CN112472722A

CN112472722A - Technical means for promoting body injury repair by using embryonic stem cell exosomes

Info

Publication number: CN112472722A
Application number: CN202011441433.2A
Authority: CN
Inventors: 刘娜; 于璐; 李宗金
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2021-03-12

Abstract

The invention relates to a technical means for promoting the repair of body injury by using exosomes secreted by embryonic stem cells, which takes acute kidney injury and skin injury caused by ischemia-reperfusion as models. The embryonic stem cells have high self-renewal and multi-directional differentiation potential, but the clinical application of the embryonic stem cells is limited due to the problems of immunological rejection, ethics, tumorigenicity and the like in medical application. Exosomes are substances encapsulated by a phospholipid bilayer secreted by cells, rich in nucleic acid and protein components, carrying intracellular active substances. In recent years, exosomes secreted by stem cells are increasingly used in the field of regenerative medicine. The invention uses the exosome secreted by the embryonic stem cell to treat acute kidney injury and skin injury of a mouse, thereby promoting the recovery of the kidney function and the healing of skin wound.

Description

Technical means for promoting body injury repair by using embryonic stem cell exosomes

Technical Field

The invention relates to a technical means for treating acute kidney injury and skin injury by using exosomes secreted by embryonic stem cells, belonging to the technical field of tissue engineering and new medicines.

Background

Embryonic stem cells are a class of cells isolated from early embryos, are highly undifferentiated cells with two very important properties: it can proliferate indefinitely in vitro culture, and has high self-renewal and multi-directional differentiation potential. Embryonic stem cells can differentiate into all types of cells and are the most ideal target cells for gene therapy. Because of these excellent characteristics, embryonic stem cells are called "seed cells" and provide a large amount of raw material for clinical tissue organ transplantation. However, the embryonic stem cells have the problems of immunological rejection, ethics, tumorigenicity and the like in medical application, so that the clinical application of the embryonic stem cells is limited. Thus, scientists are looking for other ways to replace embryonic stem cells to exert relevant therapeutic effects.

Extracellular Vesicles (EVs) are substances secreted by cells with a diameter of 30-100nm and encapsulated by a phospholipid bilayer. EVs are rich in nucleic acid and protein components, carry intracellular active substances, and are the main forms of paracrine. The extracellular vesicles have various functions, and are mainly embodied in the following aspects, and the extracellular vesicles can regulate the functions of cells, including the migration and invasion capabilities of the cells. Furthermore, EVs play a complex role in immune responses, and different types of immune cells affect some adaptive and innate immune diseases by exchanging EVs. An increasing number of studies have shown that EVs also play an important role in the development of the body and in the determination of tissue architecture, mediating cell-to-cell communication. In recent years, EVs secreted from stem cells are increasingly used in the field of regenerative medicine.

Several studies have shown that embryonic stem cells also secrete extracellular vesicles and play an important role in a variety of diseases and injuries. The exosome derived from the embryonic stem cells has an important effect on treating cardiovascular diseases, and can promote angiogenesis and myocardial cell proliferation by transferring miR-294, so that the repair after myocardial infarction is promoted. The exosomes derived from embryonic stem cells also inhibit doxorubicin and inflammation-induced necrosis of muscle cells. Research also finds that extracellular vesicles secreted by embryonic stem cells can promote the proliferation of primary mouse skin fibroblasts, and research finds that exosomes derived from embryonic stem cells can also improve the senescence-associated phenotype of senescent mesenchymal stem cells. Therefore, the exosome derived from the embryonic stem cell is considered as a good substitute of the embryonic stem cell in cell therapy, avoids the defects of the embryonic stem cell in clinical application, and greatly exerts the excellent characteristics of the embryonic stem cell.

Acute Kidney Injury (AKI) is a clinically common critical condition caused by ischemia-reperfusion, drugs, infection, surgery, contrast agents, etc., has become a serious public health problem worldwide, and brings a huge medical burden to china. AKI morbidity and mortality is high, and surviving patients also develop different degrees of chronic renal function impairment, easily progressing to chronic kidney disease or end stage renal disease. AKI is a complex pathological process, and the pathophysiological mechanisms of AKI mainly include impaired microcirculation, inflammation, oxidative damage, and immune disorders. After the AKI occurs in the organism, the organism itself has a certain damage repair mechanism, and from the disease process, the repair process of AKI is divided into physiological repair and pathological repair. For mild injury stimulation, the body undergoes physiological repair in the early stage, restoring normal epithelial morphology and renal function; when the injury factor is severe or persists, the amplification of inflammatory cascade caused by various pathological mechanisms in the acute stage leads to progressive impairment of kidney function, in which case the body undergoes adaptive changes and the disease enters the chronic stage, which is called pathological repair. Accumulation of pathological repair leads to the development of fibrosis, progressing to chronic kidney disease or end-stage renal disease (ESRD). Scientists have attempted to restore normal renal function by promoting physiological repair of the body after AKI and inhibiting pathological repair in a variety of ways.

The skin is an organ which is wrapped on the surface of the body, is directly contacted with the external environment, has the functions of protecting, excreting, regulating body temperature, sensing external stimulation and the like, and is the largest organ in human body organs. In life, skin injuries are caused by various reasons, the skin wounds are mainly formed by traumas including cuts, acupuncture, pounding and the like and internal injuries, the internal injuries refer to skin wounds caused by open fracture, and the wounds caused by the cuts are usually deep and subcutaneous tissues.

Disclosure of Invention

The invention relates to a technical means for promoting the repair of body injury by using exosomes secreted by embryonic stem cells.

The invention uses the exosome derived from the embryonic stem cell to treat the mouse AKI caused by ischemia reperfusion, promotes the physiological repair of an organism, inhibits the pathological repair, and thereby recovers the kidney function of the mouse.

The invention relates to a method for treating skin injury of mice by using exosomes derived from embryonic stem cells.

The invention can effectively treat AKI caused by ischemia reperfusion of mice and promote the recovery of kidney structure and function.

The invention can effectively promote the recovery of the skin injury of the mice.

Drawings

FIG. 1 is a graph depicting characterization of exosomes, which are embryonic stem cell-derived exosomes.

Fig. 2 shows that exosome treatment has a good treatment effect on recovery of mouse injured kidney structures as H & E staining of AKI mice caused by ischemia-reperfusion.

Figure 3 is exosome treatment of AKI mice resulting from ischemia reperfusion, Masson staining showing exosome treatment inhibits renal fibrosis.

FIG. 4 is a graph showing the effect of exosomes on the treatment of mouse skin injury.

Detailed Description

In the following examples, unless otherwise specified, all methods used are conventional and all reagents used are commercially available.

Example 1, the present invention provides a method for the extraction and identification of exosomes derived from stem cells.

Cell culture medium was purchased from Hyclone; reagents such as diabody and pancreatin are purchased from Gibco; cell culture consumables were purchased from NEST.

Serum required for cell culture is fetal bovine serum without exosomes, purchased from BI company, and processed by the following steps: fetal bovine serum was placed in an ultracentrifuge tube, after trimming, 120000g was centrifuged at 4 ℃ for 2 hours, after which the supernatant was taken out on a clean bench and filtered with a 0.22 μm needle filter and stored in a freezer at-80 ℃ for further use.

The procedures of cell biology experiment operations such as cell subculture, cryopreservation and recovery are described in animal cell culture (sixth edition).

Collection of ESC conditioned media containing exosomes: when ESCs cultured in 75cm2 cell culture flasks were in logarithmic growth phase and the confluency of cells reached 80%, the medium was aspirated off, washed twice with PBS, 10ml of prepared complete medium containing 10% exosome-free FBS was added to each cell culture flask, and after further culturing for 24h, the medium was collected in 50ml centrifuge tubes, which was conditioned medium rich in exosomes.

Separating and extracting exosome by an ultracentrifugation method: firstly, centrifuging the condition culture obtained in the step for 10min at the temperature of 4 ℃ at 500g, and removing cell debris; secondly, centrifuging the obtained supernatant at 4 ℃ for 10min at 2000g to remove large cell debris such as apoptotic bodies; thirdly, filtering the obtained supernatant by using a 0.22 mu m needle filter to remove the microvesicles with the diameter more than 200 nm; placing the filtered supernatant into an ultracentrifuge tube, centrifuging at 4 ℃ for 2h at 120000g, discarding the supernatant, adding a proper amount of PBS (phosphate buffer solution) to resuspend the sediment at the bottom of the tube, and storing at-80 ℃.

The exosome marker proteins Alix and CD63 were detected by Western blot (fig. 1).

1) Protein sample preparation: and adding RIPA lysate to the exosome precipitate obtained after ultracentrifugation to crack exosome, and adding 10ul of protease inhibitor PMSF into 1ml of RIPA lysate. The sample is lysed on ice for 30min, shaken on a vortex shaker every 5min to ensure sufficient lysis, centrifuged at 12000rpm for 15min at 4 ℃ after half an hour, and the supernatant is transferred to a new EP tube; measuring the protein concentration of the exosome by using a BCA method, adding a 5 xSDS loading buffer solution into the rest protein solution, boiling the mixture in boiling water for 5-10 minutes for denaturation, immediately putting the protein into liquid nitrogen for quick-cooling after the denaturation is finished, and storing the protein in a refrigerator at the temperature of-80 ℃ for later use.

2) Preparing glue: and (3) mounting the clean and dried glass plate on a glue making frame, enabling the bottom edges to be tightly fitted, and checking leakage by using distilled water. Preparing 10% separating gel solution according to the separating gel formula, mixing well, adding 4.5ml separating gel solution into the gap of the glass plate by using a liquid transfer device, immediately and gently adding distilled water to flatten the liquid surface of the separating gel, and solidifying the separating gel after about 30 min. Preparing 5% concentrated glue solution according to the formula, adding 1.5ml concentrated glue solution to the upper part of the separation glue, immediately inserting a comb, and using after the concentrated glue is solidified.

3) Polyacrylamide gel electrophoresis: placing the prepared rubber plate into an electrophoresis tank, paying attention to the inward side of the short glass plate, adding 1x Running buffer between the two glass plates, pulling out a comb, uniformly loading the sample according to the measured protein concentration, carrying out protein loading according to the principle of equal mass (20-50 ug), leveling the total volume of the loaded sample to 20ul by using 1xSDS, adding a protein sample into a loading hole, adding an electrophoresis solution to the mark of the electrophoresis tank, covering the cover of the electrophoresis tank, paying attention to the consistent direction of the positive electrode and the negative electrode, applying 80V voltage to the concentrated glue, adjusting the voltage to 110V when the bromophenol blue runs to the separation glue, stopping electrophoresis until the bromophenol blue is close to the bottom of the glass plate, and keeping the electrophoresis time for the whole electrophoresis to be 1.5 hours.

4) Film transfer: preparing a precooled membrane Transfer solution (1x Transfer buffer) containing 20% methanol in advance, soaking a membrane Transfer clamp, a sponge and filter paper in a precooled membrane Transfer buffer solution, carefully prying open a rubber plate, placing an electrophoresed polyacrylamide gel on one side of a black clamping plate, shearing a PVDF membrane with a proper size, placing the PVDF membrane in methanol for activation for 60s, placing the PVDF membrane on the rubber, removing bubbles between the rubber and the membrane, slightly covering the filter paper and the sponge on the membrane, clamping the membrane Transfer clamp according to the sequence of a negative electrode (black) of the membrane Transfer clamp, the sponge-the filter paper-the rubber-the PVDF membrane-the filter paper-the sponge-a positive electrode (white) of the membrane Transfer clamp, placing the PVDF membrane in a membrane Transfer groove, adding the membrane Transfer solution, placing the whole membrane Transfer groove in a foam box filled with ice, and carrying out membrane Transfer for 2h under constant pressure of 100.

5) And (3) sealing: the PVDF membrane after the membrane conversion is taken out and placed in 5 percent of skimmed milk (sealing liquid), and the horizontal shaking table is sealed for 2 hours at room temperature at 60 rpm.

6) Antibody incubation: first-antibody incubation: diluting the primary antibody with a confining liquid according to the instruction (Alix, 1:2000 dilution; CD63, 1:2000 dilution), sucking 2ml of the primary antibody, placing the primary antibody in an antibody incubation box, shearing a membrane by a control protein Marker, placing the membrane in the corresponding primary antibody, and incubating overnight at 4 ℃; and secondly, incubation with secondary antibody: the strips were placed in TBST on a horizontal shaker and washed 3 times at 120rpm for 10min each, then the corresponding secondary antibody was added, incubated on a horizontal shaker at 60rpm for 2h at room temperature, and washed 3 times with TBST for 10min each after incubation.

7) Luminescence detection: mixing the luminescent liquid A and the luminescent liquid B according to the proportion of 1:1 to prepare working liquid, and exposing by an exposure instrument.

Example 2, the present invention provides a method for establishing a mouse model of acute renal injury induced by ischemia-reperfusion.

1) Selecting C57/BL6 mice of about 8 weeks old, weighing, performing intraperitoneal injection anesthesia with 1.25% avermectin (10 microgram/g), removing back hair, and wiping with iodophor and alcohol for disinfection;

2) cutting skin and muscle at 0.5cm beside back vertebra and 0.5cm below rib, observing kidney, carefully separating renal artery of left kidney, clamping renal pedicle with non-traumatic capillary clamp, observing that the kidney changes from bright red to purple black is successful clamping, and covering window with wet gauze to keep surface of kidney wet;

3) after 45min of clamping, the vascular clamp was removed and the kidney was seen to be immediately perfused successfully and turn red;

4) after about 5min, 100ug of media (PBS, ESC-EVs) was injected into the renal cortex;

5) sequentially suturing peritoneum, muscle and skin layer by layer;

6) the mice were placed on a heating pad for resuscitation, and after waking, returned to their cages.

Example 3, the present invention provides a method of constructing a mouse skin lesion model.

1) Mice were weighed and anesthetized with 1.25% avermectin (10 μ g/g) by intraperitoneal injection;

2) removing back hair, cutting off the whole skin and flesh membrane on the back of the mouse, and exposing the muscle layer to cause a circular skin defect with the diameter of 1 cm;

3) a circular silicone splint (inner diameter: 10mm, outer diameter: 15mm) is fixed in position to prevent wound contraction and thereby restrain the wound.

4) Post-operative animals were randomized into 2 groups: PBS control group, ESC-EVs treated group, mice were injected subcutaneously with vehicle at the site of skin injury.

Claims

1. The effective component of the treatment means is exosome from stem cells, which is a paracrine component separated and extracted from cell supernatant cultured in vitro, carries various bioactive molecules such as protein, RNA and the like in donor cells, and has the function of treating injury.

2. Exosome according to claim 1, characterized in that: the exosomes are derived from embryonic stem cells.

3. A technical means for delivering exosomes and promoting recovery of kidney function after acute kidney injury, which is to treat mice with acute kidney injury caused by ischemia-reperfusion with the exosomes of claim 1 to promote recovery of kidney function and structure.

4. The technical means of claim 3, characterized in that: the technology can promote the recovery of the functions and the structures of the kidney after acute kidney injury through exosomes, including the promotion of physiological repair and the inhibition of pathological repair mechanisms.

5. A technical means for delivering exosomes and promoting wound healing after skin injury, which is to treat skin trauma caused by incised wound by using the exosomes according to claim 1 and promote the healing of the skin wound.

6. The use in tissue damage according to claim 5, wherein the tissue damage comprises kidney damage, skin damage, liver damage, lower limb ischemia, and the like.