CN112656817A - Stem cell preparation and application thereof in preparation of medicine for treating liver injury - Google Patents
Stem cell preparation and application thereof in preparation of medicine for treating liver injury Download PDFInfo
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- CN112656817A CN112656817A CN202110093604.5A CN202110093604A CN112656817A CN 112656817 A CN112656817 A CN 112656817A CN 202110093604 A CN202110093604 A CN 202110093604A CN 112656817 A CN112656817 A CN 112656817A
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Abstract
The invention discloses a stem cell preparation and application thereof in preparing a liver injury medicament, wherein the stem cell preparation is prepared from dental pulp mesenchymal stem cells, a dendrobium officinale extract and normal saline; the content of the dental pulp mesenchymal stem cells is 4 multiplied by 105~1×107Per mL; the content of the dendrobium officinale extract is 0.5-1 mg/ml. The stem cell preparation has good effect of treating liver injury, the synergy effect can be achieved by combining the dental pulp mesenchymal stem cells and the dendrobium officinale extract in the aspect of treating liver injury, and the dendrobium officinale extract can promote the dental pulp mesenchymal stem cells to be used between dental pulpThe mesenchymal stem cells migrate to the liver injury part, the anti-inflammatory effect of the dental pulp mesenchymal stem cells is enhanced, and the proliferation of the dental pulp mesenchymal stem cells can be promoted to a certain extent, so that the combination of the dental pulp mesenchymal stem cells and the liver injury part has a synergistic effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a stem cell preparation and application thereof in preparing a medicine for treating liver injury.
Background
The liver is a parenchymal organ which plays a role in important physiological functions of the human body, and when the morphology and function of liver cells, liver tissues or the whole liver organ are pathologically changed, it is called liver injury.
Mesenchymal Stem Cells (MSCs), a class of non-hematopoietic stem cells derived from early-developing mesoderm and having the potential for self-replication and multipotential differentiation, are most commonly extracted from bone marrow, and can also be obtained from various tissues such as adipose tissue, umbilical cord tissue, liver, dental pulp, and the like. Research finds that the MSCs have the effects of immunoregulation, anti-inflammation, anti-fibrosis, anti-oxidative stress, anti-apoptosis and the like on liver cells, and are adult stem cells which have the highest application value in treating liver injury by a cell replacement method.
The traditional Chinese medicine possibly contains a large amount of components for promoting cell proliferation and differentiation, and has great value in the construction of tissue engineering skin and the in vitro culture research of stem cells.
The dendrobium officinale is classified as a holy drug with the functions of lightening the body and prolonging the life in the Shennong herbal classic, is easy to be absorbed by the human body, nourishes the whole body, enables all internal organs of the body to run freely, moistens the skin, smoothens qi and blood, can get rid of troubles of diseases after a period of time, and is bright and youthful again. Modern pharmacological studies show that the dendrobium has the effects of resisting tumor, oxidation and liver injury and reducing blood sugar, and also has good curative effects on preventing and treating cataract and improving immunity.
At present, no document or patent reports that the dental pulp mesenchymal stem cells and the dendrobium officinale extract are combined to treat liver injury.
Disclosure of Invention
The invention provides a stem cell preparation and application thereof in preparing a medicament for treating liver injury.
The invention adopts the following technical scheme for solving the technical problems:
a stem cell preparation is prepared from dental pulp mesenchymal stem cells, Dendrobium officinale extract and normal saline;
the content of the dental pulp mesenchymal stem cells is 4 multiplied by 105~1×107Per mL;
the content of the dendrobium officinale extract is 0.5-1 mg/ml.
The inventor of the invention discovers in a large amount of researches that the synergistic effect can be achieved in the aspect of treating liver injury by combining the dental pulp mesenchymal stem cells and the dendrobium officinale extract, the dendrobium officinale extract can promote the migration of the dental pulp mesenchymal stem cells to the liver injury part and enhance the anti-inflammatory effect of the dental pulp mesenchymal stem cells, and can also promote the proliferation of the dental pulp mesenchymal stem cells to a certain extent, so that the combination of the dental pulp mesenchymal stem cells and the dendrobium officinale extract has the synergistic effect.
As a most preferred embodiment, the content of the dental pulp mesenchymal stem cells is 5 × 106one/mL.
As a most preferable scheme, the content of the dendrobium officinale extract is 0.8 mg/ml.
As a preferred embodiment, the preparation method of the dental pulp mesenchymal stem cell comprises the following steps:
s1, cleaning collected deciduous teeth with normal saline, crushing deciduous teeth, taking pulp tissue, and cutting into pieces of 1-3 mm3Digesting the tissue block by using tissue digestive juice with 3-6 times volume of the tissue block for 60-90 min in oscillation at the temperature of 36.5-37.5 ℃ and the rotating speed of 150-300 rpm; the tissue digestive juice is 0.05% trypsin solution;
s2, after digestion, filtering, and centrifuging at the rotating speed of 1000-1500 rpm for 6-10 min; discarding supernatant, resuspending the precipitate with culture medium I, inoculating the precipitate into T75 culture flask at 9000-12000/mL, placing the flask at 36.5-37.5 ℃ and 5% CO2Culturing in a saturated humidity incubator, carrying out first liquid change for 3-4 days, then carrying out half liquid change every 2-3 days, carrying out culture passage by using a fresh culture medium I when the cell fusion degree is more than 80%, culturing until P5 generation, and taking P3-P5 generation passage cells;
and S3, culturing the P3-P5 generation passage cells with a culture medium II for 18-30 h, and collecting the cells to obtain the dental pulp mesenchymal stem cells.
As a preferable scheme, the culture medium I comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12, and the additives comprise 5-10 mM/ml D-glucose, 0.8-1.5 mM/ml riboflavin, 8-15 mg/ml soybean lecithin, 0.3-0.8 mg/ml vitamin C, 3-6 ng/ml dry cell growth factors and 2-5 ng/ml epidermal growth factors.
As a preferable scheme, the culture medium II comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12, and the additives comprise 5-10 mM/ml D-glucose, 0.8-1.5 mM/ml riboflavin, 2-6 mg/ml psoralen, 2-6 mg/ml fibronectin, 8-15 mg/ml soybean lecithin, 0.3-0.8 mg/ml vitamin C, 3-6 ng/ml dry cell growth factors and 2-5 ng/ml epidermal growth factors.
The inventor of the invention surprisingly discovers in a large number of researches that the addition of psoralen and vitamin C in a culture medium II can further improve the efficiency of targeting the dental pulp mesenchymal stem cells to the liver injury part and improve the effects of restoring the liver function and the tissue structure by culturing the passage cells of the P3-P5 generations by using the culture medium II, so that the treatment effect on the liver injury is further improved.
As a preferred scheme, the preparation method of the dendrobium officinale extract comprises the following steps:
s11, crushing the dendrobium officinale into 60-100 meshes to obtain dendrobium officinale powder;
s12, adding the dendrobium officinale powder into deionized water, carrying out steam distillation to obtain an oil-water mixture, and separating a water phase;
s13, filtering the water phase by using an ultrafiltration membrane of 5 kilodalton;
s14, putting the filtered water phase into a freezing box, quickly freezing, quickly reducing the temperature of the freezing box to-42 to-36 ℃, starting a vacuum pump, vacuumizing the freezing box to 35-45 Pa, keeping the temperature and the vacuum degree for 50-80 min, adjusting the temperature of the freezing box to-26 to-18 ℃, and keeping the vacuum degree for 30-35 Pa for 50-80 min; the temperature of the freeze drying box is increased to-8-2 ℃, the vacuum degree is adjusted to 25-35 Pa, and the freeze drying box is kept for 50-80 min; and adjusting the temperature of the freeze-drying box to 10-20 ℃, adjusting the vacuum degree to 20-25 Pa, keeping for 70-120 min, and finally, stopping freeze-drying when the vacuum degree is restored to the atmospheric pressure to obtain the dendrobium officinale extract.
The dendrobium officinale extract with a good effect of treating liver injury is obtained through grinding, steam distillation, ultrafiltration membrane filtration and vacuum freeze drying, wherein macromolecular substances and impurities below can be intercepted through ultrafiltration membrane filtration, and effective components for treating liver injury can be effectively reserved through vacuum freeze drying.
As a preferable scheme, the weight ratio of the deionized water to the dendrobium officinale powder in the S12 is (6-10): 1.
the invention also provides a preparation method of the stem cell preparation, which comprises the steps of uniformly mixing the dendrobium officinale extract and normal saline, and re-suspending the dental pulp mesenchymal stem cells to obtain the stem cell preparation.
The invention also provides application of the stem cell preparation in preparing a medicament for treating liver injury.
The invention has the beneficial effects that: the stem cell preparation has a good effect of treating liver injury, the dental pulp mesenchymal stem cells and the dendrobium officinale extract are combined to achieve a synergistic effect in the aspect of treating liver injury, the dendrobium officinale extract can promote the migration of the dental pulp mesenchymal stem cells to a liver injury part and enhance the anti-inflammatory effect of the dental pulp mesenchymal stem cells, and can also promote the proliferation of the dental pulp mesenchymal stem cells to a certain degree, so that the combination of the dental pulp mesenchymal stem cells and the dendrobium officinale extract has the synergistic effect.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A stem cell preparation is characterized in that the stem cell preparation is prepared by supplementing 10ml of dental pulp mesenchymal stem cells, dendrobium officinale extract and normal saline;
the content of the dental pulp mesenchymal stem cells is 5 multiplied by 106one/mL.
The content of the dendrobium officinale extract is 0.8 mg/ml.
The preparation method of the dental pulp mesenchymal stem cells comprises the following steps:
s1, collecting teeth naturally falling off in the tooth changing process of the children, and signing an informed consent before collecting a sample;
s2, collectingCleaning deciduous teeth with normal saline, clamping deciduous teeth with sterile hydraulic clamp, collecting dental pulp tissue, and cutting into pieces of 1mm3Digesting the tissue block by using tissue digestive juice with 5 times volume of the tissue block for 80min in oscillation at the temperature of 37 ℃ and the rotating speed of 200 rpm; the tissue digestive juice is 0.05% trypsin solution;
s3, after digestion, filtering, and centrifuging at the rotating speed of 1200rpm for 7 min; discarding supernatant, resuspending the precipitate with medium I, inoculating into T75 culture flask at 10000/mL, standing at 37 deg.C and 5% CO2Culturing in a saturated humidity incubator for 4 days with the first liquid change, then half liquid change every 2 days, culturing and subculturing with a fresh culture medium I when the cell fusion degree is more than 80%, culturing until P5 generation, and taking P3-P5 generation subculturing cells;
and S4, culturing the P3-P5 generation passage cells with a culture medium II for 24 hours, and collecting the cells to obtain the dental pulp mesenchymal stem cells.
The culture medium I comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12, and the additives comprise 6mM/ml D-glucose, 1.2mM/ml riboflavin, 12mg/ml soybean lecithin, 0.6 mg/ml vitamin C, 5ng/ml stem cell growth factors and 4ng/ml epidermal cell growth factors.
The culture medium II comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12, and the additives comprise 8mM/ml D-glucose, 1mM/ml riboflavin, 5mg/ml psoralen, 4mg/ml fibronectin, 11mg/ml soybean lecithin, 0.7 mg/ml vitamin C, 5ng/ml stem cell growth factor and 4ng/ml epidermal cell growth factor.
The preparation method of the dendrobium officinale extract comprises the following steps:
s11, crushing the dendrobium officinale into 90 meshes to obtain dendrobium officinale powder;
s12, adding the dendrobium officinale powder into deionized water, carrying out steam distillation to obtain an oil-water mixture, and separating a water phase; the weight ratio of the deionized water to the dendrobium officinale powder is 9: 1;
s13, filtering the water phase by using an ultrafiltration membrane of 5 kilodalton;
s14, putting the filtered water phase into a freezing box, quickly freezing, quickly reducing the temperature of the freezing box to-40 ℃, starting a vacuum pump, vacuumizing the freezing box to 40Pa, keeping the temperature and the vacuum degree for 60min, adjusting the temperature of the freezing box to-20 ℃, keeping the vacuum degree at 32Pa, and keeping for 60 min; the temperature of the freeze-drying box is raised to 0 ℃, the vacuum degree is adjusted to 30 Pa, and the freeze-drying box is kept for 60 min; and adjusting the temperature of the freeze-drying box to 15 ℃, adjusting the vacuum degree to 24Pa, keeping for 90min, and finally, stopping freeze-drying when the vacuum degree is restored to the atmospheric pressure to obtain the dendrobium officinale extract.
The preparation method of the stem cell preparation comprises the steps of uniformly mixing the dendrobium officinale extract and normal saline, and re-suspending dental pulp mesenchymal stem cells to obtain the stem cell preparation.
Example 2
A stem cell preparation is characterized in that the stem cell preparation is prepared by supplementing 10ml of dental pulp mesenchymal stem cells, dendrobium officinale extract and normal saline;
the content of the dental pulp mesenchymal stem cells is 5 multiplied by 106one/mL.
The content of the dendrobium officinale extract is 0.8 mg/ml.
The preparation method of the dental pulp mesenchymal stem cells comprises the following steps:
s1, collecting teeth naturally falling off in the tooth changing process of the children, and signing an informed consent before collecting a sample;
s2, cleaning collected deciduous teeth with normal saline, clamping deciduous teeth with aseptic hydraulic clamp, collecting pulp tissue, and cutting into pieces of 1mm3Digesting the tissue block by using tissue digestive juice with 5 times volume of the tissue block for 80min in oscillation at the temperature of 37 ℃ and the rotating speed of 200 rpm; the tissue digestive juice is 0.05% trypsin solution;
s3, after digestion, filtering, and centrifuging at the rotating speed of 1200rpm for 7 min; discarding supernatant, resuspending the precipitate with medium I, inoculating into T75 culture flask at 10000/mL, standing at 37 deg.C and 5% CO2Culturing in a saturated humidity incubator for 4 days with the first liquid change, then half liquid change every 2 days, culturing and subculturing with a fresh culture medium I when the cell fusion degree is more than 80%, culturing until P5 generation, and taking P3-P5 generation subculturing cells;
and S4, culturing the P3-P5 generation passage cells with a culture medium II for 24 hours, and collecting the cells to obtain the dental pulp mesenchymal stem cells.
The culture medium I comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12, and the additives comprise 6mM/ml D-glucose, 1.2mM/ml riboflavin, 12mg/ml soybean lecithin, 0.6 mg/ml vitamin C, 5ng/ml stem cell growth factors and 4ng/ml epidermal cell growth factors.
The culture medium II comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12, and the additives comprise 5mM/ml D-glucose, 0.8mM/ml riboflavin, 2mg/ml psoralen, 2mg/ml fibronectin, 8mg/ml soybean lecithin, 0.3 mg/ml vitamin C, 3ng/ml stem cell growth factor and 2ng/ml epidermal cell growth factor.
The preparation method of the dendrobium officinale extract comprises the following steps:
s11, crushing the dendrobium officinale into 90 meshes to obtain dendrobium officinale powder;
s12, adding the dendrobium officinale powder into deionized water, carrying out steam distillation to obtain an oil-water mixture, and separating a water phase; the weight ratio of the deionized water to the dendrobium officinale powder is 9: 1;
s13, filtering the water phase by using an ultrafiltration membrane of 5 kilodalton;
s14, putting the filtered water phase into a freezing box, quickly freezing, quickly reducing the temperature of the freezing box to-40 ℃, starting a vacuum pump, vacuumizing the freezing box to 40Pa, keeping the temperature and the vacuum degree for 60min, adjusting the temperature of the freezing box to-20 ℃, keeping the vacuum degree at 32Pa, and keeping for 60 min; the temperature of the freeze-drying box is raised to 0 ℃, the vacuum degree is adjusted to 30 Pa, and the freeze-drying box is kept for 60 min; and adjusting the temperature of the freeze-drying box to 15 ℃, adjusting the vacuum degree to 24Pa, keeping for 90min, and finally, stopping freeze-drying when the vacuum degree is restored to the atmospheric pressure to obtain the dendrobium officinale extract.
The preparation method of the stem cell preparation comprises the steps of uniformly mixing the dendrobium officinale extract and normal saline, and re-suspending dental pulp mesenchymal stem cells to obtain the stem cell preparation.
Example 3
A stem cell preparation is characterized in that the stem cell preparation is prepared by supplementing 10ml of dental pulp mesenchymal stem cells, dendrobium officinale extract and normal saline;
the content of the dental pulp mesenchymal stem cells is 5 multiplied by 106one/mL.
The content of the dendrobium officinale extract is 0.8 mg/ml.
The preparation method of the dental pulp mesenchymal stem cells comprises the following steps:
s1, collecting teeth naturally falling off in the tooth changing process of the children, and signing an informed consent before collecting a sample;
s2, cleaning collected deciduous teeth with normal saline, clamping deciduous teeth with aseptic hydraulic clamp, collecting pulp tissue, and cutting into pieces of 1mm3Digesting the tissue block by using tissue digestive juice with 5 times volume of the tissue block for 80min in oscillation at the temperature of 37 ℃ and the rotating speed of 200 rpm; the tissue digestive juice is 0.05% trypsin solution;
s3, after digestion, filtering, and centrifuging at the rotating speed of 1200rpm for 7 min; discarding supernatant, resuspending the precipitate with medium I, inoculating into T75 culture flask at 10000/mL, standing at 37 deg.C and 5% CO2Culturing in a saturated humidity incubator for 4 days with the first liquid change, then half liquid change every 2 days, culturing and subculturing with a fresh culture medium I when the cell fusion degree is more than 80%, culturing until P5 generation, and taking P3-P5 generation subculturing cells;
and S4, culturing the P3-P5 generation passage cells with a culture medium II for 24 hours, and collecting the cells to obtain the dental pulp mesenchymal stem cells.
The culture medium I comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12, and the additives comprise 6mM/ml D-glucose, 1.2mM/ml riboflavin, 12mg/ml soybean lecithin, 0.6 mg/ml vitamin C, 5ng/ml stem cell growth factors and 4ng/ml epidermal cell growth factors.
The culture medium II comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12, and the additives comprise 10mM/ml D-glucose, 1.5mM/ml riboflavin, 3mg/ml psoralen, 5mg/ml fibronectin, 15mg/ml soybean lecithin, 0.6 mg/ml vitamin C, 6ng/ml stem cell growth factor and 5ng/ml epidermal cell growth factor.
The preparation method of the dendrobium officinale extract comprises the following steps:
s11, crushing the dendrobium officinale into 90 meshes to obtain dendrobium officinale powder;
s12, adding the dendrobium officinale powder into deionized water, carrying out steam distillation to obtain an oil-water mixture, and separating a water phase; the weight ratio of the deionized water to the dendrobium officinale powder is 9: 1;
s13, filtering the water phase by using an ultrafiltration membrane of 5 kilodalton;
s14, putting the filtered water phase into a freezing box, quickly freezing, quickly reducing the temperature of the freezing box to-40 ℃, starting a vacuum pump, vacuumizing the freezing box to 40Pa, keeping the temperature and the vacuum degree for 60min, adjusting the temperature of the freezing box to-20 ℃, keeping the vacuum degree at 32Pa, and keeping for 60 min; the temperature of the freeze-drying box is raised to 0 ℃, the vacuum degree is adjusted to 30 Pa, and the freeze-drying box is kept for 60 min; and adjusting the temperature of the freeze-drying box to 15 ℃, adjusting the vacuum degree to 24Pa, keeping for 90min, and finally, stopping freeze-drying when the vacuum degree is restored to the atmospheric pressure to obtain the dendrobium officinale extract.
The preparation method of the stem cell preparation comprises the steps of uniformly mixing the dendrobium officinale extract and normal saline, and re-suspending dental pulp mesenchymal stem cells to obtain the stem cell preparation.
Comparative example 1
Comparative example 1 is different from example 1 in that the stem cell preparation described in comparative example 1 does not contain the dendrobium officinale extract, and the rest is the same.
Comparative example 2
Comparative example 2 is different from example 1 in that the stem cell preparation described in comparative example 2 does not contain dental pulp mesenchymal stem cells, and the others are the same.
Comparative example 3
Comparative example 3 is different from example 1 in that the medium II described in comparative example 3 does not contain psoralen in the additive components, and the other components are the same.
In this comparative example, the medium II included a basal medium of DMEM/F12 and supplements including 8mM/ml D-glucose, 1mM/ml riboflavin, 4mg/ml fibronectin, 11mg/ml soy lecithin, 0.7 mg/ml vitamin C, 5ng/ml stem cell growth factor, 4ng/ml epidermal growth factor.
Comparative example 4
Comparative example 4 is different from example 1 in that the medium II described in comparative example 4 does not contain vitamin C as an additive component, and the other components are the same.
In this comparative example, the medium II included a basal medium of DMEM/F12 and supplements including 8mM/ml D-glucose, 1mM/ml riboflavin, 5mg/ml psoralen, 4mg/ml fibronectin, 11mg/ml soy lecithin, 5ng/ml stem cell growth factor, 4ng/ml epidermal growth factor.
Comparative example 5
Comparative example 5 is different from example 1 in that the preparation method of the dendrobium officinale extract is different from example 1, and in this comparative example, the aqueous phase is not filtered by the ultrafiltration membrane, but the rest is the same.
In the comparative example, the preparation method of the dendrobium officinale extract comprises the following steps:
s11, crushing the dendrobium officinale into 90 meshes to obtain dendrobium officinale powder;
s12, adding the dendrobium officinale powder into deionized water, carrying out steam distillation to obtain an oil-water mixture, and separating a water phase; the weight ratio of the deionized water to the dendrobium officinale powder is 9: 1;
s13, putting the filtered water phase into a freezing box, quickly freezing, quickly reducing the temperature of the freezing box to-40 ℃, starting a vacuum pump, vacuumizing the freezing box to 40Pa, keeping the temperature and the vacuum degree for 60min, adjusting the temperature of the freezing box to-20 ℃, keeping the vacuum degree at 32Pa, and keeping for 60 min; the temperature of the freeze-drying box is raised to 0 ℃, the vacuum degree is adjusted to 30 Pa, and the freeze-drying box is kept for 60 min; and adjusting the temperature of the freeze-drying box to 15 ℃, adjusting the vacuum degree to 24Pa, keeping for 90min, and finally, stopping freeze-drying when the vacuum degree is restored to the atmospheric pressure to obtain the dendrobium officinale extract.
Comparative example 6
Comparative example 6 is different from example 1 in that the method for preparing the dendrobium officinale extract is different from example 1, namely in the comparative example, the vacuum freeze-drying method is replaced by the drying method in an incubator, and the rest is the same.
In the comparative example, the preparation method of the dendrobium officinale extract comprises the following steps:
s11, crushing the dendrobium officinale into 90 meshes to obtain dendrobium officinale powder;
s12, adding the dendrobium officinale powder into deionized water, carrying out steam distillation to obtain an oil-water mixture, and separating a water phase; the weight ratio of the deionized water to the dendrobium officinale powder is 9: 1;
s13, filtering the water phase by using an ultrafiltration membrane of 5 kilodalton;
s14, drying the filtered water phase in a thermostat at 90 ℃ to obtain the dendrobium officinale extract.
Comparative example 7
The difference between the comparative example 7 and the example 1 is that the preparation method of the dendrobium officinale extract in the comparative example 7 is different from that in the example 1, namely, in the comparative example, alcohol is adopted instead of water evaporation, and the rest is the same.
The preparation method of the dendrobium officinale extract comprises the following steps:
s11, crushing the dendrobium officinale into 90 meshes to obtain dendrobium officinale powder;
s12, extracting the dendrobium officinale powder with 50% ethanol under reflux for 2 times, wherein each time lasts for 1 hour, and mixing the extracting solutions to obtain an alcohol extracting solution; the weight ratio of the ethanol to the dendrobium officinale powder is 9: 1;
s13, filtering the alcohol extract by using an ultrafiltration membrane of 5 ten thousand daltons to obtain a water phase;
s14, putting the filtered alcohol extract into a freezing box, quickly freezing, quickly reducing the temperature of the freezing box to-40 ℃, starting a vacuum pump, vacuumizing the freezing box to 40Pa, keeping the temperature and the vacuum degree for 60min, adjusting the temperature of the freezing box to-20 ℃, and keeping the vacuum degree at 32Pa for 60 min; the temperature of the freeze-drying box is raised to 0 ℃, the vacuum degree is adjusted to 30 Pa, and the freeze-drying box is kept for 60 min; and adjusting the temperature of the freeze-drying box to 15 ℃, adjusting the vacuum degree to 24Pa, keeping for 90min, and finally, stopping freeze-drying when the vacuum degree is restored to the atmospheric pressure to obtain the dendrobium officinale extract.
To further demonstrate the effect of the present invention, the following test methods were provided:
1. taking 110 rats with acute liver injury, randomly dividing the rats into 11 groups, 10 rats in each group, respectively injecting the stem cell preparations described in examples 1-3 and comparative examples 1-7 into tail veins, setting a control group, injecting single physiological saline into the control group for 1 time, measuring the injection amount of 0.4ml/100g of the body weight of the rats before and 7 days after injection, respectively, taking blood through eye sockets, and measuring ALT (alanine aminotransferase), AST (aspartate aminotransferase) and TBIL (total bilirubin) indexes in the blood by using a full-automatic biochemical analyzer, wherein the test results are shown in Table 1.
TABLE 1 test results
As can be seen from Table 1, the stem cell preparation of the present invention has a good effect of treating liver injury.
As can be seen from comparative examples 1-3, the additive components of different culture mediums II can affect the effect of treating liver injury, wherein example 1 is the optimal ratio.
Comparing example 1 with comparative examples 1 and 2, it can be seen that the dental pulp mesenchymal stem cells and the dendrobium officinale extract have good effect of treating liver injury, and the dental pulp mesenchymal stem cells and the dendrobium officinale extract have synergistic effect.
As is clear from comparison of example 1 with comparative examples 3 and 4, the addition of psoralen and vitamin C to the medium II of the present invention can improve the therapeutic effect on liver injury.
As can be seen from comparison of example 1 with comparative examples 4, 5 and 6, the preparation method of the dendrobium officinale extract of the present invention can improve the treatment effect on liver injury compared to other methods.
In light of the foregoing description of preferred embodiments according to the invention, it is clear that many changes and modifications can be made by the person skilled in the art without departing from the scope of the invention. The technical scope of the present invention is not limited to the contents of the specification, and must be determined according to the scope of the claims.
Claims (10)
1. A stem cell preparation is characterized by being prepared from dental pulp mesenchymal stem cells, a dendrobium officinale extract and normal saline;
the content of the dental pulp mesenchymal stem cells is 4 multiplied by 105~1×107Per mL;
the content of the dendrobium officinale extract is 0.5-1 mg/ml.
2. The stem cell preparation of claim 1, wherein the dental pulp mesenchymal stem cells are present in an amount of 5 x 106one/mL.
3. The stem cell preparation according to claim 1, wherein the content of the dendrobium officinale extract is 0.8 mg/ml.
4. The stem cell preparation of claim 1, wherein the preparation method of the dental pulp mesenchymal stem cells comprises:
s1, cleaning collected deciduous teeth with normal saline, crushing deciduous teeth, taking pulp tissue, and cutting into pieces of 1-3 mm3Digesting the tissue block by using tissue digestive juice with 3-6 times volume of the tissue block for 60-90 min in oscillation at the temperature of 36.5-37.5 ℃ and the rotating speed of 150-300 rpm; the tissue digestive juice is 0.05% trypsin solution;
s2, after digestion, filtering, and centrifuging at the rotating speed of 1000-1500 rpm for 6-10 min; discarding supernatant, resuspending the precipitate with culture medium I, inoculating the precipitate into T75 culture flask at 9000-12000/mL, placing the flask at 36.5-37.5 ℃ and 5% CO2Culturing in a saturated humidity incubator, carrying out first liquid change for 3-4 days, then carrying out half liquid change every 2-3 days, carrying out culture passage by using a fresh culture medium I when the cell fusion degree is more than 80%, culturing until P5 generation, and taking P3-P5 generation passage cells;
and S3, culturing the P3-P5 generation passage cells with a culture medium II for 18-30 h, and collecting the cells to obtain the dental pulp mesenchymal stem cells.
5. The stem cell preparation according to claim 4, wherein the medium I comprises a basal medium and additives, the basal medium is DMEM/F12, and the additives comprise 5-10 mM/ml D-glucose, 0.8-1.5 mM/ml riboflavin, 8-15 mg/ml soybean lecithin, 0.3-0.8 mg/ml vitamin C, 3-6 ng/ml stem cell growth factor and 2-5 ng/ml epidermal growth factor.
6. The stem cell preparation according to claim 4, wherein the culture medium II comprises a basal medium and additives, the basal medium is DMEM/F12, and the additives comprise 5-10 mM/ml D-glucose, 0.8-1.5 mM/ml riboflavin, 2-6 mg/ml psoralen, 2-6 mg/ml fibronectin, 8-15 mg/ml soybean lecithin, 0.3-0.8 mg/ml vitamin C, 3-6 ng/ml stem cell growth factors, and 2-5 ng/ml epidermal growth factors.
7. The stem cell preparation of claim 1, wherein the Dendrobium officinale extract is prepared by the method comprising:
s11, crushing the dendrobium officinale into 60-100 meshes to obtain dendrobium officinale powder;
s12, adding the dendrobium officinale powder into deionized water, carrying out steam distillation to obtain an oil-water mixture, and separating a water phase;
s13, filtering the water phase by using an ultrafiltration membrane of 5 kilodalton;
s14, putting the filtered water phase into a freezing box, quickly freezing, quickly reducing the temperature of the freezing box to-42 to-36 ℃, starting a vacuum pump, vacuumizing the freezing box to 35-45 Pa, keeping the temperature and the vacuum degree for 50-80 min, adjusting the temperature of the freezing box to-26 to-18 ℃, and keeping the vacuum degree for 30-35 Pa for 50-80 min; the temperature of the freeze drying box is increased to-8-2 ℃, the vacuum degree is adjusted to 25-35 Pa, and the freeze drying box is kept for 50-80 min; and adjusting the temperature of the freeze-drying box to 10-20 ℃, adjusting the vacuum degree to 20-25 Pa, keeping for 70-120 min, and finally, stopping freeze-drying when the vacuum degree is restored to the atmospheric pressure to obtain the dendrobium officinale extract.
8. The stem cell preparation according to claim 7, wherein the weight ratio of the deionized water to the dendrobium officinale powder in S12 is (6-10): 1.
9. the method for preparing the stem cell preparation according to claims 1-8, wherein the stem cell preparation is obtained by uniformly mixing the dendrobium officinale extract and the physiological saline, and resuspending the dental pulp mesenchymal stem cells.
10. Use of a stem cell preparation according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment of liver injury.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114621919A (en) * | 2022-05-17 | 2022-06-14 | 天九再生医学(天津)科技有限公司 | Method for enhancing immunoregulation capability of mesenchymal stem cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114621919A (en) * | 2022-05-17 | 2022-06-14 | 天九再生医学(天津)科技有限公司 | Method for enhancing immunoregulation capability of mesenchymal stem cells |
| CN114621919B (en) * | 2022-05-17 | 2022-12-06 | 天九再生医学(天津)科技有限公司 | Method for enhancing immunoregulation capability of mesenchymal stem cells |
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