CN113171379A - Application of mesenchymal stem cell exosome in preparation of drugs for treating fatty liver disease - Google Patents
Application of mesenchymal stem cell exosome in preparation of drugs for treating fatty liver disease Download PDFInfo
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- CN113171379A CN113171379A CN202110465261.0A CN202110465261A CN113171379A CN 113171379 A CN113171379 A CN 113171379A CN 202110465261 A CN202110465261 A CN 202110465261A CN 113171379 A CN113171379 A CN 113171379A
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- mesenchymal stem
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention provides application of a mesenchymal stem cell exosome in preparation of a medicament for treating fatty liver disease, wherein the fatty liver disease is alcoholic fatty liver disease. Experiments show that the mesenchymal stem cell exosome has an obvious effect in treating fatty liver diseases, particularly alcoholic fatty liver diseases; the combination of the mesenchymal stem cell exosome and the piceatannol can obviously improve the enzyme activity of the acetaldehyde dehydrogenase and delay the occurrence of alcoholic fatty liver disease and alcohol-related cancer.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to an application of a mesenchymal stem cell exosome in preparation of a drug for treating fatty liver disease.
Background
The exosome is originally found in the supernatant of sheep red blood cells cultured in vitro, and is a vesicular corpuscle which is actively secreted by cells, has uniform size, the diameter of 40-100nm and the density of 1.10-1.18 g/ml. The scholars in 1996 discovered that B cells can promote T cell proliferation and inhibit tumor growth by releasing exosomes expressing major histocompatibility complex molecules. Exosomes contain a variety of proteins and genetic material, indicating that large molecular substances such as proteins can be loaded onto exosomes. Exosomes can be widely distributed in different body fluids and have a long half-life in vivo. In addition, exosomes can penetrate the cell membrane to release mRNA into the target cell, allowing the recipient cell to translate the transfected mRNA. At present, exosomes have begun to be applied in clinical therapy.
After drinking, 90-98% of alcohol absorbed by digestive tract enters liver from portal vein, enters systemic circulation after enzyme metabolism treatment, and the rest 2-10% of alcohol is discharged in original form through urine, sweat and expiration, or transferred to saliva or milk. The activity of alcohol metabolizing enzyme varies with regional, national and individual differences, which causes different ethanol handling capacity and different alcohol consumption, and is closely related to the onset of alcohol-related diseases.
At present, the number of people with alcohol addiction or alcohol dependence in China is as high as about 2 hundred million, and the incidence rate of alcohol-related diseases is high. The first time, the alcoholic liver diseases comprise alcoholic fatty liver, alcoholic hepatitis, cirrhosis, fibrosis and liver failure and the like, and the health of people is seriously harmed.
Drinking also becomes a major risk factor in addition to Hepatitis B Virus (HBV) infection. Acetaldehyde dehydrogenase 2(ALDH2) is one of the important rate-limiting enzymes directly involved in alcohol metabolism, and directly determines the accumulation of ethanol and acetaldehyde in vivo, and the activity level thereof has a close relationship with the occurrence of alcoholic liver disease, and further influences the occurrence of liver cancer.
Disclosure of Invention
In view of this, the present invention provides an application of mesenchymal stem cell exosome in preparation of a drug for treating fatty liver disease, aiming at overcoming the defects in the prior art.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
an application of mesenchymal stem cell exosome in preparing a medicament for treating fatty liver diseases, wherein the fatty liver diseases are alcoholic fatty liver diseases.
Further, the preparation method of the mesenchymal stem cell exosome comprises the following steps: cutting the umbilical cord into small segments, fully washing and cutting into pieces, then placing the small segments into a culture dish, culturing and subculturing, placing the mesenchymal stem cells after subculturing into the culture dish, cleaning the cells when the cell fusion reaches 60-90%, replacing a culture medium without exosome serum, continuing culturing, collecting cell supernatant, centrifuging to remove the cells and cell fragments, and extracting to obtain exosomes.
An application of a composition of mesenchymal stem cell exosome and piceatannol in preparing a medicament for treating fatty liver diseases.
The composition of the mesenchymal stem cell exosomes and the piceatannol comprises the mesenchymal stem cell exosomes and the piceatannol in a mass ratio of 1: 10-500.
Preferably, the mass ratio of the mesenchymal stem cell exosome to the composition of piceatannol is 1: 100-500.
More preferably, the mass ratio of the mesenchymal stem cell exosome to the composition of piceatannol is 1: 100-350.
The preparation method of the composition of the mesenchymal stem cell exosome and piceatannol comprises the following steps: dissolving piceatannol in glycerol to obtain piceatannol solution, performing ultrasonic treatment on piceatannol, adding mesenchymal stem cell exosome, and mixing uniformly to obtain the piceatannol.
Further, the concentration of the piceatannol solution is 0.1-0.3 ug/ml.
A fatty liver drug comprises a composition of mesenchymal stem cell exosome and piceatannol.
Further, the concentration of the composition in the medicine is 1-50 ug/g.
The medicine also comprises a medicinal carrier, an auxiliary material or a diluent.
Compared with the prior art, the invention has the following advantages:
experiments show that the mesenchymal stem cell exosome has an obvious effect in treating fatty liver diseases, particularly alcoholic fatty liver diseases; the combined use of the mesenchymal stem cell exosome and the piceatannol in a certain proportion can obviously improve the enzyme activity of the acetaldehyde dehydrogenase and delay the occurrence of alcoholic fatty liver disease and alcoholic related cancer.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to examples.
Example 1 preparation of mesenchymal stem cell exosomes
Shearing the umbilical cord into small pieces with the length of about 1-2cm, washing, removing the artery and the vein of the umbilical cord, shearing the umbilical cord pieces, placing the umbilical cord pieces in a culture dish for adherence, adding a low-sugar DMEM culture solution containing 10 wt% of FBS and 5 wt% of green chain double antibody, placing the umbilical cord pieces in an incubator for culture, and adding the culture solution after 4 hours. After 3d, the culture medium was changed every 3 d. After the cell fusion reaches 60-80%, the cell is digested by pancreatin, subcultured, and P3 generation cells are taken for experiment.
Placing the mesenchymal stem cells of the P3 generation in a culture dish, when the cell fusion reaches 60-80%, cleaning the cells, replacing the culture medium without exosome serum, continuously culturing for 48-72h, collecting the cell supernatant, centrifuging and removing the cells or cell debris. Mesenchymal stem cell exosomes were isolated and extracted using the Norgen Biotek brand plasmid/Serum Exosome Purification minikit.
Example 2 preparation of composition of mesenchymal Stem cell exosomes and piceatannol
Dissolving 10mg of piceatannol in 10ml of glycerol to obtain piceatannol solution, performing ultrasonic treatment on piceatannol for 30 minutes, stopping ultrasonic treatment, adding 0.05mg of mesenchymal stem cell exosome, and uniformly mixing to obtain the composition A.
Dissolving 10mg of piceatannol in 10ml of glycerol to obtain piceatannol solution, performing ultrasonic treatment on piceatannol for 30 minutes, stopping ultrasonic treatment, adding 0.5mg of mesenchymal stem cell exosome, and uniformly mixing to obtain a composition B.
Dissolving 10mg of piceatannol in 10ml of glycerol to obtain piceatannol solution, performing ultrasonic treatment on piceatannol for 30 minutes, stopping ultrasonic treatment, adding 0.005mg of mesenchymal stem cell exosome, and mixing uniformly to obtain the composition C.
Dissolving 0.05mg of the mesenchymal stem cell exosome in 10ml of glycerol, and uniformly mixing to obtain a composition D.
10mg of piceatannol was dissolved in 10ml of glycerol and mixed well to obtain composition E.
The specific formulation of the composition of mesenchymal stem cell exosomes and piceatannol is shown in table 1.
TABLE 1 ingredient ratio of each composition
| Each composition | Mesenchymal stem cell exosomes (mg) | Piceatannol (mg) |
| Composition A | 0.05 | 10 |
| Composition B | 0.5 | 10 |
| Composition C | 0.005 | 10 |
| Composition D | 0.05 | — |
| Composition E | — | 10 |
Example 3 animal experiments
1. Experiments on alcohol effect
120 clean-grade mice are adopted, and the male bodies are 20-30g in weight. The mice are randomly divided into a model control group, dosage groups of a composition A, a composition B, a composition C, a composition D and a composition E, wherein the dosage of each mouse is calculated according to the weight of the mouse, the dosage groups of the composition A, the composition B, the composition C, the composition D and the composition E are correspondingly fed with the compositions at the corresponding dosage every day at 2-4 pm, and the model group is fed with physiological saline at the same volume. The weight is measured daily and the dosage of the composition is adjusted according to the body weight. After the last administration, fasting is carried out for 16h, and each group is subjected to one-time perfusion with 60% ethanol, and a righting experiment and a climbing experiment are carried out. During the experiment, the mice in each group are fed with common feed, water is freely drunk, and factors among the groups are not different.
The alignment experiment method comprises the following steps: the drunk mouse can keep on taking the supine position for 30s as an index of disappearance of righting reflex. The time for the loss and recovery of the righting reflex of the mice was recorded.
The experimental method comprises the following steps: after feeding ethanol, the mixture was immediately placed on a vertical wire gauze, dropped continuously for 3 times and then terminated, and the climbing time was recorded.
2. Experiment for treating alcoholic liver disease
After the last administration of 140 mice, except for the normal control group which is given physiological saline with the same volume, the model control group, the composition A, the composition B, the composition C, the composition D and the composition E dose group are given Luzhou Laojiao wine for one time, fasting is carried out for 24h, eyeballs of the mice in each group are bled, and serum is taken for detection after high-speed refrigerated centrifugation (3000r/min, 20 min). Taking the liver after dissection, washing with normal saline, draining water by filter paper, weighing the weight of the liver, fixing the liver tissue at the same position of the right lobe of the liver with liquid nitrogen, storing in an ultra-low temperature refrigerator, homogenizing with normal saline to 40% D liver when to be detected, and centrifuging (3000r/min, 20min) to obtain the supernatant.
Example 4 results of the mouse experiment
1. The data of the alcohol-relieving test are shown in table 2.
TABLE 2 Experimental data for hangover alleviation
As can be seen from table 2, compared with the composition C-E dosage groups, both the composition a and the composition B dosage groups can prolong the mouse righting reflex disappearance time, shorten the righting reflex recovery time, prolong the mouse climbing reflex time, and improve the climbing ability thereof, which indicates that the composition of the mesenchymal stem cell exosome and piceatannol has a significant antialcoholic effect, and the composition using the mesenchymal stem cell exosome alone to antialcoholize also has a certain effect compared with the model group.
2. The data of the alcoholic liver disease treatment experiment are shown in table 3.
TABLE 3 Experimental data for the treatment of alcoholic liver disease
| ALT(U/L) | AST(U/L) | ADH(U/mL) | |
| Is normal | 6.25±0.23 | 20.36±2.74 | 2.38±0.85 |
| Model (model) | 32.14±1.20 | 106.85±6.52 | 8.64±1.82 |
| Composition A | 11.25±0.28 | 48.39±3.52 | 3.84±0.59 |
| Composition B | 12.24±1.26 | 57.31±5.41 | 4.25±0.74 |
| Composition C | 17.52±2.52 | 55.47±3.85 | 5.85±0.36 |
| Composition D | 20.27±1.26 | 65.21±5.62 | 5.74±0.23 |
| Composition E | 28.74±2.39 | 89.74±12.52 | 7.58±0.24 |
As can be seen from Table 3, the dose groups of composition A and composition B both significantly reduced the serum levels of ALT, AST and ADH, and AST and ALT are common enzymological indicators reflecting liver function damage and are liver function damage sensitivity indicators. Most of alcohol entering the body is oxidized into acetaldehyde by ADH, and is finally decomposed into carbon dioxide and water by other actions, which shows that the composition of the mesenchymal stem cell exosome and the piceatannol has a remarkable effect on treating the alcoholic liver diseases.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. An application of mesenchymal stem cell exosome in preparation of drugs for treating fatty liver diseases is characterized in that: the fatty liver disease is alcoholic fatty liver disease.
2. The use of mesenchymal stem cell exosomes according to claim 1 in the preparation of a medicament for treating fatty liver disease, wherein the medicament comprises: the preparation method of the mesenchymal stem cell exosome comprises the following steps: cutting the umbilical cord into small segments, fully washing and cutting into pieces, then placing the small segments into a culture dish, culturing and subculturing, placing the mesenchymal stem cells after subculturing into the culture dish, cleaning the cells when the cell fusion reaches 60-90%, replacing a culture medium without exosome serum, continuing culturing, collecting cell supernatant, centrifuging to remove the cells and cell fragments, and extracting to obtain exosomes.
3. An application of a composition of mesenchymal stem cell exosome and piceatannol in preparing a medicament for treating fatty liver diseases.
4. A composition of mesenchymal stem cell exosomes and piceatannol, comprising: the mass ratio of the mesenchymal stem cell exosome to the composition of piceatannol is 1: 10-500.
5. The method of preparing a composition of mesenchymal stem cell exosomes and piceatannol according to claim 4, comprising: the method comprises the following steps: dissolving piceatannol in glycerol to obtain piceatannol solution, performing ultrasonic treatment on piceatannol, adding mesenchymal stem cell exosome, and mixing uniformly to obtain the piceatannol.
6. The method of preparing a composition of mesenchymal stem cell exosomes and piceatannol according to claim 5: the concentration of the piceatannol solution is 0.1-0.3 ug/ml.
7. A drug for treating fatty liver, which is characterized in that: the medicine contains the composition of mesenchymal stem cell exosome and piceatannol.
8. The fatty liver disease drug of claim 7, wherein: the concentration of the composition in the medicine is 1-50 ug/g.
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Citations (5)
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| JP2001247470A (en) * | 2000-03-03 | 2001-09-11 | Morishita Jintan Kk | Agent for protecting liver |
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Patent Citations (5)
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|---|---|---|---|---|
| JP2001247470A (en) * | 2000-03-03 | 2001-09-11 | Morishita Jintan Kk | Agent for protecting liver |
| US20120107413A1 (en) * | 2009-07-23 | 2012-05-03 | Singapore Health Services Pte Ltd | Pre-natal mesenchymal stem cells |
| WO2020172270A1 (en) * | 2019-02-19 | 2020-08-27 | Direct Biologics, Llc | Acellular intravenous infusion including mesenchymal stem cell growth factors and exosomes |
| WO2020257720A1 (en) * | 2019-06-19 | 2020-12-24 | Celularity Inc. | Exosomes for disease treatment |
| CN112352047A (en) * | 2020-09-08 | 2021-02-09 | 江苏大学 | Preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells |
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