A gene-loaded exosome, its preparation method and application

Abstract

The present invention is applicable to the field of biomedical technologies, and provides a gene-loaded exosome, a preparation method thereof, and an application. The gene-loaded exosome is obtained by loading the SMAD7 gene into an exosome vector. Among them, the exosome vector is formed by conjugating a CD34 antibody with a phospholipid polyethylene glycol succinimidyl ester and then binding it to the surface of secreted exosomes. The present invention utilizes the binding of the conjugate of the CD34 antibody and the phospholipid polyethylene glycol succinimidyl ester to the surface of secreted exosomes to synthesize an exosome vector with high endothelial cell targeting characteristics, and then loads the SMAD7 gene into the exosome vector to prepare a novel gene-loaded exosome, providing a new preclinical research strategy for the targeted treatment of HO, and also providing a certain theoretical basis for further studying and elaborating the pathogenesis of HO.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a gene-loaded exosome, a preparation method thereof, and an application thereof. Background Art

[0002] Heterotopic ossification (HO) is an abnormal bone formation process that occurs in muscle or connective tissue, mostly around large joints such as the hip and knee joints. As a complication of central nervous system diseases, it is common in patients with nerve paralysis. HO can directly reduce the range of motion of adjacent joints, limit joint movement, and even lead to joint ankylosis and loss of function. It may also cause neurological symptoms, compress and damage the surrounding nerves, and cause pressure sores, forming local pressure ulcers. Compared with normal bone, heterotopic ossification bone has higher metabolic activity, bone deposition rate, bone suture width, and osteoblast number. The disability rate of this disease is as high as 10% - 36%, and the high incidence age groups are young and middle-aged people. The pain caused by HO seriously affects the postoperative activities of patients and is the main complication and main factor affecting the postoperative functional recovery and quality of life of patients.

[0003] However, currently, the incidence of HO is affected by internal and external factors, and its pathogenesis is complex. Its pathological mechanism and related signaling pathways are currently unclear, and the targeted prevention and treatment methods are limited and the effects are often not ideal. Therefore, how to prevent and reverse the pathological process of HO not only has important clinical significance, but also has immeasurable economic and social value for patients and their families. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide a gene-loaded exosome, aiming to solve the problem that the existing prevention and treatment methods for heterotopic ossification are limited and the effects are not ideal.

[0005] The embodiments of the present invention are implemented as follows. A gene-loaded exosome is obtained by loading the SMAD7 gene into an exosome vector.

[0006] Wherein, the exosome vector is formed by conjugating a CD34 antibody with a phospholipid polyethylene glycol succinimide ester and then binding it to the surface of secreted exosomes.

[0007] Another purpose of the embodiments of the present invention is a preparation method of the above-mentioned gene-loaded exosome, including:

[0008] Reacting a phospholipid polyethylene glycol succinimide ester with a CD34 antibody to synthesize an exosome treatment solution.

[0009] Disperse secreted exosomes in a phosphate buffered saline solution, add the exosome treatment solution, and perform an incubation reaction at room temperature to obtain an exosome vector.

[0010] Incubate the exosome vector with SMAD7 plasmid to obtain the gene-loaded exosomes.

[0011] Another object of the embodiments of the present invention is the application of the gene-loaded exosomes in inhibiting the endothelial-mesenchymal transition of mouse aortic endothelial cells.

[0012] Another object of the embodiments of the present invention is the application of the gene-loaded exosomes in the preparation of drugs for preventing and / or treating heterotopic ossification.

[0013] In the embodiments of the present invention, first, CD34 antibody is conjugated with phospholipid polyethylene glycol succinimide ester and then combined with the surface of secreted exosomes to synthesize an exosome vector with high endothelial cell targeting characteristics. Then, the SMAD7 gene is loaded into the exosome vector to prepare a novel gene-loaded exosome, providing a new preclinical research strategy for the targeted treatment of HO and also providing a certain theoretical basis for further studying and elaborating the pathogenesis of HO. Description of the Drawings

[0014] Figure 1 is a transmission electron micrograph of the exosome vector provided by the embodiments of the present invention;

[0015] Figure 2 is a particle size distribution diagram of the exosome vector provided by the embodiments of the present invention;

[0016] Figure 3 is a detection result diagram of the exosome marker protein provided by the embodiments of the present invention;

[0017] Figure 4 is a detection result diagram of the CD34 antibody on the outer membrane of the exosome vector provided by the embodiments of the present invention;

[0018] Figure 5 is a detection result diagram of the biocompatibility of the exosome vector provided by the embodiments of the present invention;

[0019] Figure 6 is a detection result diagram of the stability of the exosome vector at 4°C and 37°C provided by the embodiments of the present invention;

[0020] Figure 7 is a detection result diagram of the cellular internalization of the exosome vector provided by the embodiments of the present invention;

[0021] Figure 8 is a detection result diagram of the loading amount of pcDNA3.1-SMAD7 in the gene-loaded exosomes provided by the embodiments of the present invention;

[0022] Figure 9It is a diagram showing the detection results of the mRNA expression of SMAD7 after co - culturing gene - loaded exosomes provided by the embodiments of the present invention with mouse aortic endothelial cells for 48 hours;

[0023] Figure 10 It is a diagram showing the detection results of the protein expression of SMAD7 after co - culturing gene - loaded exosomes provided by the embodiments of the present invention with mouse aortic endothelial cells for 48 hours;

[0024] Figure 11 It is a diagram showing the detection results of the mRNA expression of genes related to the EndMT pathway, namely CD31, VE - cadherin, N - cadherin, and vimentin, provided by the embodiments of the present invention;

[0025] Figure 12 It is a diagram showing the detection results of the protein expression of genes related to the EndMT pathway, namely CD31, VE - cadherin, N - cadherin, and vimentin, provided by the embodiments of the present invention. Detailed implementation manners

[0026] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Previous studies have shown that mesenchymal stem cells (MSCs) in local soft tissues after trauma gradually differentiate into two phenotypes, chondrocytes and osteoblasts, secrete osteoid and then gradually mineralize, and finally form mature bone tissue to produce HO. The endothelial - to - mesenchymal transition (EndMT) pathway is an important pathogenesis of HO. The EndMT pathway widely exists in embryonic development, heart diseases, kidney diseases, tumors and other physiological or pathological processes.

[0028] Vascular endothelial cells break through the vascular barrier under pathological or physiological conditions, become free in tissues, they dedifferentiate into mesenchymal stem cells, and then differentiate into specific cells and tissues under local conditions. Endothelial markers are expressed in chondrocytes and osteocytes of the HO rat model, and they are different from normal osteocytes and chondrocytes, indicating that endothelial cells are closely related to the occurrence and development of HO. In addition, under the stimulation of local tissue inflammatory mediators and signals, vascular endothelial cells are transformed into chondrocytes and osteocytes through the EndMT pathway, and form heterotopic ossification tissue through the mechanisms of intimal osteogenesis and chondrogenic osteogenesis. This shows that the EndMT pathway of endothelial cells has the potential to develop prevention and treatment methods for HO.

[0029] Based on previous studies on the molecular mechanisms and signaling pathways related to HO, we investigated targeted gene drugs that can be used for the treatment of HO. SMAD7 is an inhibitory gene (I-SMADs) in the EndMT signaling pathway and has the potential to prevent the transformation of myofibroblasts and the progression of other EndMT. The transcriptional product of the SMAD7 gene can competitively bind to the type II TGF-β receptor, thereby inhibiting the TGF-β signaling pathway and playing a negative regulatory role. Therefore, overexpression of SMAD7 in vascular endothelial cells can effectively block the TGF-β signaling pathway and inhibit HO mediated by the EndMT pathway. Overexpression of SMAD7 in vascular endothelial cells requires a gene vector with high biocompatibility, good sustained-release performance, high plasmid loading capacity, and good targeting properties. Lentiviral vectors are a widely used gene vector. However, due to their limitations in targeting and biocompatibility, it is difficult to apply them in clinical treatment. Exosomes are vesicles with a diameter of 40 - 100 nm secreted by living cells, which have high biological activity, biocompatibility, and can stably transport drugs, therapeutic genes, proteins, etc. They are important mediators for information transmission and material exchange between cells. Drugs loaded with exosomes, as natural endogenous substance transporters, have the advantages of low toxicity, non-immunogenicity, and good permeability. Currently, exosomes have been successfully loaded with small molecule chemical drugs and gene drugs for the treatment of cancer, Alzheimer's disease, and other diseases. Therefore, if the SMAD7 gene is loaded into exosomes, it can help it enter cells smoothly and achieve high expression to a certain extent, thus providing a new idea for the clinical prevention and treatment of HO.

[0030] In the embodiments of the present invention, to solve the problem that the existing prevention and treatment methods for heterotopic ossification are limited and the effects are not ideal, first, an exosome vector with high endothelial cell targeting characteristics is synthesized by conjugating CD34 antibody with phospholipid polyethylene glycol succinimide ester and then binding it to the surface of secreted exosomes. Then, the SMAD7 gene is loaded into this exosome vector to prepare a novel gene-loaded exosome, providing a new preclinical research strategy for the targeted treatment of HO and also providing a certain theoretical basis for further studying and elaborating the pathogenesis of HO.

[0031] The embodiments of the present invention provide a gene-loaded exosome, which is obtained by loading the SMAD7 gene into an exosome vector;

[0032] wherein, the exosome vector is formed by conjugating CD34 antibody with phospholipid polyethylene glycol succinimide ester and then binding it to the surface of secreted exosomes.

[0033] The embodiments of the present invention also provide a preparation method of the above gene-loaded exosome, including the following steps:

[0034] Step S1: React phospholipid-polyethylene glycol succinimide ester with CD34 antibody to synthesize exosome treatment solution.

[0035] Step S2: Disperse secreted exosomes in phosphate buffered saline solution, add the exosome treatment solution, and perform an incubation reaction at room temperature to obtain exosome carriers.

[0036] In the embodiment of the present invention, the exosome membrane is mainly composed of a lipid membrane. Therefore, lipophilic compounds are easily integrated into the exosome membrane. In the present invention, DSPE-PEG-AbCD34 is synthesized by conjugating CD34 antibody with phospholipid-polyethylene glycol succinimide ester (DSPE-PEG-NHS). Then, exosomes are treated with DSPE-PEG-AbCD34 to fuse the lipophilic DSPE onto the exosome membrane, and PEG-AbCD34 is thus carried to the surface of the exosomes to obtain exosome carriers (Exosome-DSPE-PEG-AbCD34).

[0037] Step S3: Add SMAD7 plasmid to the exosome carriers and perform an incubation reaction to obtain gene-loaded exosomes.

[0038] In the embodiment of the present invention, the purity and structural integrity of exosomes are detected by transmission electron microscope (TEM).

[0039] In the embodiment of the present invention, the viability of cells in the logarithmic growth phase is detected by the CCK-8 method. The specific method is as follows:

[0040] The concentration of cells in the logarithmic phase is adjusted to 5×10 4 cells / mL. Add 100 uL of cell suspension to each well of a 96-well culture plate and culture at 37 °C in a 5% CO 2 incubator for 24 h. After drug treatment, discard the supernatant, add 100 uL of fresh medium to each well, and then add 10 uL of CCK-8 solution. After culturing at 37 °C for 4 h, detect the absorbance (OD) value of each well at 450 nm by an enzyme reader. Each experiment is repeated three times.

[0041] In the embodiment of the present invention, cells are seeded into a 96-well plate at an initial density of 1×10 5 cells per well, incubated with Exosome-DSPE-PEG-AbCD34 at 37 °C for 2 h, then cultured in 60 nM hemolysin red medium for 30 min, washed 3 times with cold PBS, and fixed with 4% paraformaldehyde for 20 min. Then, the cells are treated with DAPI for 10 min and washed twice with PBS, and then observed and photographed using a laser confocal scanning microscope.

[0042] In the embodiment of the present invention, total cellular RNA was extracted according to the instructions of the Trizol kit, and then mRNA was reverse-transcribed into cDNA according to the operation instructions of the TaqMan microRNA reverse transcription kit. The expression of each gene was detected by Applied Biosystems 7500 fluorescence quantitative PCR. The primer sequences used are shown in Table 1.

[0043] Table 1

[0044]

[0045] In the embodiment of the present invention, AbCD34 on Exosome-DSPE-PEG-AbCD34 was detected by SDS-PAGE gel electrophoresis. After centrifugation at 120,000 g, an appropriate amount of RIPA cell lysate was added to the precipitate of exosomes, followed by ice bath for 25 min and centrifugation at 10,000 g for 20 min. The BCA protein quantification kit was used to quantify the protein concentrations of exosomes and cell lysates. Lysates containing equal amounts of protein were mixed with SDS-PAGE sample loading buffer and heated at 100 °C for 5 min. Proteins in the above mixture were separated by SDS-PAGE electrophoresis, and then stained with R250 gel staining solution and decolorized with decolorizing solution. Exosome marker proteins (CD9, CD63), SMAD7, CD31, VE-cadherin, N-cadherin, vimentin and GAPDH were detected by Western blotting. Protein samples were separated by SDS-PAGE and then transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 1 h, and then the first antibody corresponding to the protein to be detected was added and incubated at 4 °C for 4 h. After reaction with the HRP-labeled second antibody and washing, ECL luminescence solution was used for exposure and recording of photos.

[0046] In the embodiment of the present invention, the secreted exosomes are exosomes of mouse aortic endothelial cells. The exosomes of mouse aortic endothelial cells were extracted according to the instructions of the EXoCap Iltracentrifugation / storage Booster kit in the present invention.

[0047] In the embodiment of the present invention, the method for culturing mouse aortic endothelial cells is as follows:

[0048] Mouse aortic endothelial cells were placed in DMEM medium containing 10% FBS and 5% CO 2 and cultured at 37 °C. When the cell density reached 85%, they were digested with trypsin and passaged.

[0049] In a preferred embodiment of the present invention, step S1 includes:

[0050] Step S11: React 5 mL of phospholipid-polyethylene glycol succinimide ester with 10 μL of CD34 antibody at a concentration of 1 mg / mL at room temperature for 24 hours to synthesize the exosome treatment solution.

[0051] In a preferred embodiment of the present invention, the step S2 includes:

[0052] Step S21: Disperse the secreted exosomes in phosphate buffered saline and adjust the protein content to 50 mg / mL to obtain an exosome dispersion;

[0053] Step S22: Add 0.1 mL of the exosome treatment solution at a concentration of 20 mg / mL to 2 mL of the exosome dispersion, gently blow and mix the mixture, and incubate at room temperature for 1 hour. After centrifugation, an exosome carrier is obtained.

[0054] In a preferred embodiment of the present invention, the step S3 includes:

[0055] Step S31: Add 100 μL of SMAD7 (1 μg / μL) plasmid to 2 mL of the exosome carrier at a concentration of 1 mg / mL and carry out an incubation reaction (overnight) for 12 - 16 hours to obtain the gene-loaded exosomes.

[0056] The embodiment of the present invention also provides an application of the above-mentioned gene-loaded exosomes in inhibiting the endothelial-mesenchymal transition of mouse aortic endothelial cells.

[0057] The embodiment of the present invention also provides an application of the above-mentioned gene-loaded exosomes in the preparation of drugs for preventing and / or treating heterotopic ossification.

[0058] The following gives examples of certain embodiments of the present invention, and the purpose is not to limit the scope of the present invention.

[0059] Example 1 Preparation and Characterization of Exosome-DSPE-PEG2000-AbCD34

[0060] In this example, exosomes were isolated from the culture medium of mouse aortic endothelial cells. The exosome treatment solution (DSPE-PEG2000-AbCD34) was synthesized by reacting 5 mL of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-N-hydroxysuccinimide (DSPE-PEG2000-NHS) (0.1 M) with 10 μL of excess CD34 antibody (AbCD34) (1 mg / mL) at room temperature for 24 h. Then, the prepared exosomes were dispersed in 5 mL of PBS, and their protein content was adjusted to 50 mg / mL. 0.1 mL of the DSPE-PEG2000-AbCD34 solution with a concentration of 20 mg / mL was added to 2 mL of the exosome dispersion, and the mixture was gently blown and incubated at room temperature for 1 h. Then, the dispersion was transferred to a special centrifuge tube and ultracentrifuged at 120,000 rpm and 4 °C for 70 min. The upper solution was aspirated to remove the unbound DSPE-PEG2000-AbCD34, and the resulting precipitate was the exosome carrier conjugated with AbCD34 (Exosome-DSPE-PEG2000-AbCD34). The pre-cooled PBS solution was added to the precipitate, blown evenly, and dissolved again.

[0061] After preparation, the exosome carrier of the present invention was characterized by TEM ( Figure 1 ) and a zeta potential particle size analyzer ( Figure 2 ). The specific operation was as follows: The protein content of the exosome carrier dispersion was diluted to 100 mg / ml using HEPES buffer, and then 10 μL was taken and added to a 200-mesh copper grid (coated with a carbon film) at room temperature for transmission electron microscopy observation until the copper grid was completely dry. Then, the sample was observed and photographed simultaneously at an accelerating voltage of 75 kV. The protein content of the exosome carrier dispersion was adjusted to 50 mg / mL to detect its particle size. 20 μL was taken and added to 980 μL of ddH 2 O, and then the particle size of the exosome carrier was measured by a zeta potential particle size analyzer based on the light scattering method.

[0062] The present invention also detected exosome marker proteins (CD9, CD63) ( Figure 3 ) by Western blotting and the CD34 antibody loaded on the outer membrane of exosomes ( Figure 4 ) by SDS-PAGE gel electrophoresis. The results showed that the exosomes of the present invention were successfully isolated and extracted from mouse aortic endothelial cells, and Exosome-DSPE-PEG2000-AbCD34 with CD34 targeting characteristics was synthesized.

[0063] Example 2 Detection of biocompatibility, stability, targeting, and cellular internalization of Exosome-DSPE-PEG2000-AbCD34

[0064] Biocompatibility, stability, targeting, and cellular internalization are important properties of gene carriers. In this invention, the CCK-8 method was used to detect the effect of different concentrations of Exosome-DSPE-PEG2000-AbCD34 on the viability of mouse aortic endothelial cells. The results showed that all concentrations of Exosome-DSPE-PEG2000-AbCD34 had no effect on the survival rate of these cells ( Figure 5 ).

[0065] Furthermore, the stability of Exosome-DSPE-PEG2000-AbCD34 at 4 °C and 37 °C was tested in this invention. The results showed that Exosome-DSPE-PEG2000-AbCD34 could be stably stored at 4 °C and 37 °C for at least 8 days ( Figure 6 ).

[0066] Furthermore, the targeting effect of Exosome-DSPE-PEG-2000AbCD34 on mouse aortic endothelial cells was observed by laser confocal microscopy. The results showed that after incubating the exosome carrier with the cells for 48 h, more FITC-labeled Exosome-DSPE-PEG2000-AbCD34 could be observed on the surface and inside of mouse aortic endothelial cells, indicating that FITC-labeled Exosome-DSPE-PEG2000-AbCD34 had better cell targeting and cellular internalization than Exosome-DSPE-PEG ( Figure 7 ).

[0067] Example 3 Synthesis of Exosome-DSPE-PEG2000-AbCD34-SMAD7 and Its Effect on the Expression of SMAD7 in Mouse Aortic Endothelial Cells

[0068] In this example, 100 μL of SMAD7 plasmid (pCDNA3.1-SMAD7, 200 ng / μL) was incubated with 2 mL of Exosome-DSPE-PEG2000-AbCD34 (1 mg / mL) to synthesize Exosome-DSPE-PEG2000-AbCD34-SMAD7, and then DNA gel electrophoresis was used to detect the loading effect of pcDNA3.1-SMAD7 in Exosome-DSPE-PEG2000-AbCD34-SMAD7 ( Figure 8) Gel electrophoresis was performed using a 2% agarose gel pretreated with EB for separation. The gel electrophoresis buffer was 1*ATE gel buffer, the electrophoresis voltage was 120V, and the electrophoresis time was 25 min. After the preparation of Exosome-DSPE-PEG2000-AbCD34-SMAD7, it was co-cultured with mouse aortic endothelial cells for 48 h, and then the expression of SMAD7 in the cells was detected by qPCR and Western blot methods.

[0069] Among them, the specific steps of qPCR are as follows:

[0070] Total RNA was extracted using Trizol total RNA extraction reagent. After centrifuging and collecting the cells, 1 mL of trizol reagent was added, and it was incubated at 15 - 30 °C for 15 min to fully lyse. At 4 °C, centrifuge at 12,000 rpm for 5 min, and discard the precipitate. (Prepare an EP tube in the laminar flow hood. After centrifugation, aspirate the supernatant into the prepared EP tube.) Add chloroform at a ratio of 200 μL of chloroform / mL of Trizol, shake vigorously for 15 s (can be shaken by hand), and incubate at 15 - 30 °C for 2 - 3 min. Note: Do not use a vortex oscillator to avoid breaking genomic DNA. Centrifuge at 4 °C, 12,000 g for 15 min. Aspirate the upper aqueous phase into another centrifuge tube. Add isopropanol at a ratio of 500 μL of isopropanol / mL of Trizol, shake by hand, and incubate at 15 - 30 °C (room temperature is fine) for 10 min. Centrifuge at 2 - 8 °C, 12,000 g for 10 min, discard the supernatant, and the RNA precipitates at the bottom of the tube. Add 75% ethanol at a ratio of 1 mL of 75% ethanol / mL of Trizol, vortex to mix well, and suspend the precipitate. Centrifuge at 4 °C, 7500 rpm for 5 min, and try to discard as much supernatant as possible. (Note: After using the centrifuge, turn off the power, open the centrifuge lid for 30 minutes to cool down, and then close it.) Air dry at room temperature or vacuum dry for 5 - 10 min. (Note: Do not use centrifugation to dry the RNA. The RNA sample should not be overly dry, otherwise it will be difficult to dissolve.) Dissolve the RNA precipitate with 30 μL of 1×RNAsafe (or 30 μL of 1‰ DEPC water), and incubate in a 60 °C water bath for 20 min. (Note: DEPC water diluted 1 / 100 with distilled water needs to be autoclaved before use. 20×RNAsafe is diluted with DEPC water.) Measure the OD value to quantify the RNA concentration (using a DU800 ultraviolet spectrophotometer). If the extracted RNA is not reverse transcribed, it can be stored at -80 °C.

[0071] Reverse transcribe mRNA into cDNA according to the operating instructions of the reverse transcription kit. Add the components for RNA reverse transcription into cDNA (the reaction system is 20 μl) in sequence, and add 1 μl of Oligo(dT). Make up the remaining volume of RNA (the volume calculated in the previous step) to 12 μl with RNase-free water, and incubate in a water bath at 65 °C for 5 min. Then add 4 μl of 5×Reaction Buffer for reverse transcription reagent, 1 μl of RNaseInhibiter, 2 μl of 10 mM dNTP mix, and 1 μl of reverse transcriptase. Use Applied Biosystems 7500 fluorescence quantitative PCR to detect the expression of each gene, and the primer sequences used are shown in Table 1. The primers used were provided by Tiangen Biotech (Shanghai) Co., Ltd.

[0072] The specific qPCR procedure is as follows: Amplify the target gene of PCR cDNA (the reaction system is 20 μl, and make up with sterile water if insufficient).

[0073] For the following experiments, use autoclaved pipette tips and autoclaved ordinary water. Sampling: ① Add 10 μl of Mix; ② Add 1 μl of primer; ③ The total volume of cDNA and water is 9 μl; Mixing: Just centrifuge briefly with a small centrifuge; Put into the PCR instrument: The conditions of the PCR instrument are set in advance, and the reaction conditions are set according to the Mix instructions; Preservation of PCR products: Generally store in a 4 °C refrigerator, and can be frozen at -20 °C for long-term storage. Each experiment is repeated three times.

[0074] The results showed that Exosome-DSPE-PEG2000-AbCD34 could transport pcDNA3.1-SMAD7 into mouse aortic endothelial cells and successfully overexpress SMAD7 in these cells ( Figure 9 and Figure 10 ).

[0075] Example 4 Effect of Exosome-DSPE-PEG2000-AbCD34-SMAD7 on the EndMT pathway in mouse aortic endothelial cells

[0076] The EndMT pathway has been proven to be an important mechanism in the pathogenesis of HO. In its molecular pathophysiological mechanism, the transforming growth factor-β (TGF-β) superfamily signaling pathway is the main signaling pathway, and SMAD7 is an important inhibitory gene in this signaling pathway.

[0077] To determine the effect of SMAD7 overexpression on the phenotype of RAOECS, in the embodiments of the present invention, RAOECS were stimulated with 10 ng / ml of TGF-β1 for 48 h to simulate the effect of TGF-β1 on local EndMT at the cellular level. Furthermore, the effect of Exosome-DSPE-PEG2000-AbCD34-SMAD7 on the TGF-β1-mediated EndoMT pathway in RAOECS was detected by qPCR and Western blot methods. The results showed that TGF-β1 could downregulate CD31 and VE-cadherin and upregulate N-cadherin and vimentin. Compared with the control group, the Exosome-DSPE-PEG2000-AbCD34-SMAD7 transfection group could upregulate the expression of CD31 and VE-cadherin, thereby antagonizing the effect of TGF-β1. At the same time, the transfection of Exosome-DSPE-PEG2000-AbCD34-SMAD7 also downregulated the expression of N-cadherin and vimentin ( Figure 11 and Figure 12 ). In summary, the overexpression of the SMAD7 gene induced by Exosome-DSPE-PEG2000-AbCD34-SMAD7 can effectively reverse the TGF-β1-mediated EndMT pathway of RAOECS.

[0078] In summary, the present invention first utilizes the binding of DSPE-PEG2000-AbCD34 (CD34 antibody) to the surface of secreted exosomes to synthesize an exosome vector with high endothelial cell targeting characteristics (Exosome-DSPE-PEG2000-AbCD34), and studies its physical, chemical and biological characteristics. It is found that this exosome vector has no effect on the viability of mouse aortic endothelial cells at high concentrations and can be stably stored at 4 °C and 37 °C for at least 8 days. Compared with Exosome-DSPE-PEG2000, Exosome-DSPE-PEG2000-AbCD34 has better targeting to mouse aortic endothelial cells and can enter the cells more effectively. Furthermore, the SMAD7 plasmid is loaded into Exosome-DSPE-PEG2000-AbCD34 to study its effect on the TGF-β-mediated EndMT pathway. The results show that Exosome-DSPE-PEG2000-AbCD34-SMAD7 can significantly increase the expression level of SMAD7 in mouse aortic endothelial cells, and the overexpression of the SMAD7 gene induced by Exosome-DSPE-PEG2000-AbCD34-SMAD7 can up-regulate the expression of CD31 and VE-cadherin, and down-regulate the expression of N-cadherin and vimentin, thus offsetting the effect of TGF-β1 and effectively reversing the EndMT pathway mediated by RAOECS through TGF-β1. Therefore, Exosome-DSPE-PEG2000-AbCD34-SMAD7 has the potential to prevent and treat HO based on SMAD7, and the findings of the present invention provide a certain theoretical basis and preclinical research strategy for the prevention and treatment of HO.

[0079] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

[0080] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0081]

[0082]

[0083] Sequence Listing <110> Zhong Biao, Wei Zhenyuan <120> A Gene-Loaded Exosome and Its Preparation Method and Application <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 ggaggtgaca gaaggtggg 19 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <400> 2 gcttggcagc gaaacaacta acagg 25 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <400> 3 cttcacccag accaagtaca ca 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 aatggtgaaa gcgtcctggt 20 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <400> 5 gtgccattag ccaagggaat tcagc 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <400> 6 gcgttcctgt tccactcata ggagg 25 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 ggaccagcta accaacgaca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 aaggtcaaga cgtgccagag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 agtgccagcc tcgtctcata 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 gagaaggcag ccctggtaac 20

Claims

1. A method for preparing gene-loaded exosomes, characterized in that, comprising: Reacting 5 mL of DSPE-PEG2000-NHS with 10 μL of CD34 antibody (AbCD34) at a concentration of 1 mg / mL at room temperature for 24 hours to synthesize a DSPE-PEG2000-AbCD34 treatment solution; Disperse the secreted exosomes in phosphate buffered saline and adjust the protein content to 50 mg / mL to obtain an exosome dispersion; Add 0.1 mL of the DSPE-PEG2000-AbCD34 treatment solution at a concentration of 20 mg / mL to 2 mL of the exosome dispersion, gently blow and mix the mixture and incubate at room temperature for 1 hour, and obtain an exosome carrier after centrifugation; Add 100 μL of SMAD7 plasmid to 2 mL of the exosome carrier at a concentration of 1 mg / mL and incubate for 12-16 hours to obtain the gene-loaded exosomes; The secreted exosomes are exosomes of mouse aortic endothelial cells.

2. The method for preparing gene-loaded exosomes according to claim 1, characterized in that, The culture method of the mouse aortic endothelial cells is: Mouse aortic endothelial cells were placed in DMEM medium containing 10% FBS and 5% CO 2 and cultured at 37°C. When the cell density reached 85%, the cells were digested with trypsin and passaged.

3. A gene-loaded exosome, characterized in that, The gene-loaded exosomes are prepared by the method for preparing gene-loaded exosomes according to any one of claims 1-2.

4. Use of the gene-loaded exosomes according to claim 3 in the preparation of drugs for preventing and / or treating heterotopic ossification.

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