing the tissue blocks and collagenase I solution according to a weight ratio of 1:0.28, and reacting for 30min to obtain a mixture;
s3, centrifuging the mixture at 1000r/min for 25min, passing through a 0.4 mu m sterile filter membrane to obtain a cell precipitate, and washing with PBS buffer solution for 3 times;
s4, washing the cell sediment for 3 times by using a balanced salt buffer solution, re-suspending the cells by using a balanced salt buffer solution with the volume of 2 times, and purifying the cell suspension by using a density gradient centrifugation method to obtain umbilical cord mesenchymal primordial stem cells;
s5, inoculating umbilical cord mesenchymal primordial stem cells into a culture medium at a density of 1X 10/ml, placing into an incubator for culturing, and culturing at 37 ℃ under 5% CO 2 Culturing for 3d under saturated humidity, controlling the passage times within P10, and collecting culture solution supernatant, namely first supernatant, when the cell fusion rate reaches 85%;
s6, centrifuging the first clear liquid for 20min at the temperature of 4 ℃ and the speed of 1800r/min, collecting the supernatant, ultrafiltering, concentrating, removing small molecules, and filtering and sterilizing by a sterile filter membrane with the thickness of 0.3 mu m to obtain umbilical mesenchymal cell sediment;
s7, washing umbilical cord mesenchymal cell sediment with PBS buffer solution for 3 times, inoculating into serum-free culture medium at a density of 1×10/ml, placing into an incubator for culturing, culturing at 37deg.C under 5% CO 2 Culturing for 45h under saturated humidity, and collecting culture solution supernatant, namely second supernatant;
s8, transferring the second clear liquid onto a sucrose heavy water density pad with the mass concentration of 30%, centrifuging for 120min at the temperature of 4 ℃ by a density gradient of 120000g, and collecting a bottom buffer pad;
s9, washing the bottom buffer cushion for 3 times by using PBS solution, performing ultrafiltration concentration to remove small molecules, and performing filter sterilization by using a sterile filter membrane with the diameter of 0.25 mu m to obtain umbilical mesenchymal stem cell exosomes.
The PBS buffer of this example contained 2wt% penicillin and 2.4wt% streptomycin sulfate; the medium of this example was DMEM medium containing 10wt% fbs.
The preparation method of the kudzuvine root extract of the embodiment comprises the following steps:
s21, cleaning the kudzuvine root with deionized water, carrying out ultrasonic treatment at 100W and 20 ℃ for 15min, drying, and crushing to 30 meshes to obtain kudzuvine root powder;
s22, mixing radix puerariae powder and 75% ethanol solution according to a weight ratio of 1:7, mixing, leaching at 45deg.C for 60min, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with a filter membrane with pore diameter of 0.3 μm to obtain a first extractive solution;
s23, mixing the first extracting solution and activated carbon according to the weight ratio of 1:0.012, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain second extractive solution;
s24, mixing the second extracting solution with 1.5% chitosan solution according to the weight ratio of 1: mixing at 0.006, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain third extractive solution;
s25, introducing the third extracting solution into an adsorption column filled with macroporous resin, eluting at a speed of 2.5BV/h, flushing the adsorption column with deionized water with a volume which is 3 times that of the resin column at a flow speed of 2BV/h and an ethanol solution with a volume which is 2 times that of 50wt% of the resin column at a flow speed of 2BV/h, and collecting the ethanol solution to obtain the radix puerariae extract.
The macroporous adsorbent resin of this example was HPD-100.
The compound vitamin of the embodiment consists of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 according to the weight ratio of 1.5:0.5:1:2:1.
The preparation method of the composition containing stem cell exosomes of the present embodiment comprises the following steps:
s31, dissolving honey, compound vitamins, olive oil, starch, glutamic acid, cysteine, glycine, glucuronolactone and sodium carboxymethyl cellulose in deionized water, and stirring at 45 ℃ and 320rmp for 38min to completely dissolve the mixture to obtain a mixture A;
s32, adding umbilical mesenchymal stem cell exosomes and radix puerariae extract into the mixture A, and stirring at 40 ℃ and 150rmp for 22min to obtain a mixture B;
s33, refluxing the mixture B at 37 ℃ for 1.51h, filtering, concentrating to a relative density of 1.5, and freeze-drying to obtain the composition containing the stem cell exosomes.
Comparative example 1.
Unlike example 3, umbilical cord mesenchymal stem cell exosomes were not contained.
Comparative example 2.
Unlike example 3, the extract of Pueraria lobata was not contained.
Comparative example 3.
Unlike example 3, the umbilical cord mesenchymal stem cell exosomes were prepared by the following methods:
s1, taking umbilical cord tissue, putting the umbilical cord tissue into PBS buffer solution for cleaning for 3 times for 9min each time, removing artery and vein to obtain Walton gum, shearing the Walton gum into tissue blocks with the size of 5mm, flushing the tissue blocks with deionized water for 22min, and then cleaning the tissue blocks with the PBS buffer solution for 5 times for 8min each time to obtain tissue blocks;
s2, mixing the tissue blocks and collagenase I solution according to a weight ratio of 1:0.28, and reacting for 30min to obtain a mixture;
s3, centrifuging the mixture at 1000r/min for 25min, passing through a 0.4 mu m sterile filter membrane to obtain a cell precipitate, and washing with PBS buffer solution for 3 times;
s4, washing the cell sediment for 3 times by using a balanced salt buffer solution, re-suspending the cells by using a balanced salt buffer solution with the volume of 2 times, and purifying the cell suspension by using a density gradient centrifugation method to obtain umbilical cord mesenchymal primordial stem cells;
s5, inoculating umbilical cord mesenchymal primordial stem cells into a culture medium at a density of 1X 10/ml, placing into an incubator for culturing, and culturing at 37 ℃ under 5% CO 2 Culturing under saturated humidity for 3d, controlling passage times within P10, collecting culture supernatant when cell fusion rate reaches 85%, namely the first supernatant；
S6, centrifuging the first clear liquid for 20min at 4 ℃ under 1800r/min, collecting the supernatant, ultrafiltering, concentrating to remove small molecules, filtering and sterilizing by a sterile filter membrane of 0.3 mu m, transferring to a sucrose heavy water density pad with the mass concentration of 30%, centrifuging for 120min at 4 ℃ with a density gradient of 120000g, and collecting a bottom buffer pad;
s7, washing the bottom buffer cushion for 3 times by using PBS solution, performing ultrafiltration concentration to remove small molecules, and performing filter sterilization by using a sterile filter membrane with the diameter of 0.25 mu m to obtain umbilical mesenchymal stem cell exosomes.
The PBS buffer of this example contained 2wt% penicillin and 2.4wt% streptomycin sulfate; the medium of this example was DMEM medium containing 10wt% fbs.
Comparative example 4.
Unlike example 3, the preparation method of the kudzuvine root extract is as follows:
s21, cleaning the kudzuvine root with deionized water, carrying out ultrasonic treatment at 100W and 20 ℃ for 15min, drying, and crushing to 30 meshes to obtain kudzuvine root powder;
s22, mixing radix puerariae powder and 75% ethanol solution according to a weight ratio of 1:7, mixing, leaching at 45deg.C for 60min, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain radix Puerariae extract;
s23, rotary evaporating the radix puerariae extract by a rotary evaporator at 45 ℃ to concentrate and retract to recover ethanol, extracting the rotary evaporation product by ethyl acetate, centrifuging the obtained product, rotary evaporating again to concentrate, and freeze-drying to obtain the radix puerariae extract.
Comparative example 5.
Unlike example 3, the preparation method of the kudzuvine root extract is as follows:
s21, cleaning the kudzuvine root with deionized water, carrying out ultrasonic treatment at 100W and 20 ℃ for 15min, drying, and crushing to 30 meshes to obtain kudzuvine root powder;
s22, mixing radix puerariae powder and 75% ethanol solution according to a weight ratio of 1:7, mixing, leaching at 45deg.C for 60min, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with a filter membrane with pore diameter of 0.3 μm to obtain a first extractive solution;
s23, mixing the first extracting solution and activated carbon according to the weight ratio of 1:0.012, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain second extractive solution;
s24, mixing the second extracting solution with 1.5% chitosan solution according to the weight ratio of 1: mixing at 0.006, centrifuging at 25deg.C and 12000r/min for 20min, and filtering with 0.3 μm filter membrane to obtain third extractive solution;
s25, introducing the third extracting solution into an adsorption column filled with macroporous resin, eluting at a speed of 2.5BV/h, flushing the adsorption column with deionized water with a volume which is 3 times that of the resin column at a flow speed of 2BV/h and an ethanol solution with a volume which is 2 times that of 60wt% of the resin column at a flow speed of 2BV/h, and collecting the ethanol solution to obtain the radix puerariae extract.
The macroporous adsorbent resin of this example was HPD-100.
Since the pathogenic mechanism of liver injury of mice and human and the action targets of drugs are similar, the treatment effect of a plurality of drugs is similar, so the experimental data of the drugs on mice are used for deducing the treatment effect of the drugs on liver injury of human, and the drugs obtained in examples 1-3 and comparative examples 1-5 are tested, and the specific experimental operation is as follows:
1. 55 healthy mice with a weight of 20+/-2 g were selected, and after one week of adaptive feeding, the mice were randomly divided into a normal group, a model group, a positive group and a dosing group, wherein the dosing group comprises examples 1-3 and comparative examples 1-5, 8 mice were allocated to each group, and the experimental results of each group are the average value of the experimental results of the mice of the group.
Wherein, the positive group is given with diammonium glycyrrhizinate aqueous solution, and the mice are irrigated with 0.1mL diammonium glycyrrhizinate aqueous solution per 10g body weight twice a day, once a day in the morning and in the evening; normal group and model group irrigate corresponding volume of normal saline, irrigate stomach once a day in the morning; the administration group irrigates the stomach once a day in the morning with the corresponding volumes of the drugs of the examples and the comparative examples.
In addition to the normal groups, the groups were intraperitoneally injected with carbon tetrachloride olive oil solution twice a week, 10 μl of carbon tetrachloride olive oil solution was intraperitoneally injected per 10g of body weight mice to cause liver injury; the normal group was injected with olive oil, 10 μl olive oil per 10g body weight mice were intraperitoneally injected.
2. Six weeks after administration, blood and liver tissue of each group of mice were collected, serum was obtained after centrifugation of the blood, and the contents of ATL and AST were detected to evaluate the liver function of each group of mice, and the liver tissue staining tube was used to wipe the liver injury degree of each group of mice. Wherein, the ATL and AST contents of the model group are 1, and the liver staining condition of the mice in the normal group is used as a reference index, and the model group is classified into 1 grade, 2 grade and 3 grade according to the damage degree from light to heavy.
3. The test results are shown in the following table:
from the table above, it can be seen that the components of the product of the invention synergistically increase, so that inflammatory cells in the injured liver can be reduced, the cell morphology can be improved, and liver injury can be effectively treated.
From the embodiment 1-3, the composition containing the stem cell exosome disclosed by the invention has the advantages that the umbilical cord mesenchymal stem cell exosome and the radix puerariae extract are matched, and the composition can be used together with the components such as amino acid, vitamin and the like to promote the repair and proliferation of liver cells, relieve inflammation and treat injury.
Comparative example 3 and comparative examples 1 and 3 show that the umbilical cord mesenchymal stem cell exosomes are not added and only one cell culture is performed on the umbilical cord mesenchymal stem cell exosomes, so that the metabolism and regulation information of the liver cells cannot be fully expressed, and the therapeutic effect on liver injury is limited.
As is clear from comparative example 3 and comparative examples 2, 4 and 5, the effective components in Pueraria are not sufficiently extracted without adding Pueraria extract or by distinguishing the extraction methods. According to the invention, after ethanol leaching, target products are separated by means of macroporous resin elution through a series of decolorization and impurity removal reactions, so that the purity of the kudzuvine root extract is improved, the activity of liver cells is regulated, and liver injury is relieved. Wherein example 3 is the optimal ratio, other conditions have respective preference under different numerical experiments.
While the foregoing is directed to the preferred embodiment of the present invention, it is to be understood that the invention is not limited to the precise form set forth herein, and that various other changes and modifications may be affected therein by one skilled in the art without departing from the scope or spirit of the invention.
Claims (10)
1. A composition containing stem cell exosomes, characterized in that the composition containing stem cell exosomes comprises, in parts by weight: 0.1-0.5 part of umbilical mesenchymal stem cell exosome, 3-6 parts of kudzu root extract, 0.2-1 part of honey, 0.06-0.32 part of compound vitamin, 0.3-0.7 part of olive oil, 0.08-0.2 part of starch, 0.03-0.15 part of glutamic acid, 0.03-0.15 part of cysteine, 0.03-0.15 part of glycine, 0.05-0.35 part of glucuronolactone, 0.05-0.18 part of sodium carboxymethyl cellulose and 22-37 parts of deionized water.
2. The stem cell exosome-containing composition according to claim 1, wherein the stem cell exosome-containing composition comprises in parts by weight: 0.2-0.4 part of umbilical mesenchymal stem cell exosome, 4-5 parts of kudzu root extract, 0.4-0.8 part of honey, 0.18-21 parts of compound vitamin, 0.4-0.6 part of olive oil, 0.12-0.16 part of starch, 0.07-0.11 part of glutamic acid, 0.07-0.11 part of cysteine, 0.07-0.11 part of glycine, 0.17-0.23 part of glucuronolactone, 0.09-0.14 part of sodium carboxymethyl cellulose and 27-32 parts of deionized water.
3. The stem cell exosome-containing composition according to claim 1, wherein the stem cell exosome-containing composition comprises in parts by weight: 0.3 part of umbilical mesenchymal stem cell exosome, 4 parts of radix puerariae extract, 0.6 part of honey, 0.2 part of compound vitamin, 0.5 part of olive oil, 0.14 part of starch, 0.09 part of glutamic acid, 0.09 part of cysteine, 0.09 part of glycine, 0.21 part of glucuronolactone, 0.12 part of sodium carboxymethyl cellulose and 29 parts of deionized water.
4. The stem cell exosome-containing composition according to claim 1, wherein the umbilical mesenchymal stem cell exosome is prepared by the following steps:
s1, taking umbilical cord tissue, placing the umbilical cord tissue into PBS buffer solution for cleaning for 3 times, each time for 8-10min, removing the artery and vein to obtain Walton gum, shearing the Walton gum into tissue blocks with the size of 4-6mm, flushing the tissue blocks with deionized water for 15-30min, and then cleaning the tissue blocks with the PBS buffer solution for 5 times, each time for 5-8min to obtain the tissue blocks;
s2, mixing the tissue blocks and collagenase I solution according to a weight ratio of 1: (0.2-0.28) and reacting for 15-30min to obtain a mixture;
s3, centrifuging the mixture at 800-1200r/min for 20-30min, passing through a 0.4 mu m sterile filter membrane to obtain cell sediment, and washing with PBS buffer solution for 3 times;
s4, washing the cell sediment for 3 times by using a balanced salt buffer solution, re-suspending the cells by using a balanced salt buffer solution with the volume of 2 times, and purifying the cell suspension by using a density gradient centrifugation method to obtain umbilical cord mesenchymal primordial stem cells;
s5, inoculating umbilical cord mesenchymal primordial stem cells into a culture medium at a density of 1X 10/ml, placing the culture medium in an incubator for culturing for 2-4d, controlling the passage times within P10, and collecting culture solution supernatant, namely first supernatant, when the cell fusion rate reaches 80-90%;
s6, centrifuging the first clear liquid for 10-30min at the temperature of 4 ℃ and the speed of 1800r/min, collecting the supernatant, ultrafiltering, concentrating to remove small molecules, and filtering and sterilizing by a sterile filter membrane with the thickness of 0.3 mu m to obtain umbilical mesenchymal cell sediment;
s7, washing umbilical cord mesenchymal cell sediment 3 times by using PBS buffer solution, inoculating the umbilical cord mesenchymal cell sediment into a serum-free culture medium at the density of 1X 10/ml, placing the umbilical cord mesenchymal cell sediment into an incubator for culturing for 42-48 hours, and collecting culture solution supernatant, namely second supernatant;
s8, transferring the second clear liquid to a sucrose heavy water density pad with the mass concentration of 30%, centrifuging for 120min at 4 ℃ by a density gradient of 80000-160000g, and collecting a bottom buffer pad;
s9, washing the bottom buffer cushion for 3 times by using PBS solution, performing ultrafiltration concentration to remove small molecules, and performing filter sterilization by using a sterile filter membrane with the diameter of 0.25 mu m to obtain umbilical mesenchymal stem cell exosomes.
5. A combination according to claim 4 comprising stem cell exosomesWherein the PBS buffer comprises 1-3wt% penicillin and 1.2-3.6wt% streptomycin sulfate; the culture medium is DMEM culture medium containing 8-12wt% FBS; the culture conditions of the incubator are 36-38 ℃ and 4-6% CO 2 Saturated humidity.
6. The stem cell exosome-containing composition according to claim 1, wherein the pueraria extract is prepared by the following steps:
s21, cleaning the kudzuvine root with deionized water, carrying out ultrasonic treatment at 100W and 20 ℃ for 15min, drying, and crushing to 20-40 meshes to obtain kudzuvine root powder;
s22, mixing radix puerariae powder and 65-85% ethanol solution according to a weight ratio of 1: (5-8) mixing, leaching at 40-50deg.C for 30-90min, centrifuging, and filtering to obtain first extractive solution;
s23, mixing the first extracting solution and activated carbon according to the weight ratio of 1: (0.006-0.018), centrifuging, and filtering to obtain second extractive solution;
s24, mixing the second extracting solution with 1.5% chitosan solution according to the weight ratio of 1: (0.002-0.01), mixing, centrifuging, and filtering to obtain a third extractive solution;
s25, introducing the third extracting solution into an adsorption column filled with macroporous resin, eluting at the speed of 2-3BV/h, flushing the adsorption column with deionized water with the volume of 3 times of the resin column at the flow rate of 1.5-2.5V/h and ethanol solution with the volume of 2 times of 50wt% of the resin column at the flow rate of 1-3BV/h, and collecting the ethanol solution to obtain the radix puerariae extract.
7. The composition of claim 6, wherein the centrifugation conditions are 25 ℃ and 12000r/min, and the centrifugation time is 20min; the pore diameter of the filter membrane is 0.3 mu m; the macroporous adsorption resin is HPD-100.
8. The stem cell exosome-containing composition according to claim 1, wherein the multivitamin consists of vitamin a, vitamin C, vitamin E, vitamin B6 and vitamin B12 in a weight ratio of 1.5:0.5:1:2:1.
9. The stem cell exosome-containing composition according to claim 1, wherein the stem cell exosome-containing composition is prepared by a method comprising:
s31, dissolving honey, compound vitamin, olive oil, starch, glutamic acid, cysteine, glycine, glucuronolactone and sodium carboxymethyl cellulose in deionized water, and stirring at 45 ℃ and 240-360rmp for 30-45min to completely dissolve the mixture to obtain a mixture A;
s32, adding umbilical mesenchymal stem cell exosomes and radix puerariae extract into the mixture A, and stirring at 40 ℃ and 120-180rmp for 15-30min to obtain a mixture B;
s33, refluxing the mixture B at 37 ℃ for 1-2h, filtering, concentrating to a relative density of 1.5, and freeze-drying to obtain the composition containing stem cell exosomes.
10. A composition comprising stem cell exosomes according to any one of claims 1-9 and its use in the preparation of a medicament having a role in the treatment of liver injury.
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