CN112656816A - Exosome-loaded fibrin glue and application thereof - Google Patents
Exosome-loaded fibrin glue and application thereof Download PDFInfo
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Abstract
The invention discloses an exosome-loaded fibrin glue and application thereof, and a preparation method of the exosome-loaded fibrin glue comprises the following steps: loading the exosome obtained by extraction and separation into fibrin glue to obtain the fibrin glue loaded with exosome. The invention also discloses application of the exosome-loaded fibrin glue in preparation of a preparation for treating spinal cord injury. The fibrin glue loaded with exosome has significant promotion effect on motor function recovery after spinal cord injury.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to an exosome-loaded fibrin glue and application thereof in preparation of a preparation for treating spinal cord injury.
Background
The central nervous system plays an important role in normal life activities of the human body, and the spinal cord plays an important role as an important bridge between the brain and peripheral nerves, and plays roles in conduction, reflection and the like. However, with the frequent occurrence of traffic accidents and the rise of various high-risk extreme sports in recent years, the incidence of spinal cord injuries has also increased year by year. Once traumatic spinal cord injury occurs, the patient is at risk of hemiplegia, paraplegia, or even death. In addition, due to the lack of an effective nerve regeneration strategy, tissue repair after spinal cord injury is still a difficult problem, patients are frequently bedridden, the quality of life is low, the patients seriously depend on the accompanying of family members, and serious physical and psychological and economic burdens are brought to individuals and families.
After spinal cord injury occurs, a great amount of neuron necrosis and vascular rupture are generated at the injury part, so that local severe bleeding is caused, and a severe oxidative stress environment is formed. In addition, the pathological tissue can activate microglia cells to cause severe inflammatory reaction, further induce necrosis of normal nervous tissue around the pathological tissue and seriously obstruct regeneration and bridging of nerves.
In recent years, research on exosomes has been reported as late spring shoots. The exosome is an extracellular vesicle with the particle size of only 50-150 nm and has the characteristics of low immunogenicity, high biocompatibility and the like. The inside of the cell carries abundant substances, such as lipid, protein, DNA, various RNAs and the like, and the substance exchange and information communication are carried out among cells, so that the life activities of receptor cells can be regulated.
According to the research reports, some exosomes produced by specific kinds of cells (such as mesenchymal stem cells) can regulate the oxidative stress environment of diseased tissues, improve inflammatory response, maintain the survival of neurons, promote axon elongation and local vascular remodeling, and greatly promote the repair of motor function and nerve regeneration after spinal cord injury (systematic administration of exosomes from cardiac tissue cells and neural tissue regeneration) [ J ]. Journal of cellular Blood Flow & Metabolism,2013 ]. Therefore, it is promising to use exosomes as a safe and effective means to treat spinal cord injury.
The Chinese patent application with publication number CN111454895A specifically provides an experimental method for repairing spinal cord by inducing microglial cell polarization by exosome, and proves that spinal cord injury can be more effectively repaired by transferring microglial cell M1/M2 polarization to an exon processed by adipose-derived stem cells containing lncGm 37494.
However, the microenvironment regulation of exosomes requires efficient delivery strategies to transport them to the damaged area before effective performance. In addition, the damaged spinal cord tissue forms tissue cavities, and therefore, it is necessary to establish an effective extracellular matrix supplementing strategy and carry exosomes in the supplemented extracellular matrix.
Disclosure of Invention
The invention aims to promote the regeneration of nerves at spinal cord injury and provides a preparation method of fibrin glue loaded with exosomes; the prepared fibrin glue loaded with exosomes has high biological safety and compatibility, can effectively carry and deliver exosomes to spinal cord injury parts, promotes the obvious regeneration of injured nerves, and finally realizes the recovery of motor functions and various physiological functions of spinal cord injury patients.
The technical scheme provided by the invention is as follows:
an exosome-loaded fibrin glue, the preparation method of which comprises the following steps: loading the exosome obtained by extraction and separation into fibrin glue to obtain the fibrin glue loaded with exosome.
In the technical scheme, the exosome-loaded fibrin glue is a composite biological material assembled by exosomes of specific cells and fibrin glue.
Exosome is an important means for generating cells and exchanging substances and communicating information with the outside, the composition of the content of exosome and the regulation effect of exosome on acceptor cells are related to the types of donor cells, and cells such as mesenchymal stem cells, neural stem cells, schwann cells, astrocytes and the like all have the promotion effect on the regeneration of neurons. Meanwhile, the regeneration action of different cell exosomes can be synergistically generated, so that the nerve repair is accelerated.
In addition, fibrin glue has been widely used in clinical practice, and has been demonstrated to have good biocompatibility and biodegradability; and fibrin glue was shown to be beneficial for nerve regeneration after spinal cord injury. The fibrin glue is a two-component system, mainly comprises fibrinogen and thrombin, can simulate the final step of the in vivo blood coagulation process, and the fibrinogen is decomposed into fibrin under the action of the thrombin and rapidly forms a net structure to finally finish the blood coagulation.
The exosome is derived from one or more of mesenchymal stem cells, neural stem cells, human umbilical vein endothelial cells, Schwann cells, astrocytes, oligodendrocytes or pericytes; preferably, the exosomes are derived from mesenchymal stem cells.
Mesenchymal stem cells are multipotent stem cells, have the potential of multidirectional differentiation, are widely researched and used in clinical application due to the advantages of wide sources, capability of large-scale amplification, low immunogenicity, no ethical dispute and the like. More importantly, the mesenchymal stem cells can secrete various substances to promote the regeneration of damaged tissues, and the exosomes generated by the mesenchymal stem cells are proved to be beneficial to the regeneration of nerves and the recovery of motor functions after spinal cord injury.
Loading the exosome obtained by extraction and separation into fibrin glue under the action of thrombin, which specifically comprises the following steps: mixing and respectively dispersing the suspension of the exosome in a fibrinogen solution and a thrombin solution, and mixing and gelling the two solutions to obtain the exosome-loaded fibrin glue;
or mixing the thrombin solution and the fibrinogen solution for gelation to obtain fibrin glue, and then incubating the exosomes and the fibrin glue together to obtain the fibrin glue loaded with exosomes.
The co-incubation of the exosome and the fibrin glue is specifically to inject the exosome into the fibrin glue.
The suspension mixture of the exosomes is that the exosomes are resuspended in PBS buffer solution, and the concentration of the suspension mixture of the exosomes is 1 mu g/mL-10 mg/mL.
The fibrinogen solution is a fibrinogen aqueous solution, and the concentration is 10 mg/mL-1 g/mL; CaCl with thrombin solution as thrombin2The concentration of the solution is 100 IU/mL-1000 IU/mL.
The feeding ratio of the exosome, the fibrinogen and the thrombin is 50-1000 mu g: 0.1-100 mg: 10-100000 IU; preferably 50 to 150 μ g: 1.0-2.0 mg: 10 to 20 IU.
The regeneration of exosomes mainly depends on the substances contained in the exosomes, and a sufficient amount of exosomes are needed for treatment, but the large-scale extraction of exosomes is still difficult; the enzyme has high catalytic activity, but an excessive amount of the enzyme may affect the body function to cause unnecessary burden. Therefore, the amount of exosome and enzyme used should be controlled within a certain range.
The invention also provides application of the exosome-loaded fibrin glue in preparation of a preparation for treating spinal cord injury.
Compared with the prior art, the invention has the beneficial effects that:
(1) the used exosome has the advantages of high safety and low risk, and has good prospect of in vivo application.
(2) The combined treatment by adopting a plurality of exosomes is expected to produce stronger and faster regeneration effect.
(3) Fibrin glue is available on the market and has good biological safety.
(4) The exosome is dispersed in the fibrin glue to treat spinal cord injury, so that the exosome can be efficiently delivered, extracellular matrix can be filled, and the spinal cord tissue regeneration and function recovery are facilitated.
(5) The preparation method provided by the invention is simple and easy to control.
Drawings
Fig. 1 is a transmission electron micrograph of the mesenchymal stem cell exosomes collected in example 1.
Fig. 2 is a three-dimensional distribution diagram of exosomes in fibrin glue as prepared in example 3.
Fig. 3 shows the results of scoring the motor function of rats with spinal cord injury after the application of the control group, the blank fibrin glue group and the exosome-fibrin glue group in example 1.
FIG. 4 shows the results of NF and GFAP immunofluorescence staining of spinal cord injury sites of rats with spinal cord injury of control group, blank fibrin glue group and exosome-fibrin glue group in application example 1.
FIG. 5 shows the results of immunofluorescence staining of HNE and OHdG in spinal cord injury sites of rats with spinal cord injury in the control group, the blank fibrin glue group and the exosome-fibrin glue group in application example 1.
FIG. 6 shows the results of HE staining of rats with spinal cord injury in control group, blank fibrin glue group and exosome-fibrin glue group according to application example 1 on heart, liver, spleen, lung and kidney.
Fig. 7 shows the results of the serum liver function and renal function tests of rats with spinal cord injury in the control group, the blank fibrin glue group and the exosome-fibrin glue group in application example 1.
Detailed Description
The invention is further illustrated by the following specific examples and description. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.
Example 1 extraction and isolation of exosomes
Culturing human umbilical cord mesenchymal stem cells (purchased from Tianjin Changsha biotechnology limited) by using an alpha-MEM culture medium (10% of serum without exosomes is added according to volume percentage, and growth factors bFGF and EGF are added according to the concentration of 20 ng/mL), collecting a culture solution after the cell coverage rate reaches 90%, removing cell debris and large vesicles in the culture solution through primary centrifugation (3,000g,30min,4 ℃), further ultracentrifuging the culture solution (100,000g,70min,4 ℃), and collecting bottom precipitates to obtain exosomes.
The appearance of the collected exosomes is characterized by a transmission electron microscope, and the result is shown in fig. 1. The exosome obtained by extraction and separation is in a cup-shaped or bowl-shaped structure, the particle size is about 150nm, the outline is clear, the structure is complete, and impurities are less.
Example 2: exosome extraction and separation
Culturing neural stem cells (purchased from Seike Biotech Co., Ltd.) in DMEM/F12 medium (2% B27 SUPPLEMENT, 1% glutamine, 1% penicillin-streptomycin double antibody, heparin sodium at a concentration of 4. mu.g/mL, growth factors bFGF and EGF at a concentration of 20 ng/mL), collecting the culture solution after passage for 5 days, centrifuging (3,000g,30min,4 ℃; 10,000g,30min,4 ℃) to collect the supernatant, concentrating the supernatant by using a 100KD ultrafiltration tube, and separating the concentrated solution by a qEV separation column to obtain the exosome.
Example 3: preparation of exosome-loaded fibrin glue
Dissolving 1.5mg of fibrinogen in 15 mu L of water for injection to obtain a fibrinogen solution of 100 mg/mL; dissolving thrombin in CaCl2The concentration of the resulting solution was adjusted to 1000 IU/mL. Exosomes were resuspended in PBS to a concentration of 100 μ g/30 μ L.
Sucking 15 mu L of fibrinogen solution (100mg/mL) and 15 mu L of the exosome suspension extracted in the example 1, and uniformly mixing to obtain 50mg/mL of fibrinogen solution containing exosomes; sucking 15 mu L of thrombin (1000IU/mL) and 15 mu L of exosome suspension, and uniformly mixing to obtain 500IU/mL thrombin solution containing exosomes; the two solutions were mixed rapidly and after about 10 seconds an exosome-loaded fibrin glue was obtained.
To examine the distribution of exosomes in the gel, exosomes were labeled and stained with CM-DiI dye before preparation of exosome-loaded fibrin glue. The exosome-loaded fibrin glue was prepared as described above, placed in a confocal dish and Z-stack scanned using a confocal microscope. The result is shown in fig. 2, the exosomes marked by fluorescence are uniformly distributed in the fibrin glue, and the fluorescence signals of a large number of exosomes can be observed from three-dimensional multi-angle observation.
Example 4: preparation of exosome-loaded fibrin glue
Dissolving 3.0mg of fibrinogen in 30 mu L of water for injection to obtain a fibrinogen solution of 100 mg/mL; dissolving thrombin in CaCl2The concentration of the resulting solution was adjusted to 1000 IU/mL. 30 mu L of fibrinogen solution (100mg/mL) and 30 mu L of thrombin solution (1000IU/mL) are respectively taken, and the two solutions are quickly mixed and completely gelled to obtain the fibrin glue. Mu.g of exosomes extracted in example 1 were injected into a gel scaffold to obtain exosome-loaded fibrin glue.
Application example 1:
female SD rats with the body weight of 220-250 g are selected, anesthetized with barbiturate sodium, and the back hair of the rats is shaved with thoracic vertebrae T9 and T10 as the center. After iodophor sterilization, an opening of about 5cm was cut with a scalpel, exposing the muscles on both sides of the free spinous process behind the fascia. T9, T10 spinal cages were cut with rongeur, the spinal cord exposed and cut. Equal volumes of PBS (control group), fibrin glue blank (fibrin glue blank group), and the exosome-loaded fibrin glue prepared in example 3 (exosome-fibrin glue group) were transplanted to the spinal cord injury site, respectively, and paraspinal muscle and skin were sutured in sequence. Within one week after operation, 8 million U of penicillin is injected daily, and urination is performed twice. Thereafter, urine was excreted once a day.
The rats are subjected to Basso Beattie Bresnahan (BBB) test and scoring at 1-4 weeks after the operation, the spinal cord recovery condition of the rats is evaluated, and the result is shown in figure 3, and the fibrin glue loaded with exosomes has a significant promotion effect on the motor function recovery of the rats with spinal cord injury.
The rat spinal cord was immunofluorescent stained at the fourth postoperative week for the mature neuronal markers Neurofilament (NF), activated astrocyte marker Glial Fibrillary Acidic Protein (GFAP), 4-Hydroxynonenal (HNE) and 8-hydroxy-2' -deoxyguanosine (OHdG), markers for oxidation products of lipids and DNA, respectively. The immunostaining results of the spinal cord injury are shown in fig. 4 and fig. 5, and the fibrin glue loaded with exosome can effectively inhibit the generation of HNE and OHdG, and can also inhibit the formation of glial scar tissue, thereby finally promoting the regeneration of neurons.
The exosome and the fibrin glue used in the research have good biocompatibility, so histopathological detection is carried out on main visceral organs and serum of the rat at the experimental end point, the results are shown in fig. 6 and 7, the visceral organ tissues mainly comprising heart, liver, spleen, lung and kidney have normal forms and do not generate obvious pathological changes, and the staining results are similar to the staining results of normal rats. Glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), Blood Urea Nitrogen (BUN) and creatinine index (CR) in serum were detected, and no significant difference was found compared with normal rats. The above results demonstrate that the exosome-fibrin glue therapeutic system has good biosafety.
The embodiments described above have been described in detail to illustrate the technical solutions and advantages of the present invention, and it should be understood that any modifications, additions, and equivalents within the spirit and scope of the present invention are encompassed by the following claims.
Claims (9)
1. An exosome-loaded fibrin glue, characterized in that it is prepared by a method comprising the steps of: loading the exosome obtained by extraction and separation into fibrin glue to obtain the fibrin glue loaded with exosome.
2. An exosome-loaded fibrin glue according to claim 1, wherein the exosomes are derived from one or more of mesenchymal stem cells, neural stem cells, human umbilical vein endothelial cells, schwann cells, astrocytes, oligodendrocytes or pericytes.
3. An exosome-loaded fibrin glue according to claim 1, wherein the exosomes obtained by extraction and isolation are loaded into the fibrin glue, in particular:
mixing and respectively dispersing the suspension of the exosome in a fibrinogen solution and a thrombin solution, and mixing and gelling the two solutions to obtain the exosome-loaded fibrin glue;
or mixing the thrombin solution and the fibrinogen solution for gelation to obtain fibrin glue, and then incubating the exosomes and the fibrin glue together to obtain the fibrin glue loaded with exosomes.
4. The exosome-loaded fibrin glue of claim 3, wherein said suspension of exosomes is resuspended in PBS buffer and the concentration of exosome suspension is between 1 μ g/mL and 10 mg/mL.
5. An exosome-loaded fibrin glue according to claim 3, wherein said fibrinogen solution is an aqueous solution of fibrinogen, at a concentration of 10mg/mL to 1 g/mL; CaCl with thrombin solution as thrombin2The concentration of the solution is 100 IU/mL-1000 IU/mL.
6. An exosome-loaded fibrin glue according to claim 3, wherein the dosage ratio of exosomes, fibrinogen and thrombin is 50-1000 μ g: 0.1-100 mg: 10 to 100000 IU.
7. An exosome-loaded fibrin glue according to claim 6, wherein the dosage ratio of exosomes, fibrinogen and thrombin is 50-150 μ g: 1.0-2.0 mg: 10 to 20 IU.
8. An exosome-loaded fibrin glue according to claim 3, wherein co-incubating exosomes with fibrin glue is specifically injecting exosomes into fibrin glue.
9. Use of an exosome-loaded fibrin glue according to any one of claims 1-8 in the preparation of a formulation for treating spinal cord injury.
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Application publication date: 20210416 |