CN118028224A - Engineering exosome and preparation and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biological medicine, in particular to an engineering exosome and preparation and application thereof. The invention combines the mesenchymal stem cell-derived exosome with the small molecule Sephin1 with endoplasmic reticulum steady-state maintenance function by an intermittent ultrasonic technology to construct an engineering exosome. The engineering exosome prepared by the invention solves the problems of dysregulated endoplasmic reticulum steady state, damaged stem cell function and poor bone tissue healing effect in the high-sugar microenvironment of diabetes, and the invention introduces small molecules Sephin with the function of maintaining the endoplasmic reticulum steady state into the mesenchymal stem cell-derived exosome by using an intermittent ultrasonic technology, thereby effectively reducing the expression of related gene proteins of the dysregulated stem cell endoplasmic reticulum in the high-sugar environment, promoting the proliferation of stem cells in the high-sugar environment, down regulating the apoptosis level of the stem cells, promoting the osteogenic differentiation of the stem cells and effectively optimizing the treatment efficiency of the mesenchymal stem cell-derived exosome.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to an engineering exosome and preparation and application thereof.
Background
Extracellular vesicles play an important role in intercellular communication, and can be used as transport carriers of signal molecules to transfer various functional molecules such as contained DNA, RNA, proteins, lipids and the like to receptor cells. The mesenchymal stem cell-derived exosome is an extracellular vesicle secreted by mesenchymal stem cells, and has a diameter of about 40-160 nm. Mesenchymal stem cell-derived exosomes have attracted intense interest and have been widely studied due to their regenerative-promoting properties similar to their parental cells. And as a natural nano vesicle, compared with an artificial nano material, the mesenchymal stem cell-derived exosome has the obvious advantages of low immunogenicity, good biocompatibility, easy uptake by receptor cells, easy engineering and the like. However, the regenerative therapeutic effect of the exosomes alone is limited. Therefore, there is an urgent need to enhance the functionalization of exosomes to increase the therapeutic efficiency.
Endoplasmic reticulum is a critical intracellular membrane organelle with a variety of basic functions including protein synthesis, folding, assembly and transport, regulation of lipid synthesis, ion signaling, and regulation of cellular stress, among others. The endoplasmic reticulum interacts with almost all other organelles, and maintenance of endoplasmic reticulum homeostasis is important for the proper functioning of cellular functions. Previous studies have shown that diabetic hyperglycemia can induce sustained and intense stress in the cytoplasmic reticulum in a variety of tissues, leading to impaired cellular function and subsequent pathophysiological changes.
Therefore, it is important to provide an engineered exosome capable of restoring the regenerative function of high sugar environment damaged stem cells.
Disclosure of Invention
In order to solve the above problems, the present invention provides an engineering exosome and its preparation and application. The invention combines the mesenchymal stem cell-derived exosome with the small molecule Sephin1 with the steady-state maintenance function of the endoplasmic reticulum by the intermittent ultrasonic technology to construct an engineering exosome, thereby functionalizing the mesenchymal stem cell-derived exosome and improving the treatment efficiency of the mesenchymal stem cell-derived exosome on the premise of ensuring the safety.
The aim of the invention can be achieved by the following technical scheme:
A first object of the present invention is to provide an engineered exosome into which Sephin (hereinafter referred to as "Sep") is introduced; the engineered exosomes have endoplasmic reticulum steady-state maintenance functions.
The second object of the present invention is to provide a method for preparing an engineered exosome, comprising the steps of:
(S1) placing mesenchymal stem cells in an alpha-MEM culture medium containing FBS for primary culture and amplification, and then placing in an exosome-free culture medium for secondary culture to obtain a culture solution;
(S2) continuously centrifuging the culture solution obtained in the step (S1), collecting supernatant, and performing aftertreatment to obtain mesenchymal stem cell-derived exosomes;
(S3) introducing Sep into the mesenchymal stem cell-derived exosome prepared in the step (S2) by intermittent ultrasonic, and performing post-treatment to obtain an engineering exosome for maintaining the steady state of the endoplasmic reticulum of the cell: sep@Exo.
In one embodiment of the invention, in the step (S1), the cells are cultured in a 5% CO 2 incubator at 37 ℃ until the cell confluence reaches 70% -80% in the primary culture process.
In one embodiment of the present invention, in step (S1), the secondary culturing process is carried out in a 5% CO 2 incubator at 37℃for 40-60 hours.
In one embodiment of the present invention, in step (S2), the continuous centrifugation is specifically as follows:
the culture solution is centrifuged with 200 g to 500g, 1500 g to 2500g and 8000 g to 12000g in sequence, and sediment is discarded after each centrifugation is finished, and the supernatant is collected.
In one embodiment of the present invention, in step (S2), the post-treatment is to filter the supernatant, centrifuge, then wash in PBS, and re-suspend in PBS after centrifugation.
In one embodiment of the present invention, in the step (S3), the ratio of the amount of Sep to the amount of mesenchymal stem cell-derived exosomes is 10 to 15 μg:1mg.
In one embodiment of the invention, in step (S3), the ultrasonic parameters are 10% -30% amplitude, 5-15 seconds on/off, 1-3 minutes duration, 4-8 cycles, and 1-3 minutes cooling period between each cycle.
In one embodiment of the present invention, in step (S3), the post-treatment is a purification treatment after incubation at 37 ℃.
A third object of the present invention is to provide the use of an engineered exosome for restoring the regenerative function of a high sugar environment damaged stem cell.
In one embodiment of the invention, the agent is an agent that reduces expression of a gene protein associated with a disorder of the endoplasmic reticulum of a stem cell in a high sugar environment.
In one embodiment of the invention, the agent is an agent that promotes proliferation of stem cells in a high sugar environment.
In one embodiment of the invention, the agent is an agent that down-regulates the level of stem cell apoptosis.
In one embodiment of the invention, the agent is an agent that promotes osteoblast differentiation of stem cells.
Compared with the prior art, the invention has the following beneficial effects:
The engineering exosome prepared by the invention solves the problems of dysregulation of endoplasmic reticulum steady state, impaired stem cell function and poor bone tissue healing effect in a high-sugar microenvironment of diabetes, and the invention introduces small molecule Sep with the function of maintaining endoplasmic reticulum steady state into the mesenchymal stem cell-derived exosome by an intermittent ultrasonic technology, thereby effectively reducing the expression of related gene proteins of the dysregulation of the endoplasmic reticulum, promoting stem cell proliferation under the high-sugar environment, down regulating the apoptosis level of stem cells, promoting the osteogenic differentiation of the stem cells and effectively optimizing the treatment efficiency of the mesenchymal stem cell-derived exosome.
Drawings
FIG. 1 scanning electron microscope image of exosomes and engineered exosomes.
FIG. 2 particle size distribution of exosomes and engineered exosomes.
FIG. 3 is a WB map of an exosome and an engineered exosome.
FIG. 4 is a schematic representation of stem cell proliferation activity after exosomes are added to the high sugar medium and engineered exosomes.
FIG. 5 is a schematic representation of stem cell apoptosis levels after exosomes are added to the high sugar medium and engineered exosomes.
FIG. 6 is a schematic diagram showing the expression level of genes related to the disturbance of the endoplasmic reticulum of stem cells after exosomes are added to the high sugar medium and the exosomes are engineered.
FIG. 7 is a schematic representation of protein expression levels associated with dysregulated stem cell endoplasmic reticulum after addition of exosomes and engineered exosomes to the high-sugar medium.
FIG. 8 schematic representation of stem cell osteoinductive alkaline phosphatase staining (100 μm) after exosomes were added to the high-sugar medium and engineered.
FIG. 9 schematic representation of stem cell osteo-induced alizarin red staining (100 μm) after exosomes were added to the high-sugar medium and engineered.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
In the examples below, unless otherwise specified, all reagents used were commercially available, and all detection means and methods used were conventional in the art.
Example 1
This example provides an engineered exosome (Sep@Exo) and a method of making the same.
(S1) culturing mesenchymal stem cells in an alpha-MEM medium containing 10% FBS (37 ℃,5% CO 2 cell incubator), replacing the original medium (the alpha-MEM medium containing 10% FBS) with an exosome-free alpha-MEM medium when cell confluence reaches 70-80%, culturing (37 ℃,5% CO 2 incubator) for 48 hours, and collecting the supernatant;
(S2) separating exosomes from the collected supernatant by a continuous centrifugation method; specifically, the above collected supernatant was centrifuged at 300g for 10 minutes at 4℃in order, the precipitate was discarded, the supernatant was collected, centrifuged at 2000g for 20 minutes, the precipitate was discarded, the supernatant was collected, centrifuged at 10000g for 30 minutes, the precipitate was discarded, and the supernatant was collected to remove cells and debris; next, the supernatant finally collected was filtered using a 0.22 μm filter (sterile), and then centrifuged at 100000g for 90 minutes using an ultracentrifuge, the exosome pellet was washed in PBS, and again ultracentrifuged at 100000g for 90 minutes; then resuspending the obtained exosome precipitate in PBS to obtain exosome product;
(S3) introducing Sep into Exo by a batch ultrasonic method; specifically, 12. Mu.g of Sep was mixed with 1mg of the exosome product in 1mL of PBS to obtain a suspension; the suspension was then subjected to intermittent and gentle sonication using a sonicator with a 20% amplitude, 10 seconds on/off, 2 minutes duration, 6 cycles with a 2 minute cooling period between each cycle. After sonication, incubation was carried out at 37 ℃ for 2 hours to restore the exosome membrane structure, and finally the unloaded Sep molecules were removed using an ultrafiltration tube to obtain purified sep@exo.
Performance analysis:
(1) Characterization of Exo (an exosome product obtained in step (S2) above) and sep@exo: for morphological analysis, exosomes were fixed with 2.5% glutaraldehyde and placed on an electron microscope grid, then stained with 1% uranyl acetate solution for 2 minutes, washed with distilled water and dried, and images captured using a transmission electron microscope at 120 kV; the results are shown in FIG. 1, where Exo and Sep@Exo exhibit cup-like or spheroid-like morphology typical of exosomes. The exosomes were suspended in PBS and the size distribution of the exosomes was measured using a particle size analyzer; as shown in FIG. 2, exo and Sep@Exo have similar particle size distributions and are concentrated in the range of 40-160 nm. The exosome marker proteins were detected using Western Blot (WB) and as shown in fig. 3, exo and sep@exo specifically contained the exosome marker proteins CD9, CD81, alix and TSG101, with little expression of the negative marker protein Calnexin.
(2) Cell culture: mesenchymal stem cells were isolated using femur and tibia of 3 week old male Sprague Dawley (SD) rats. Specifically, after removal of both ends of femur and tibia, cells were washed from bone marrow and cultured in normal glucose medium (α -MEM containing 5.5mM D-glucose) containing 10% FBS and 1% penicillin-streptomycin at 37 ℃ in an incubator containing 5% CO 2. Mesenchymal stem cells of passage 2-4 with a growth density of 80-90% confluence were used for subsequent experiments. To simulate hyperglycemia, HG medium was prepared by adding 29.5mM D-glucose to normal glucose medium using a High Glucose (HG) concentration of 35 mM. Commercial Cyagen osteogenic media was used for Osteoinduction (OI). Exo and Sep@Exo were used at a concentration of 50. Mu.g/mL.
(3) Cell proliferation activity assay: after 1,4,7 days of cell culture, cell viability was assessed using a cell counting kit. Specifically, the medium was discarded, 110. Mu.L of CCK-8 working solution was added, and incubated at 37℃for 2 hours, and OD was measured at a wavelength of 450nm in an microplate reader. The results are shown in FIG. 4 (CON is a control group, HG is a high glucose concentration group, HG-Exo is an Exo group used at a high glucose concentration, HG-Sep@Exo is a Sep@Exo group used at a high glucose concentration), and the high sugar environment impairs cell proliferation activity; the HG-Exo group showed increased cell viability compared to the HG group, while the HG-Sep@Exo group showed better therapeutic effects.
(4) Apoptosis level detection: after 7 days of cell culture, the apoptosis rate of each group of cells was analyzed using an Annexin V-FITC/PI apoptosis detection kit. Specifically, the harvested cells were digested, washed 1 time with PBS buffer, 1 time with binding buffer, and then resuspended in 195. Mu.L of binding buffer supplemented with 5. Mu.L of Annexin V-FITC and 10. Mu.L of PI staining solution. Incubate for 15 minutes in the dark at room temperature, after buffer wash, analyze using flow cytometry. The results are shown in FIG. 5 (CON is the control group, HG is the high glucose concentration group, exo is the high glucose concentration group, HG-Sep@Exo is the high glucose concentration group, sep@Exo) and the high glucose environment increased the apoptosis level of the cells. In comparison with the HG group, the HG-Exo group showed a down-regulated apoptosis rate, whereas the HG-Sep@Exo group could almost return to normal.
(5) RT-PCR detection of gene expression: after 7 days of cell culture, total cellular RNA was extracted using RNAiso Plus and then reverse transcribed into cDNA by the PRIMESCRIPT first strand cDNA synthesis kit. UsingGREEN MASTER Mix kit prepares Real-time PCR reaction, and Real-time PCR reaction is carried out in a PCR system LightCycler 480. The target genes are GRP78, CHOP, GRP94, PERK, ATF6 and ENR1.GAPDH was used as a reference gene and relative gene expression levels were analyzed using the 2 -ΔΔCT method. As a result, as shown in FIG. 6 (CON is a control group, HG is a high glucose concentration group, HG-Exo is an Exo group used at a high glucose concentration, HG-Sep@Exo is a Sep@Exo group used at a high glucose concentration), the level of gene expression associated with the disturbance of the endoplasmic reticulum in a high sugar environment is increased; the HG-Exo group showed a down-regulation trend and the HG-Sep@Exo group showed a more remarkable effect as compared to the HG group.
(6) WB detection protein expression: cells or exosome pellet were lysed with RIPA lysis buffer supplemented with protease inhibitors and sonicated in an ice bath for 30 min. Protein concentration was quantified and consistently adjusted using BCA protein assay kit. The sample was placed in a metal bath at 100℃for 7 minutes. Each histone sample was separated by 4% -20% gradient SDS-PAGE gel and subsequently transferred onto a polyvinylidene fluoride membrane. Next, the membranes were incubated successively with 5% BSA for 1 hour, specific primary antibody for 4 ℃ overnight, washed 3 times with PBS on a shaker, incubated with secondary antibody for 1 hour, and washed 3 times with PBS on a shaker. After the membrane has reacted with ECL reagent, the signal is captured using a membrane imaging system. As a result, as shown in FIG. 7 (CON is a control group, HG is a high glucose concentration group, HG-Exo is an Exo group used at a high glucose concentration, HG-Sep@Exo is a Sep@Exo group used at a high glucose concentration), the expression level of the protein associated with the disturbance of the endoplasmic reticulum in a high sugar environment was increased. Compared with the HG group, the HG-Exo group showed a down-regulation trend, while the HG-Sep@Exo group showed a more significant down-regulation trend, consistent with the results of FIG. 6; the Exo itself has a certain maintenance function of endoplasmic reticulum steady state, and the engineered exosome Sep@exo strengthens the function due to the introduction of Sep.
(7) Alkaline phosphatase staining: after 7 days of Osteoinduction (OI), the samples were stained using the BCIP/NBT alkaline phosphatase chromogenic kit. Specifically, the medium was discarded, the cells were washed 3 times with PBS, fixed at 4% PFA for 20 minutes, washed 3 times with PBS, incubated with alkaline phosphatase chromogenic working solution for 30 minutes, washed 3 times with PBS, and images were collected by a scanner and a microscope. As shown in FIG. 8, the results of the important markers for osteogenic differentiation by alkaline phosphatase showed that the OI-HG group had a very light coloration compared to the OI group, and the high sugar environment destroyed the osteogenic differentiation function of stem cells, which was improved in the OI-HG-Exo group, while the OI-HG-Sep@Exo group was more deeply colored.
(8) Alizarin red staining: after 21 days of Osteoinduction (OI), the medium was discarded, the cells were washed 3 times with PBS, fixed for 20 minutes in 4% PFA, and washed 3 times with double distilled water. 1% alizarin red was used for 20 minutes and images were acquired with a scanner and microscope. The results are shown in FIG. 9, and alizarin red staining was used to detect calcium salt deposition in osteogenic differentiation. The results show that calcium salt deposition is less common in the OI-HG group than in the OI group, which is improved in the OI-HG-Exo group, while alizarin red staining is darker in the OI-HG-Sep@Exo group. The engineering exosomes are shown to effectively save the osteoblast differentiation capacity of stem cells while maintaining the steady state of the endoplasmic reticulum.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the explanation of the present invention, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. An engineered exosome, wherein the engineered exosome is an exosome introduced Sephin a 1; the engineered exosomes have endoplasmic reticulum steady-state maintenance functions.
2. A method of preparing an engineered exosome of claim 1, comprising the steps of:
(S1) placing mesenchymal stem cells in an alpha-MEM culture medium containing FBS for primary culture and amplification, and then placing in an exosome-free culture medium for secondary culture to obtain a culture solution;
(S2) continuously centrifuging the culture solution obtained in the step (S1), collecting supernatant, and performing aftertreatment to obtain mesenchymal stem cell-derived exosomes;
(S3) introducing Sephin1 into the mesenchymal stem cell-derived exosome prepared in the step (S2) through intermittent ultrasonic, and performing post-treatment to obtain an engineering exosome for maintaining the steady state of the endoplasmic reticulum of the cell: sep@Exo.
3. The method of claim 2, wherein in step (S2), the continuous centrifugation is performed as follows:
the culture solution is centrifuged with 200 g to 500g, 1500 g to 2500g and 8000 g to 12000g in sequence, and sediment is discarded after each centrifugation is finished, and the supernatant is collected.
4. The method of claim 2, wherein in the step (S3), the dosage ratio of Sephin1 to mesenchymal stem cell-derived exosomes is 10-15 μg:1mg.
5. The method of claim 2, wherein in step (S3), the ultrasonic parameters are 10% -30% amplitude, 5-15 seconds on/off, 1-3 minutes duration, 4-8 cycles, and 1-3 minutes cooling period between each cycle.
6. Use of the engineered exosome of claim 1 in the manufacture of a medicament for restoring regenerative function to high sugar environment damaged stem cells.
7. The use of an engineered exosome according to claim 6 for the manufacture of a medicament for restoring regenerative function to stem cells damaged in a high sugar environment, wherein the medicament is a medicament for reducing expression of a gene protein associated with dysregulation of the endoplasmic reticulum of stem cells in the high sugar environment.
8. The use of an engineered exosome of claim 6 in the manufacture of a medicament for restoring regenerative function to stem cells damaged in a high sugar environment, wherein the medicament is a medicament for promoting proliferation of stem cells in a high sugar environment.
9. The use of an engineered exosome of claim 6 in the manufacture of a medicament for restoring regenerative function to stem cells damaged in a high glycemic environment, wherein the medicament is a medicament for down regulating apoptosis levels in stem cells.
10. The use of an engineered exosome of claim 6 in the manufacture of a medicament for restoring regenerative function to stem cells damaged in a high sugar environment, wherein the medicament is a medicament for promoting osteogenic differentiation of stem cells.
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