CN114748506B - Application of bone marrow mesenchymal stem cell exosomes - Google Patents

Abstract

The invention provides application of bone marrow mesenchymal stem cell exosomes. Liver function damage can be alleviated by injecting BMSCs-Exo into HIRI kunming mice. Wherein miR-25-3p is significantly down-regulated in HIRI liver, BMSCs and BMSCs-Exo both contain miR-25-3p, when miR-25-3p mimics (miR-25-3 p agomir and miR-25-3p mimic) are respectively transferred into HIRI mice and hypoxia/reoxygenation AML12 liver cells, miR-25-3p expression quantity is increased, apoptosis and inflammation related factor expression reduction is detected through qRT-PCR and Western blotting, and liver injury is reduced through measuring aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) content in serum and hematoxylin-eosin (HE) staining. In addition, the MAPK/ERK signaling pathway and PI3K/AKT signaling pathway were tested for reduced expression of genes and related proteins by qRT-PCR and Western blotting. Therefore, miR-25-3p inhibits hepatic cell apoptosis induced by HIRI injury by inhibiting MAPK/ERK and PI3K/AKT signal paths, so that HIRI is improved, and a new thought is provided for treatment of HIRI.

Description

Application of bone marrow mesenchymal stem cell exosomes

Technical field

The present invention relates to the biological and medical fields, and in particular to the application of bone marrow mesenchymal stem cell exosomes.

Background technique

Hepatic ischemia reperfusion injury (HIRI) refers to a pathological phenomenon in which the blood flow supply to the liver is blocked for a period of time during liver surgery and then the blood flow supply is restored. Liver dysfunction and structural damage are not alleviated but worsened. . HIRI is a common clinical problem in liver trauma treatment, liver lobectomy, liver transplantation, etc. It can lead to organ failure and increase the incidence of rejection, which has a great impact on the prognosis and survival of patients. Up to now, there is still a lack of clinically effective intervention measures. During ischemia-reperfusion, cells are in a state of oxidative stress, with endoplasmic reticulum stress and calcium overload, mitochondrial swelling and outer membrane rupture, and cytochrome C release, thereby stimulating the apoptotic signaling pathway and leading to cell apoptosis. occur.

Mesenchymal stem cells (MSCs) are one of the most widely used stem cell types in clinical practice for immune regulation, organ reconstruction, and tissue repair. They can respond to resting or hypoxia, stress, radiation, and oxidative damage. Secrete exosomes (Exo) under other stimuli. Exosomes are considered to be a new therapeutic tool for delivering functional proteins, mRNA, miRNA and lncRNA, transporting bioactive molecules to recipient cells to regulate cell functions, which have higher biological efficiency compared with stem cells. Safety and more stable signal transduction efficiency, it can serve as a strong candidate for cell-free therapy. Studies have shown that mesenchymal stem cell exosomes can regulate cell apoptosis and reduce liver ischemia-reperfusion injury. However, stem cell exosomes contain a variety of bioactive substances, which have negative effects on the role of stem cell exosomes in reducing liver ischemia-reperfusion injury. The mechanism requires further study.

Contents of the invention

In order to overcome the shortcomings of the existing technology, one of the purposes of the present invention is to provide an application of bone marrow mesenchymal stem cell exosomes.

One of the objectives of the present invention is achieved by adopting the following technical solution: application of bone marrow mesenchymal stem cell exosomes in the preparation of drugs for treating liver ischemia-reperfusion injury, and the drugs are used to inhibit the MAPK/ERK signaling pathway and PI3K/AKT signaling pathway.

As an embodiment, the bone marrow mesenchymal stem cell exosomes contain miR-25-3p.

The second object of the present invention is to provide an application of miRNA derived from bone marrow mesenchymal stem cell exosomes in the preparation of drugs for treating liver ischemia-reperfusion injury.

The second object of the present invention is achieved by the following technical solution: the application of a bone marrow mesenchymal stem cell exosome-derived miRNA in the preparation of a drug for treating liver ischemia-reperfusion injury, the drug is used to inhibit MAPK/ ERK signaling pathway and PI3K/AKT signaling pathway.

As an embodiment, the miRNA is miR-25-3p.

A third object of the present invention is to provide a drug for treating liver ischemia-reperfusion injury.

The third object of the present invention is achieved by adopting the following technical solution: a drug for treating liver ischemia-reperfusion injury, the drug contains components that inhibit the MAPK/ERK signaling pathway and the PI3K/AKT signaling pathway.

As an embodiment, the drug contains miR-25-3p.

Compared with the existing technology, the beneficial effect of the present invention is that: the present invention finds that human bone marrow mesenchymal stem cell exosome-derived miRNAmiR-25-3p inhibits HIRI injury-induced hepatocytes by inhibiting the MAPK/ERK and PI3K/AKT signaling pathways. Apoptosis, thereby reducing liver ischemia-reperfusion injury. Therefore, the present invention provides new ideas for the treatment of HIRI.

Description of the drawings

Figure 1A is a diagram showing the diameter size results of ZetaView detection of exosomes provided by an embodiment of the present invention;

Figure 1B is a diagram showing the morphological characteristics of exosomes using a transmission electron microscope according to an embodiment of the present invention;

Figure 1C is a diagram showing the results of detecting exosome marker proteins by Western blotting provided by an embodiment of the present invention;

Figure 2 is a graph showing the results of ALT and AST content in the serum after the BMSCs exosomes provided by the embodiment of the present invention were injected into HIRI mice and reperfused for 3h, 6h, and 9h respectively;

Figure 3 shows the qRT-PCR detection of miR-25-3p in the liver of HIRI mice and its control CK, BMSCs, BMSCs-Exo, BMSCs-Exo treated HIRI mouse group and its control PBS-treated HIRI mouse provided by the embodiment of the present invention. Plot of content results in the group;

Figures 4A and 4B are diagrams showing the results of Tunel detection of cell apoptosis in liver tissue provided by embodiments of the present invention; wherein, the white cells in Figure 4A represent apoptotic cells, and the gray cells represent normal cells;

Figure 4C is a diagram showing the results of qRT-PCR detection of the contents of GSDMD and NLRP3 mRNA in liver tissue provided by the embodiment of the present invention;

Figure 4D and Figure 4E are the results of Western blot detection of Caspase1 content in liver tissue provided by the embodiment of the present invention; lanes 1 and 2 of Figure 4D are physiological saline NS, and lanes 3 and 4 are miR-25-3p agomir. ;

Figure 4F is a diagram showing the results of qRT-PCR detection of inflammation-related gene content in AML12 cells provided by the embodiment of the present invention;

Figure 5A is a graph showing the results of the levels of ALT and AST in the serum of mice with ischemia-reperfusion injury treated with the miR-25-3p mimic provided by the embodiment of the present invention;

Figure 5B is a diagram showing the results of HE staining of liver tissue after the miR-25-3p mimic provided by the embodiment of the present invention treated mice with liver ischemia and reperfusion;

Figure 6A and Figure 6B are the results of Western blot detection of p-AKT and p-ERK expression in liver tissue provided by the embodiment of the present invention; wherein, lanes 1 and 2 in Figure 6A represent NS, and lanes 3 and 4 represent agomir. ;

Figure 6C is a diagram showing the results of qRT-PCR detection of PI3K, Raf, Ras, and MEK gene mRNA contents in liver tissue provided by the embodiment of the present invention;

Figure 6D is a graph showing the results of qRT-PCR detection of PI3K and MEK gene mRNA contents in AML12 hypoxia-reoxygenated liver cells provided by the embodiment of the present invention.

Detailed ways

Below, the present invention will be further described with reference to the accompanying drawings and specific implementation modes. It should be noted that, on the premise that there is no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. . In the following examples, unless otherwise stated, all reagents used are of analytical grade and can be obtained from commercial sources. Experimental methods that do not indicate specific conditions in the article usually follow conventional conditions such as those described in the book "Molecular Cloning Experimental Guide" edited by J. Sambrook et al. published by Science Press in 2002, or as recommended by the manufacturer. conditions of. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as familiar to one skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the present invention.

1. Experimental methods

1. Collection, purification and identification of BMSCs exosomes

Human bone marrow mesenchymal stem cells (BMSCs) were purchased from Wuhan Pronosei Life Technology Co., Ltd. (CP-H166) and cultured in DMEM medium containing 10% fetal calf serum. When the cells reached 70%-80% confluence, Wash with PBS, inoculate into serum-free medium at a ratio of 1:3 and culture for 48 hours. Collect the supernatant at 4°C, centrifuge at 300g for 10 minutes, and retain the supernatant; centrifuge again at 2,000g for 10 minutes, retain the supernatant; centrifuge at 10,000g for 30 minutes, retain the supernatant. Clear; filter with a 0.22 μm filter, centrifuge at 100,000g for 2 hours, and retain the precipitate. Add PBS, mix well, and centrifuge again at 100,000g for 2 hours to retain the precipitate, which is considered to be relatively pure exosomes. Add 100 μL PBS to the exosomes and freeze them in a -80°C refrigerator. Particle analyzer detects exosome diameter. The morphology of exosomes was observed by transmission electron microscopy. Western blot identification of exosome surface markers.

2. Experimental animals

SPF grade male Kunming mice, weighing 20-25g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (production license: SCXK (Hunan) 2021-0002). Feed ordinary mouse chow and filtered tap water ad libitum. Room temperature 22℃±1℃, humidity 65%±5%, 12h light/dark alternation. The animal experiment protocol was reviewed and approved by the Animal Ethics Committee of Guilin Medical College. The experimental process was strictly implemented in accordance with the National Standards of the People's Republic of China (GB/T 35892-2018) for the Ethical Review Guidelines for Laboratory Animal Welfare.

3. Source of miR-25-3p mimics

Purchase the MiR-25-3p mimic for cells, MiR-25-3pmimic, and its control, MiR-25-3p mimic NC, as well as the MiR-25-3p mimic for animals from Guangzhou Ribo Biotechnology Co., Ltd. miR-25-3pagomir. Physiological saline (NS) was used as a control for miR-25-3p agomir. .

4. Preparation of mouse liver ischemia-reperfusion injury model

Mice were divided into experimental group (agomir) and control group (NS), with 3 mice in each group. 24 h before surgery, each mouse was injected with 100 μl of 13 nmol agomir diluted in normal saline or 100 μl of normal saline through the tail vein. No food was allowed 12 hours before surgery and water was freely available. 4% chloral hydrate was anesthetized by intraperitoneal injection at 0.1ml/10g, and the limbs were fixed with tape. Disinfect with 70% alcohol, make a longitudinal opening in the middle of the abdomen, and cut the skin layer and muscle layer up to the xiphoid process. The left lobe and middle lobe of the liver were exposed, and the hepatic artery and portal vein of the middle lobe and left lobe were clamped with non-invasive vascular clips. During the clamping process, the incision was covered with wet gauze, and the mouse was placed on a 37°C constant-temperature heating pad to keep warm. After 60 minutes of continuous ischemia, the vascular clamp was quickly removed to restore the ischemic liver blood flow, the abdominal muscles and skin were sutured layer by layer, and the incision was disinfected. The mice were harvested 6 hours after reperfusion.

5. Cellular hypoxia-reoxygenation model

Mouse hepatocytes (AML12) were purchased from the Type Culture Collection Cell Bank of the Chinese Academy of Sciences (SCSP-550). Cells were cultured in DMEM medium containing 10% fetal calf serum. Cells were plated in a 24-well plate 24 hours in advance at a density of 1.5 × 105 to 2.5 × 105 cells/well. When the cell coverage reached about 60% to 70%, the cells were transfected with 100nM of miR-25-3p mimic and its control miR-25-3p mimic NC. After 24 hours, the cells were cultured with PBS for 6 hours under hypoxic conditions (5% CO2, 1% O2, and 94% N2, 37°C), and then for 12 hours under normal conditions.

6. Serological indicator detection

Blood was collected from the eyeballs of the mice at 6 hours of reperfusion, left to stand at room temperature for 30 minutes, and then centrifuged at 1800g for 15 minutes at 4°C. The upper serum was taken, and the aspartate aminotransferase (AST) in the serum was measured using a kit from Nanjing Jiancheng Bioengineering Institute. ) and alanine aminotransferase (ALT) levels, the OD value of each well was measured with a microplate reader with a wavelength of 510 mm.

7. Tunel experiment

Liver tissue was fixed with 4% paraformaldehyde for 48 hours, dehydrated with graded alcohol, embedded in paraffin, and sectioned with a paraffin microtome at a thickness of 4 μm. Bake in an oven at 60°C for 4.5 hours, dewax, and hydrate with gradient alcohol. Treat the tissue with Proteinase K at 37°C for 20 minutes. Add 50 μl of TUNEL reaction mixture to each tissue, and react for 1 hour in a humid box at 37°C in the dark. Add 100 ul of DAPI staining solution dropwise, and incubate in a constant temperature incubation box at room temperature for 20 minutes in the dark. Water-soluble mounting medium Cover the slide. Use a fluorescence microscope with a green wavelength of 488nm and a red wavelength of 596nm to observe and take pictures, and calculate the TUNEL positive rate.

8. Hematoxylin-eosin (HE) staining experiment

Liver tissue was fixed with 4% paraformaldehyde for 48 hours, dehydrated with graded alcohol, embedded in paraffin, and sectioned with a paraffin microtome at a thickness of 4 μm. Bake in an oven at 60°C for 4.5 hours, dewax, and hydrate with gradient alcohol. Hematoxylin staining for 5 min, hydrochloric acid ethanol differentiation for 30 sec, and eosin staining for 5 min. Dehydrated with graded alcohol and mounted with neutral gum. Optical microscopy observation.

9. Real-time quantitative PCR (qRT-PCR) experiment

Use the UNIQ-10 column Trizol total RNA extraction kit from Sangon Bioengineering (Shanghai) Co., Ltd. to extract mRNA and miRNA from AML12 cells or liver tissue. The OD260 value and OD260/280 ratio were measured using a spectrophotometer, the RNA concentration was calculated based on the OD260, and the integrity of the total RNA was identified by 1% agarose gel electrophoresis. miRNA and mRNA were reverse transcribed into cDNA using Maxima ReverseTranscriptase (Thermo Scientific). 2X SG Fast qPCRMaster Mix (High Rox, B639273, BBI, ABI) was used to perform qRT-PCR on mRNA, and RiboBio miRNA detection was performed on miRNA according to the instruction manual. The relative expression of genes was determined using the 2-DDCT method. Endogenous controls for mRNA and miRNA expression are GAPDH and U6, respectively. The sequences of each primer are as follows:

10. Western blot experiment

Add protease inhibitors and RIPA lysis buffer to the liver tissue or cells to extract total protein. Protein concentration was determined using BCA protein quantification kit (Solarbio). Select different concentration (10%-15%) separation gels according to the protein molecular weight for electrophoresis separation, activate the PVDF membrane with methanol, install a "sandwich" structure, transfer the protein to the PVDF membrane through electrophoresis, and block with 5% skim milk at room temperature for 1 hour. Wash the membrane three times with PBST, incubate with primary antibody at 4°C overnight, and incubate with secondary antibody at 37°C for 1 hour. The membrane was washed three times with PBST, and protein bands were detected using an ECL kit (Millipore). The gel imaging system scans and quantifies the optical density values of the bands.

11. Transmission electron microscope

Hepatocytes prefixed with 2% paraformaldehyde-2.5% glutaraldehyde, rinsed with cold buffer for 15 min The 812 resin mixture was soaked overnight, embedded in Epon 812 resin, polymerized at 35°C for 24h, 45°C for 12h, 55°C for 12h and 60°C for 24h. After positioning the semi-thin sections, 50nm ultra-thin sections were made with a glass knife, uranyl acetate and citrate. Lead double electron staining, observed with JEM-1230 transmission electron microscope. After the cells are digested, the cells can be made into a suspension before embedding and slicing. Exosomes are dropped on a carrier copper grid with a pore size of 2 nm under exosome electron microscopy, left to stand for 2 minutes, and then negatively stained with 3% phosphotungstic acid for 5 minutes. JEM-1230 type transmission Electron microscopy observation.

12. Statistical analysis

SPSS 25.0 statistical software was used for analysis, and P<0.05 was considered a statistically significant difference.

2. Experimental results

1. Isolation and identification of BMSCs exosomes

The cell culture supernatant of P3 generation BMSCs was collected and subjected to differential centrifugation. The particle size of the exosomes obtained was detected with a particle analyzer. Figure 1A shows the results of ZetaView detection of the diameter size of exosomes; Figure 1B shows the morphological characteristics of exosomes using transmission electron microscopy; Figure 1C shows the results of detection of marker proteins by Western blot. The specific results are as follows: The particle diameter of the purified exosomes detected by ZetaView is approximately 60-200nm. Transmission electron microscopy shows that the diameter of exosomes is about 60-200nm, low electron density components can be seen in the cells, they are round or oval, and the cell membrane structure is intact. Westernblot shows that CD9 and CD63 are positive in exosomes, but Calnexin is negative.

2. BMSCs exosomes can improve liver damage caused by HIRI

In order to detect the impact of BMSCs exosomes on HIRI mice, 100 μg BMSCs exosomes were dissolved in 100 μL PBS, injected into HIRI mice through the tail vein, and reperfused for 3h, 6h, and 9h respectively. ALT in the serum was detected by eyeball blood sampling. , AST content. Please refer to Figure 2. The results show that at three time points of 3h, 6h, and 9h reperfusion, the ALT and AST of the BMSCs exosome injection group were lower than those of the PBS injection group.

3. miR-25-3p participates in BMSCs exosomes to reduce HIRI damage

In order to clarify the correlation between miR-25-3p and BMSCs exosomes in reducing HIRI injury, qRT-PCR was used to detect the expression of miR-25-3p in the livers of HIRI mice and their control CK, BMSCs, BMSCs-Exo, and BMSCs-Exo treatments. The content in the HIRI mouse group and its control PBS-treated HIRI mouse group. Please refer to Figure 3. It can be seen that the expression of miR-25-3p in the HIRI liver is lower than that in the CK group; both BMSCs and BMSCs-Exo contain miR-25-3p; when BMSCs-Exo was transferred into HIRI mice through the tail vein , the level of miR-25-3p in liver tissue increased. Therefore, miR-25-3p exists in BMSCs and their exosomes and participates in the reduction of HIRI injury by BMSCs exosomes.

4.miR-25-3p inhibits apoptosis

The miR-25-3p mimic agomir can overexpress miR-25-3p in animals. It was injected into HIRI mice through the tail vein. At 6 hours after reperfusion, the livers were taken and paraffin sections were made. Tunel experiment was used to detect cell apoptosis. DAPI was used to Label cell nuclei. Please refer to Figure 4A and Figure 4B. The results show that the rate of Tunel-positive cells in the experimental group (agomir group) was lower than that in the physiological saline injection group (NS group). Please refer to Figure 4C. qRT-PCR detection showed that the levels of GSDMD and NLRP3 mRNA in the liver tissue of the experimental group were also lower than those of the saline group. Please refer to Figure 4D and Figure 4E. The content of Caspase1 protein in the liver tissue of the experimental group was lower than that of the NS group. The miR-25-3p mimic mimic can overexpress intracellular miR-25-3p. The miR-25-3p mimic and control NC were transferred into AML12 hypoxia-reoxygenation hepatocytes, and the content of inflammation-related genes was detected by qRT-PCR. , please refer to Figure 4F, it was found that the contents of TNF-α, IL-6, IL-1b and IL-18 were lower than those in the control group, and TNF-α and IL-1b reached significant differences.

5.miR-25-3p reduces liver damage

Serum ALT and AST levels generally reflect the degree of liver cell damage. After the miR-25-3p agomir was transferred into HIRI mice, blood was collected from the eyeballs to detect the levels of ALT and AST in the serum. Please refer to 5A, the results showed that the ALT and AST contents of the miR-25-3p group were lower than those of the control NS group, and the ALT contents decreased significantly. Please refer to 5B. Observing the liver tissue structure, it was found that the liver lobules in the experimental group had a complete structure, the liver cell cords were arranged neatly, and the cytoplasm was uniform. However, in the control NS group, the liver lobules had an unclear structure, the liver cells were arranged disorderly, enlarged to varying degrees, and vacuoles appeared. degeneration and inflammatory infiltration.

6. miR-25-3p regulates MAPK/ERK and PI3K/AKT signaling pathways

In order to elucidate the signaling pathway by which miR-25-3p regulates cell apoptosis, liver tissues of mice from the experimental group and the control group were collected for detection. Please refer to Figure 6A and Figure 6B. Western blot results show that overexpression of miR-25-3p significantly inhibits the expression of p-AKT and p-ERK. Please refer to Figure 6C, the qRT-PCR results showed that the levels of PI3K, Raf, Ras and MEK were also lower than those in the control group. When AML12 cells were transferred into miR-25-3p mimic, please refer to Figure 6D. qRT-PCR also showed that the levels of PI3K and MEK mRNA were reduced. Among them, Raf, Ras, MEK and ERK are related factors of the MAPK/ERK signaling pathway, and PI3K and AKT are related factors of the PI3K/AKT signaling pathway. This shows that miR-25-3p inhibits the MAPK/ERK signaling pathway and the PI3K/AKT signaling pathway.

7. Summary

In the embodiments of the present invention, through the above experiments and analysis, it can be seen that the expression of miR-25-3p is down-regulated in the liver of HIRI mice. When BMSCs exosomes are injected into HIRI mice, HIRI can be alleviated, and experimental verification miR-25-3p exists in BMSCs and their exosomes, and is involved in BMSCs exosomes reducing HIRI damage.

After transfecting miR-25-3p into mouse hepatocyte AML12 hypoxia-reoxygenation model, qRT-PCR showed that the expression of inflammatory factors TNF-α, IL-6, IL-1β, and IL-18 decreased. miR-25-3p also causes a decrease in the expression of apoptosis-related factors Caspase-1, NLRP3, and GSDMD in the liver tissue of HIRI mice. This shows that miR-25-3p plays a role in inhibiting inflammation and apoptosis in HIRI. By detecting ALT and AST levels in serum, and observing liver tissue structure, it can be concluded that miR-25-3p can slow down liver damage. .

Some studies have found that downregulation of cellular communication network factor 1 (CCN1) can reduce ALT, AST levels, MPO activity, IL-6 and TNF-α levels in C57BL/6HIRI mice, inhibit the MEK/ERK signaling pathway, and reduce inflammation. reactions and liver damage. Some studies have also found that cerebroprotein hydrolysate-I (CH-I) inhibits the expression of MEK-ERK-CREB after cerebral ischemia/reperfusion injury and increases the expression of BDNF, thus playing a neuroprotective role and improving the brain function. Neurobehavioral function in rats with arterial embolization. Therefore, it shows that inhibiting the MEK/ERK signaling pathway can reduce the inflammatory response and play a protective role in ischemia/reperfusion organs. Another study found that octreotide (OCT) can inhibit p62 expression by inhibiting the PI3K/AKT/mTOR/S757-ULK1 signaling pathway, and increase the expression of Beclin-1, ATG7 and LC3, leading to liver autophagy and maintaining a series of antioxidants. and anti-inflammatory cascade, protecting the liver from ischemia-reperfusion injury. At the same time, the PI3K/AKT pathway is involved in the regulation of cell apoptosis. Some studies have found that TLR4 is activated after spinal cord injury, promoting the expression of lncRNA-F630028O10Rik. This lncRNA, as the ceRNA of the miR-1231-5p/Col1a1 axis, enhances the apoptosis of microglia after spinal cord injury by activating the PI3K/AKT pathway. Therefore, it shows that inhibiting the PI3K/AKT signaling pathway can reduce the inflammatory response and play a protective role in ischemia/reperfusion organs. Therefore, inhibiting the MEK/ERK signaling pathway and PI3K/AKT signaling pathway can reduce the inflammatory response and protect organs from ischemia/reperfusion.

This application found through experiments that miR-25-3p reduces the contents of Raf, Ras, MEK and ERK in the MAPK/ERK signaling pathway, and also reduces the contents of PI3K and AKT in the PI3K/AKT signaling pathway. Therefore, miR-25-3p inhibits the MAPK/ERK signaling pathway and the PI3K/AKT signaling pathway.

In summary, miR-25-3p inhibits hepatocyte apoptosis induced by HIRI injury by inhibiting the MAPK/ERK and PI3K/AKT signaling pathways, thereby improving HIRI and providing new ideas for the treatment of HIRI.

The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantive changes and substitutions made by those skilled in the art on the basis of the present invention fall within the scope of the present invention. Scope of protection claimed.

Claims (1)

1. The application of miRNA derived from bone marrow mesenchymal stem cell exosome in preparing in-vitro hepatic cell MAPK/ERK signal pathway and PI3K/AKT signal pathway inhibiting reagent is characterized in that the miRNA is miR-25-3p.