CN111088214A

CN111088214A - Liver-like cell exosome of stem cell source, preparation method and application thereof

Info

Publication number: CN111088214A
Application number: CN201911219571.3A
Authority: CN
Inventors: 杨博; 王兰; 赵圆圆; 朱琳; 代辰; 赵光远; 陈栋; 魏来; 陈知水
Original assignee: Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-05-01

Abstract

The present invention relates to a method for preparing exosomes, the exosomes are obtained by using in vitro stem cells to induce and differentiate into liver-like cells to secrete and obtain the exosomes, the liver-like exosomes prepared by the method, and the The application of liver-like cell exosomes in the preparation of drugs for treating liver ischemia-reperfusion injury and in preventing hypoxic injury of isolated cells. The invention induces stem cells to differentiate into hepatocyte-like cells and obtains hepatocyte-like cell exosomes. The exosomes can reduce liver I/R injury in vivo and hepatocyte injury caused by hypoxia culture in vitro, and its protective effect is better than that of extrahepatic cells. exosomes. Meanwhile, exosomes of hepatocyte-like cells induced by BMSCs led to increased autophagy in liver tissue in vivo and in hepatocytes in vitro. Therefore, the hepatocyte-like cell exosomes of the present invention can be used as a therapeutic drug for liver I/R injury and an agent for preventing hypoxic injury of isolated cells.

Description

Liver-like cell exosome of stem cell source, preparation method and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a method for preparing exosomes, a liver-like exosome prepared by the method, application of the liver-like exosome in preparing a medicine for treating liver ischemia-reperfusion injury and application in preventing isolated cell hypoxia injury.

Background

Hepatic ischemia/reperfusion (I/R) injury is a serious complication during surgical procedures, such as hepatectomy and Orthotopic Liver Transplantation (OLT), which can adversely affect patient and graft outcome. Impaired autophagy, mitochondrial dysfunction and consequent disruption of cellular homeostasis are characteristic of liver I/R damage. Although the mechanism of liver I/R injury has been extensively studied, there is no effective way to reduce or prevent liver I/R injury. Therefore, there is an urgent need to develop effective therapeutic agents to treat I/R induced liver damage.

Exosomes (exosomes) are membrane-derived nanovesicles (30-150nm) that are released by many types of cells under normal and pathological conditions, including hepatocytes. The effects of exosomes on cell function are associated with a variety of physiological processes, including: cell-cell communication, cancer metastasis, immunomodulatory activity, and spread of infectious agents. Recent studies have shown that exosomes have a protective effect in tissue ischemia-reperfusion injury. Exosomes can reduce the extent of myocardial infarction and ameliorate renal ischemia reperfusion injury by inhibiting inflammatory responses, reducing oxidative stress, inhibiting fibrosis and promoting angiogenesis. Recent studies have also found that hepatocytes can secrete exosomes and play an important role in the regeneration and repair of hepatocytes. The clinical advantages of exosomes compared to cell therapy are enormous. By intravenous infusion of cells, most of the cells adhere to the alveolar capillaries and the survival rate of the host is low. However, exosomes do not present problems such as cell embolization or stem cell differentiation. In addition, by enriching for some proteins and RNA, exosomes may be better protected than cell therapies.

Recent studies have shown that hepatocyte-derived exosomes form sphingosine-1-phosphate (S1P) in hepatocytes, leading to cell proliferation and liver regeneration following I/R injury or partial hepatectomy. More importantly, exosomes secreted by hepatocytes have been shown to be of great interest for new therapeutic approaches to acute and chronic liver diseases. However, there are still many limitations in the acquisition and in vitro culture of primary hepatocytes. Therefore, there is a need for new drugs for the treatment of I/R injury.

Disclosure of Invention

To solve the above problems, the present invention provides a method for preparing exosomes obtained by inducing secretion of differentiated liver-like cells using an ex-vivo stem cell.

In a specific embodiment, the method comprises the steps of:

s1: inducing the ex vivo stem cells into hepatocyte-like cells;

s2: allowing the hepatocyte-like cell to secrete exosomes;

s3: collecting the exosomes.

In a specific embodiment, the ex vivo stem cell is a bone marrow mesenchymal stem cell.

In a preferred embodiment, S1 is used to differentiate the ex vivo stem cells into the hepatocyte-like cells by adding fibroblast growth factor 4, hepatocyte growth factor, oncostatin M, insulin-transferrin-sodium selenite and dexamethasone for incubation in the culture environment of the ex vivo stem cells.

In a preferred embodiment, S1 includes the steps of:

s11: fibroblast growth factor 4 was added on induction day 0;

s12: adding hepatocyte growth factor on day 3;

s13: adding hepatocyte growth factor, tumor suppressor M, insulin-transferrin-sodium selenite and dexamethasone on day 6, and culturing until the stem cells are differentiated into liver-like cells.

In a preferred embodiment, in S2, the hepatocyte-like cells are cultured in a serum environment without exosomes for 24 hours, allowing the hepatocyte-like cells to secrete exosomes.

The invention also provides a liver-like cell exosome prepared by the method.

The invention also provides application of the liver-like cell exosome in preparing a medicament for treating liver ischemia-reperfusion injury.

The invention also provides application of the liver-like cell exosome in preventing isolated cell hypoxia injury.

Preferably, the ex vivo cell is an ex vivo hepatocyte.

The invention can reduce the in vivo liver I/R damage and the in vitro liver cell damage caused by hypoxia culture by inducing the stem cells to differentiate into the liver-like cells and obtaining the liver-like cell exosomes, and the protective effect of the exosomes is better than that of the liver cell exosomes. Meanwhile, exosomes of hepatocyte-like cells induced by BMSCs resulted in increased autophagy of liver tissues in vivo and liver cells in vitro. Therefore, the liver-like cell exosome can be used as a therapeutic drug for liver I/R injury and an agent for preventing hypoxia injury of isolated cells.

Drawings

FIG. 1 shows the expression of CD29, CD44, CD105, Sca-1, CD11b, CD31, CD45 and CD117 on the surface of isolated BMSC cells;

FIG. 2 is a photomicrograph of liver-like cells after 21 days of induced BMSC culture, showing epithelioid differentiation;

FIG. 3 is a fluorescent photograph of induced liver-like cells stained with AFP antibody and ALB antibody, respectively;

FIG. 4 is a statistical plot of the expression levels of HNF-3 β in liver-like cells after induction;

FIG. 5 is an electron micrograph of liver-like cell exosomes;

FIG. 6 is a statistical plot of the particle size distribution of liver-like cellular exosomes;

FIG. 7 is a statistical plot of serum ALT and AST levels in mice from the I/R control group, MSC-Heps-Exo group, and Heps-Exo group;

FIG. 8 is a photograph of HE staining and TUNEL staining of liver sections of mice of the I/R control group, MSC-Heps-Exo group and Heps-Exo group;

FIG. 9 is a statistical chart of cell viability of control group, MSC-Heps-Exo group and Heps-Exo group in the isolated cell hypoxia experiment;

FIG. 10 is the apoptosis flow distribution graph of control group, MSC-Heps-Exo group and Heps-Exo group in the isolated cell hypoxia experiment;

FIG. 11 is the apoptosis rate according to FIG. 10;

FIG. 12 is a photograph of immunoblotting of liver tissues of mice in I/R control group and MSC-Heps-Exo group to detect LC3-II and Beclin-1 (left) and a statistical chart of the expression amounts of LC3-II and Beclin-1 (right) converted from the immunoblotting photograph;

FIG. 13 is an electron micrograph (lower) of liver sections of mice in the I/R control group and the MSC-Heps-Exo group and a result of counting autophagosomes obtained according to the electron micrograph (upper), and arrows indicate partial autophagosomes.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

1. Laboratory animal

C57BL/6 mouse, male, weighing 22. + -.2 g, purchased from Beijing HFK bioscience, Inc. (Beijing, China), housed in the animal house of the organ transplant research institute of the affiliated college of Tongji medical, Huazhong university. All procedures involved in animal use in this study were conducted and monitored according to the guidelines of the Chinese animal protection Committee and approved by the animal protection and use Committee of the affiliate of the college of Tongji medical college, university of science and technology, Huazhong.

2. Isolation, culture and hepatic differentiation of mesenchymal stem cells

BMSCs were obtained from C57BL/6 mice for subculture. The BMSCs obtained had a typical mesenchymal stem cell phenotype with positive CD29, CD44, CD105 and Sca-1, negative CD11b, CD31, CD45 and CD117, isotype control indicated as shaded curves (fig. 1), and we can see that we obtained typical BMSC cells.

The BMSCs of the third generation were induced to differentiate into hepatocyte-like cells by adding fibroblast growth factor 4(FGF-4) at a final concentration of 10ng/ml on day 0, Hepatocyte Growth Factor (HGF) on day 6, HGF (20 ng/ml) on day 6, oncostatin M (OSM) at a final concentration of 10ng/ml, 1 × insulin-transferrin-sodium selenite (ITS) treatment and dexamethasone (Dex) at a final concentration of 20 μ g/l. After 21 days of culture, epithelial-like cells morphologically resembling hepatocytes (fig. 2).

The expression of AFP and ALB was observed to be higher in cells induced by BMSC as shown in FIG. 3, while HNF-3 β gene expression was measured by qPCR, and as shown in FIG. 4, the level of HNF-3 β in the induced cells was lower than that of AML12 hepatocytes but significantly higher than that of BMSC (. times.P < 0.01).

3. Isolation of exosomes

The resulting hepatocyte-like cells were induced to change to a cell culture environment without Exosome serum, and after 24 hours of culture, cell supernatants were collected to extract exosomes, which were isolated from BMSC-derived hepatocyte-like cells using Total Exosome Isolation Kit (Invitrogen, California, USA):

0.5ml of total exosome-separating agent was added to each 1ml of filtered conditioned medium and mixed well by inversion. After overnight incubation at 4 ℃, the mixture was centrifuged at 12,000 × g for 70 minutes at 4 ℃. All supernatants were then removed by aspiration and the exosome pellet resuspended with a convenient amount of Phosphate Buffered Saline (PBS). The size and concentration of exosomes were determined using a Zetasizer Nano (Malvern instruments, Malvern, UK) and electron microscopy.

An electron micrograph of the exosomes is shown in fig. 5, which is in the form of a cup, and the size and concentration of the exosomes were determined using Zetasizer Nano, and the results are shown in fig. 6, with an average vesicle diameter of 144nm and a concentration of 4.9 × 10⁸particles/mL.

4. Modeling of liver injury model and exosome therapy

4.1 modeling of liver I/R injury

A70% liver I/R animal model was established according to the following method: mice were anesthetized with sodium pentobarbital (60mg/kg) by intraperitoneal injection. The arteries and portal vein were then blocked with atraumatic clamps for 60 minutes to stop blood flow to the left and middle lobes of the liver. During surgery, the temperature of the mice was maintained at 37 ℃ using a heating pad and incubator. After 6 hours of reperfusion, mice were sacrificed to collect blood and liver tissue. Mice in the treatment group were injected with 100 μ g of liver-like cell exosomes derived from mesenchymal stem cells (MSC-Heps-Exo group) or hepatocyte exosomes (Heps-Exo group), respectively, before and after surgery through the tail vein. At the same time point, the control group (I/R) was injected with an equal volume of saline. The control group was subjected to laparotomy only, and no vessel was occluded.

4.2 hepatic cell hypoxia modeling

The model of cellular hypoxia is by cobalt chloride (CoCl)₂Sigma, st louis, missouri) process. The mouse liver cell line AML12 was used in this experiment. For cell propagation, the cell lines were cultured in Dulbecco's modified Eagle's Medium/F-12 (DMEM/F12, Hyclone, Logan City, UT, USA) and supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), ITS broth supplement and 40ng/mL Dex. Cells were maintained at 37 ℃ and 5% CO₂In a moist incubator. In the model of cellular hypoxia, AML12 cells were incubated with 200. mu.M CoCl in FBS-free DMEM/F12 when the confluency reached 60% -70%₂The culture was carried out for 24 hours.

5. Analysis of liver-like cell exosome (MSC-Heps-Exo) treatment status

5.1 serum ALT/AST levels and histological observations

Serum ALT and AST levels were measured using a standard clinical automatic analyzer in the clinical laboratory of the affiliated college hospital of college medical college of huazhong university of science and technology. As shown in FIG. 7, serum ALT and AST levels were significantly reduced in both the MSC-Heps-Exo group and the Heps-Exo group compared to the I/R control group, and serum ALT and AST levels in the MSC-Heps-Exo group were only about 1/5 in the I/R group, significantly lower than in the Heps-Exo group.

The histopathological analysis was performed by the following method: after liver harvesting, fresh liver tissue was fixed in 4% paraformaldehyde for paraffin embedding. The embedded tissue was then cut into 5 μm sections and stained with hematoxylin-eosin (H & E). Cell death in liver paraffin sections was detected by TUNEL assay. The detection was carried out according to the instructions of the manufacturer's in situ cell death detection kit (Basel Roche, Switzerland). Staining photographs were taken using an optical microscope.

Fig. 8 shows HE and TUNEL stained photographs of liver tissue sections. HE staining of the liver tissue section shows that a large amount of bleeding and necrosis occur in the liver tissue of the I/R control group, the structure of liver lobes is seriously damaged, and TUNEL staining shows that a large area of apoptosis area is found in the liver tissue of the I/R control group. The liver injury of the exosome treatment group is obviously reduced, wherein the treatment effect of the MSC-Heps-Exo group is obviously better than that of the Heps-Exo group, the apoptosis area is minimum, and the liver lobe structure is more complete.

5.2 in vitro validation of hepatocyte protection

Cell viability was determined by CCK-8 kit (Dojindo, Japan). Briefly, cells were seeded at a density of 5000 cells/well in 96-well plates and incubated for 24 hours. Thereafter, the cells were subjected to the above-described hypoxia treatment, and then 10. mu.l of a CCK8 solution was added to each well and incubated at 37 ℃ for another 100 minutes. Finally, the absorbance value at a wavelength of 450nm was measured using a microplate reader. As shown in FIG. 9, the survival rate of AML12 cells was significantly higher in the exosome-treated group than in the I/R control group, whereas the survival rate of AML12 cells was about twice as high in the MSC-Heps-Exo group as in the control group, significantly higher than in the Heps-Exo group.

Apoptosis was analyzed using Annexin V-FITC and Propidium Iodide (PI) staining kit. Briefly, AML12 cells were digested and centrifuged at 300 × g for 5 minutes, then incubated with annexin V-FITC and PI in the dark for 5 minutes. The percentage of apoptotic cells was quantified by flow cytometry on a facscalibur (bd biosciences). Data analysis was performed using FlowJo software (oregon, usa). As shown in FIGS. 10 and 11, the apoptosis of the exosome-treated group was lower than that of the control group, especially late apoptotic cells (Annexin-V +/PI +), which were significantly reduced after MSC-Heps-Exo treatment. These results also demonstrate that MSC-Heps-Exo can increase the tolerance of hepatocytes to hypoxia more than Heps-Exo.

5.3, MSC-Heps-Exo enhanced autophagy during liver I/R injury

Autophagy is one of the most recent research hotspots, and the body eliminates damaged mitochondria and inhibits excessive ROS production by autophagy, which becomes an important guarantee against I/R damage. Elderly livers have difficulty tolerating I/R injury due to impaired autophagy, which in turn promotes the onset of Mitochondrial Permeability Transition (MPT) and cell death.

1) Detection of autophagy marker proteins LC3-II and Beclin-1

Expression of LC3-II in liver tissue was detected using immunoblotting by determining protein content using the Bradford assay (Sigma, USA) normalized for bovine serum albumin, separating the protein sample by SDS-PAGE gel, then electrotransferring onto PVDF membrane (Millipore, USA.) nonspecific binding sites were blocked with 5% bovine serum albumin TBST at room temperature for 1 hour, incubating the membrane with anti- β -actin primary antibody (Cell Signaling technology, Beverly, MA, USA) at 100000 dilution at 4 ℃ overnight, LC3, Beclin-1(Cell Signaling technology) at 1: 1000 dilution, then washing the membrane and incubating with secondary antibody at room temperature for 1 hour, then washing and detecting the signal.

As shown in FIG. 12, the level of LC3-II (standard indicator of autophagy activity) was increased in MSC-Heps-Exo-treated I/R mice compared to I/R mice, and the PI3K complex component Beclin-1 required for autophagy also appeared in a similar phenomenon to that of LC 3-II.

2) Effect of MSC-Heps-Exo on autophagosome number

Exosomes in PBS were fixed in 1.5M sodium cocoate buffer (ph7.4) adsorbed onto copper mesh formvar grids (Electron microscopy Sciences, Hatfield, PA) and negatively stained with 2% uranyl acetate using Electron microscopy. Liver tissue was dissected with 2.5% glutaraldehyde ultrathin sections and double stained with uranyl acetate and lead citrate. The sample was observed and an image was taken using an H7700 transmission electron microscope (hitachi, tokyo, japan) for data collection. The number of exosomes and autophagosomes was counted randomly at the same magnification from each sample.

As a result, as shown in FIG. 13, the number of autophagosomes in the liver of the MSC-Heps-Exo group mice was significantly increased compared to the I/R control mice.

The above results indicate that MSC-Heps-Exo treatment can enhance autophagy corpuscle formation and autophagy during liver I/R injury.

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

1. A method for preparing exosomes, characterized in that, the exosomes are obtained by using in vitro stem cells to induce and differentiate into hepatocyte-like cells to be secreted.

2. method according to claim 1, is characterized in that, comprises the following steps:

S1: inducing the isolated stem cells into hepatocyte-like cells;

S2: make the liver-like cells secrete exosomes;

S3: Collect the exosomes.

3. The method according to claim 2, wherein the ex vivo stem cells are bone marrow mesenchymal stem cells.

4. The method according to claim 2, characterized in that, S1 is performed by adding fibroblast growth factor 4, hepatocyte growth factor, oncostatin M, insulin-transferrin in the culture environment of the isolated stem cells - Sodium selenite and dexamethasone incubation to differentiate the ex vivo stem cells into the hepatoid cells.

5. method according to claim 4, is characterized in that, S1 comprises the following steps:

S11: Fibroblast growth factor 4 was added on day 0 of induction;

S12: Add hepatocyte growth factor on day 3;

S13: On the 6th day, hepatocyte growth factor, oncostatin M, insulin-transferrin-sodium selenite and dexamethasone were added, and the cells were cultured until the stem cells were differentiated into hepatoid cells.

6 . The method according to claim 1 , wherein in S2, the hepatoid cells are cultured in a serum environment without exosomes for 24 hours, so that the hepatoid cells secrete exosomes. 7 .

7. A liver-like cell exosome, characterized in that, prepared by the method according to any one of claims 1-6.

8. Use of the hepatocyte-like cell exosome of claim 7 in the preparation of a medicament for treating liver ischemia-reperfusion injury.

9. The application of the hepatoid cell exosome of claim 7 in preventing hypoxic injury of isolated cells.

10. The use according to claim 9, wherein the isolated cells are isolated hepatocytes.