Engineered exosome containing gp96 antigen information as well as preparation method and application of engineered exosome

Abstract

The invention discloses an engineered exosome containing gp96 antigen information as well as a preparation method and application of the engineered exosome, and belongs to the technical field of exosomes. The invention solves the problem of how to further improve the effect of the exosome in the aspect of treating diseases. The exosome disclosed by the invention is derived from stem cells which are subjected to gp96 pretreatment and over-express gp96 antigen information. When the gp96 is combined with the exosome, the function of the exosome can be enhanced, so that the antigen information molecules carried by the gp96 can be more effectively transmitted into specific cells, and the physiological function of recipient cells is adjusted. The combination has potential application value in treatment of inflammation-related diseases such as osteoarthritis and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosomes, and specifically relates to an engineered exosome containing gp96 antigen information, and a preparation method and application thereof. Background Art

[0002] Exosomes are small vesicles secreted by cells with a diameter of approximately 30-150 nanometers. They can carry molecules such as proteins, RNA and DNA, and play an important role in intercellular communication.

[0003] In the prior art, the invention patent with patent number CN112190592B discloses the use of miRNA in the preparation of drugs for preventing and treating osteoarthritis, exosomes with high expression of miRNA and their use. It discloses the use of exosomes with high expression of miRNA to prevent and treat osteoarthritis, but miR-155-5p is a type of microRNA that plays a regulatory role; there are differences in miRNA expression in patients with rheumatoid arthritis (RA), leading to the occurrence of autoimmune arthritis, so miR-155-5p only performs a single repair from the aspect of inflammation, and the therapeutic effect on osteoarthritis is very limited. How to further improve the effect of exosomes in treating diseases is a difficult problem that needs to be explored in this field. Summary of the invention

[0004] In response to the problem of how to further improve the effect of exosomes in treating diseases in the prior art, the present invention provides an engineered exosome containing gp96 antigen information and a preparation method and application thereof.

[0005] The technical solution adopted by the present invention is as follows:

[0006] An engineered exosome containing gp96 antigen information, the exosome is derived from a stem cell that has been pre-treated with gp96 and then over-expressed the gp96 antigen information.

[0007] After adopting this technical solution, when gp96 is combined with exosomes, the function of exosomes can be enhanced, enabling them to more effectively deliver antigen information molecules carried by gp96 to specific cells, thereby regulating the physiological functions of recipient cells. This combination has potential application value in inflammation-related diseases.

[0008] Furthermore, the stem cells are human placental mesenchymal stem cells, gp96 is encoded by the HSP90B1 gene, and the protein sequence number is: P14625.1.

[0009] Furthermore, the exosomes are cup-like structures with a particle size of 30-200 nm.

[0010] A method for preparing engineered exosomes containing gp96 antigen information comprises the following steps:

[0011] S1: Isolate the tissue mass containing stem cells and culture the tissue mass containing stem cells with the gp96 antigen complex;

[0012] S2: Change the medium at the set time, subculture when the cell density reaches the set value, and collect the culture supernatant of the 3rd to 5th generations;

[0013] S3: Exosomes are separated from the culture supernatant to obtain engineered exosomes containing gp96 antigen information.

[0014] After adopting this technical solution, from the morphological point of view, as the number of generations increases during the passage process, stem cells may gradually lose their original morphology and growth characteristics. The stem cells of the P3 generation usually have a more uniform morphology and a faster growth rate. After the P5 generation, the morphology of the cells may become more diverse, and the growth rate may also slow down. From a functional point of view, the stem cells of the P3 generation usually have a higher pluripotency and can differentiate into various types of cells. After the P5 generation, the pluripotency of stem cells may be affected to a certain extent, and its differentiation ability may be subject to certain restrictions. From the perspective of stability, the stem cells of the P3 generation are usually more stable and are not prone to mutation or abnormal differentiation. Too high or too low generations will affect the activity and stability of stem cells, especially too low generations (such as P1, P2) may be accompanied by the instability and inadaptability of cells, so the 3rd to 5th generations of stem cells are selected in the present invention.

[0015] Furthermore, the gp96 antigen-containing complex is a protein complex derived from human placental tissue, and the antigen information contained in the gp96 antigen-containing complex includes ARHGAP33, ACTR5, MAML1, MUC3B, MAMDC4, LRP2, KCNT1, ITPR2, FBXO34, FLRT3, DHCR24, and C1orf115.

[0016] Furthermore, the specific steps of S1 are as follows:

[0017] S101: Obtain a fresh intact placenta from a pregnant woman, wash the placenta, and then remove the amniotic membrane tissue from the placenta;

[0018] S102: Clean the amniotic membrane tissue and cut it into pieces of 0.5 to 1.0 cm 2 of tissue blocks;

[0019] S103: Spread the tissue block into a T75 culture flask and place it in a cell culture incubator for 1-3 hours to allow the tissue block to adhere to the wall;

[0020] S104: Add 10 to 15 mL of serum-free stem cell culture medium supplemented with gp96 antigen complex and culture together with the tissue pieces.

[0021] Furthermore, the culture medium in S104 includes the following components: 450-475 mL of stem cell serum-free culture medium, 25-50 mL of 5-10% platelet lysate by volume, 2.5-10 mL of 0.5-2% penicillin-streptomycin, and the final concentration of the gp96 antigen complex in the culture medium is 3 ug / mL to 15 ug / mL.

[0022] Furthermore, the medium was changed every 3-5 days in S2, and the adherent cells around the tissue block were subcultured when the density reached 80-90%. During the subculture, the cells were recovered by trypsin and the subcultured cells were plated at 1.5×10 4 / cm 2 The density of culture was 25cm 2 In the culture flask, the medium was changed every 3-5 days, and the cells were passaged at a ratio of 1:2 or 1:3 when the cell growth density reached 80%-90%.

[0023] Furthermore, the specific steps of S3 are as follows:

[0024] S301: centrifuge the supernatant obtained in S2 at 2-8°C, 300-500g for 5-10 minutes, and take the supernatant;

[0025] S302: centrifuge the supernatant obtained in S301 at 2000-4000 g for 10-20 minutes at 2-8°C, and take the supernatant;

[0026] S303: centrifuging the supernatant obtained in S302 at 2-8°C and 10,000-13,000 g for 30-60 minutes, and taking the supernatant;

[0027] S304: performing ultrafiltration and concentration treatment on the supernatant obtained in S303 to obtain an ultrafiltrate;

[0028] S305: centrifuging the ultrafiltrate at 2-8°C, 5000-10000 g for 20-30 minutes to obtain a supernatant;

[0029] S306: The supernatant obtained in S305 is filtered and sterilized to obtain a concentrated solution of engineered exosomes containing gp96 antigen information.

[0030] After adopting this technical solution, the extraction and concentration operation is convenient, and no ultracentrifugation equipment and ultracentrifugation consumables are required to extract and concentrate, which can meet the basic needs of ordinary laboratories and has a relatively high enrichment efficiency. The entire operation can be carried out under the GMP environment, ensuring a sterile environment. While no exogenous reagents are mixed in the extraction process, the residual risk is controllable, and the safety of later applications is guaranteed.

[0031] Application of engineered exosomes containing gp96 antigen information in the treatment of osteoarthritis.

[0032] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0033] Exosomes from placental amniotic stem cells rich in gp96 antigen information intervene in osteoarthritis in many aspects. First, gp96 triggers the natural and specific defense system of pathogen defense in a synergistic and effective way. gp96 has the function of a natural immune adjuvant: gp96 can non-specifically activate antigen-presenting cells to express cytokines such as TNF-α and IL-12 and upregulate CD86, CD80 co-stimulatory molecules and MHC molecules, providing cytotoxic T cells with a pro-inflammatory cytokine environment and co-stimulatory molecules, stimulating immune response and improving the body's own anti-inflammatory response; secondly, the rich tumor antigen information carried by gp96 can actively activate T lymphocytes, target the pathological site for treatment, and improve the killing activity; finally, exosomes from placental amniotic mesenchymal stem cells have the same properties as placental amniotic mesenchymal stem cells. Compared with stem cells from other sources, they can secrete more anti-inflammatory factors and tissue repair factors, inhibit the activity of degradation enzymes such as matrix metalloproteinases (MMPs), reduce the destruction of cartilage matrix, and promote chondrocyte proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The morphology of exosomes.

[0035] Figure 2 Characterization diagram of exosome particle size and particle concentration.

[0036] Figure 3 This is a graph showing the results of detecting the content of stem cell factors in exosomes.

[0037] Figure 4 This is the electrophoresis band diagram of gp96 protein.

[0038] Figure 5 This is the result of IL-1β content detection.

[0039] Figure 6 This is a graph showing the results of TNF-α content detection. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of this application clearer, the technical solution in the embodiment is clearly and completely described below. Obviously, the described embodiment is only a part of the embodiment of this application, not all the embodiments. Based on the embodiment of this application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application.

[0041] The preparation method of the gp96 antigen-containing complex is as follows:

[0042] 1. Pretreatment of human ex vivo placental tissue

[0043] (1) If the placenta has been frozen, it needs to be thawed first and then the encapsulation and connective tissue can be removed.

[0044] (2) Lysis solution preparation: pre-cooled NaHCO3 and phenylmethylsulfonyl fluoride (PMSF), pH 7.0, molar ratio of the two is 30:1.

[0045] (3) The encapsulation material and connective tissue of the isolated placenta tissue are removed, the tissue is chopped into pieces, a lysis buffer is added, and the mixture is ground into a homogenate.

[0046] 2. Centrifugation of homogenate:

[0047] (1) The prepared homogenate was centrifuged at 10,000 rpm for 1 hour at 4°C, and then centrifuged again at the same conditions for 0.5 hour. After the centrifugation, the supernatant was collected for later use.

[0048] 3. GP96 monoclonal antibody-Sepharose 4B affinity chromatography:

[0049] (1) The supernatant was loaded onto a GP96 monoclonal antibody-Sepharose 4B affinity chromatography column at a flow rate of 0.8 ml / min.

[0050] (2) The column was eluted with a 0.1 M Tris-HCl buffer solution containing 0.5 M NaCl, and then with glycine-HCl as the eluent.

[0051] (3) Add NaHCO3 to the collected eluate to neutralize to pH = 7.0.

[0052] 4.HiTrap Q Sepharose ion exchange chromatography:

[0053] (1) The eluate neutralized to pH = 7.0 was loaded onto a HiTrap Q Sepharose ion exchange chromatography column at a flow rate of 1.5 ml / min.

[0054] (2) Wash the column with 0.02 M Tris-HCl buffer solution containing 0.2 M NaCl, and then gradually increase the NaCl concentration to 0.8 M for elution.

[0055] 5. Ultrafiltration concentration:

[0056] (1) The eluate is ultrafiltered and concentrated through a 30 kD membrane package to obtain a solution containing the gp96 antigen complex.

[0057] An engineered exosome containing gp96 antigen information, the preparation method of which is as follows:

[0058] S1: Isolate a tissue block containing stem cells, and culture the tissue block containing stem cells with the gp96 antigen-containing complex prepared above, as follows:

[0059] S101: obtaining a fresh and complete placenta of a pregnant woman, storing the placenta aseptically after obtaining it, and using the placenta within 3 hours after obtaining it; when using it, first use a physiological saline with a concentration of 1-3% penicillin-streptomycin double antibiotic (a physiological saline with a concentration of 2% penicillin-streptomycin double antibiotic is used in this embodiment) to rinse the placental tissue 3 times to remove residual blood and blood clots as much as possible, and after washing, bluntly peel off the amniotic tissue on the placenta;

[0060] S102: Wash the stripped amniotic membrane tissue three times with PBS buffer to remove the blood clots adhering to the surface of the amniotic membrane. Cut the washed amniotic membrane tissue into 0.5 cm pieces with sterilized ophthalmic scissors. 2 Tissue blocks were cultured with 15 mL of serum-free stem cell culture medium containing gp96 antigen complex;

[0061] S103: Spread the cut tissue blocks into a T75 culture flask until the bottom of the T75 culture flask is covered, and place the T75 culture flask in a 5% CO2, 37°C cell culture incubator for 2 hours to allow the tissue blocks to adhere to the wall;

[0062] S104: then adding 15 mL of serum-free stem cell culture medium supplemented with gp96 antigen complex and culturing together with the tissue pieces;

[0063] S2: During the growth process of adherent cells appearing at the edge of the tissue block, the medium was changed every 3 days. When the density of adherent cells around the tissue block reached 80%, the cells were subcultured and 0.25% trypsin was used to recover the cells. The subcultured cells were plated at 1X10 5 / cm 2 The density of culture was 25cm 2 In the culture flask, the medium was changed every 3 days. When the cell growth density reached 80%, the cells could be passaged at a ratio of 1:2. The third to fifth generations of placental mesenchymal stem cells were used in the experiment, and the culture supernatant was collected.

[0064] S3: Exosomes are separated from the culture supernatant to obtain engineered exosomes containing gp96 antigen information, as follows:

[0065] S301: Centrifuge the supernatant obtained in S2 at 4°C, 300 g for 5 minutes and take the supernatant;

[0066] S302: centrifuge the supernatant obtained in S301 at 4°C and 2000 g for 10 minutes, and take the supernatant;

[0067] S303: centrifuge the supernatant obtained in S302 at 10,000 g for 30 minutes at 4°C, and take the supernatant;

[0068] S304: The supernatant obtained in S303 is subjected to ultrafiltration and concentration treatment using a 100 kd ultrafiltration tube to obtain an ultrafiltrate;

[0069] S305: centrifuging the ultrafiltrate to obtain a supernatant;

[0070] S306: The supernatant obtained in S305 is filtered and sterilized using a 0.22 μm filter to obtain a concentrated solution of engineered exosomes containing gp96 antigen information, which is used immediately or stored at -80°C.

[0071] (1) Phenotype detection of placental amniotic mesenchymal stem cells:

[0072] 0.5 mL of the obtained placental mesenchymal stem cells were taken, and HLA-DR\CD34\CD19\CD14\CD45\CD90\CD105\CD73 flow cytometry fluorescent antibodies were added thereto for labeling, incubated at 4°C in the dark for 30 min, washed twice with PBS and resuspended in 400 μL PBS, and analyzed by flow cytometry. The test results are shown in Table 1. It can be seen from Table 1 that the proportion of stem cell positive expression markers such as CD90\CD105\CD73 is more than 99%, and the proportion of negative expression markers such as HLA-DR\CD34\CD19\CD14\CD45 is less than 0.5%, which meets the property requirements of stem cells.

[0073] Table 1

[0074]

[0075] (2) Identification of exosomes containing gp96 antigen information:

[0076] The morphology of exosomes was detected by electron microscopy, and the test results were as follows: Figure 1 As shown, from Figure 1 It can be seen that the exosomes are cup-like structures with clear membrane structures;

[0077] NTA was used to detect the particle size and number of exosomes. The test results were as follows: Figure 2 As shown, from Figure 2 It can be seen that the particle size of exosomes is 30-200nm;

[0078] After the exosomes were broken by ultrasound, the protein concentration was detected by BCA, and the test results are shown in Table 2. As can be seen from Table 2, the protein concentration in this batch of exosomes was 1.30 mg / mL;

[0079] Table 2

[0080] serial number OD562 OD562 OD562 avg concentration mg / ml stdev concentration mg / ml 240903 0.232 0.232 0.234 1.304773 0.013553

[0081] The ELISA method was used to detect the content of stem cell factor. The test results were as follows Figure 3 As shown; the electrophoresis method was used for detection, and the detection results were as follows Figure 4 As shown, from Figure 4 It can be seen that there is a 96kDa protein band in the electrophoresis;

[0082] The gene sequence of the protein submitted for inspection and the monitoring results are shown in Table 3. From Table 3, it can be seen that the protein gene is HSP90B1, the molecular weight is 92.468 kDa, the number of amino acids is 803, and the purity accounts for 73.53% of the total protein.

[0083] Table 3

[0084]

[0085] The exosomes prepared above are used to treat osteoarthritis, and the method of use and the therapeutic effect are as follows:

[0086] 1. Animal Experimentation:

[0087] (1) KM Kunming mouse modeling (30 mice, 6 weeks old, 18-20g, SPF level)

[0088] The experimental mice were anesthetized, the surgical area was shaved, and the left hind limb was fixed in a supine position with the knee flexed 90°, and then fully disinfected. The skin was cut to expose the patellar ligament, and the capsule was opened along the inner edge of the patellar ligament with sharp scissors. The intra-articular adipose tissue was bluntly cleaned. The medial meniscus was connected to the tibial plateau through the medial meniscus tibial collateral ligament (MMTL). After the MMTL was severed and the wound was flushed, the joint cavity was sutured with 7-0 surgical sutures, the skin wound was closed, and amoxicillin was applied locally to prevent wound infection. The sham group (sham) only opened the capsule and sutured layer by layer.

[0089] (2) Usage

[0090] The experimental group (10 rats) was injected with exosomes at 5-10 μL / joint (specifically 6 μL / joint in this experiment); the positive control group (10 rats) was injected with triamcinolone acetonide (TAA) at 6-10 μg / joint (specifically 6 μL / joint in this experiment), and the negative control group (10 rats) was injected with an equal amount of saline. The entire experiment lasted for 14 days.

[0091] Effectiveness data ( Figure 5 and Figure 6 ) showed that the inflammatory symptoms in the exosome treatment group were significantly improved, and the expression levels of inflammatory-related factors IL-1β and TNF-α in the serum were lower than those in the positive control group and significantly lower than those in the negative control group.

[0092] Safety data showed that the exosome-treated mice had no gastrointestinal symptoms such as nausea and vomiting compared with the normal mice without modeling; blood biochemistry monitoring showed no electrolyte disorders and liver and kidney function damage, no adverse drug reactions such as adrenal cortex dysfunction, obesity, and diabetes; there were no complications such as peri-articular calcification, subcutaneous fat tissue atrophy, and skin depigmentation.

[0093] 2. Clinical trials

[0094] (1) Inclusion criteria: 30 patients with moderate to severe arthritis, Kellgren-Lawrence (KL) grade 2 or above, i.e., grade 2-4 (grade 0 is normal joint and grade 4 is the most severe), with bone injuries in the knee, shoulder, elbow, hip, ankle, or wrist.

[0095] The method of use is: inject this exosome into the joint cavity 1 to 3 times, 2 to 4 ml / joint (in this embodiment, inject 3 times, with an interval of 1 month between each injection, and 2 ml / joint each time).

[0096] The treatment results were: at six months of follow-up, 83% of patients had improved inflammation compared to before treatment, 86% of patients had improved BPI (Brief Pain Inventory), 76% of patients had improved ODI (Oswestry Disability Index), 72% of patients had improved LEFS (Lower Limb Function Score), and 81% of patients had improved QD (Quick Disability Assessment Scale). All improvements were statistically significant with a p value of less than 0.001. No complications or adverse events were reported in the study, and no patient had accelerated osteoarthritis progression due to the injection of exosomes.

[0097] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. An engineered exosome containing gp96 antigen information, characterized in that: Exosomes are derived from stem cells that have been pretreated with gp96 and then overexpressed the gp96 antigen information.

2. The engineered exosome containing gp96 antigen information according to claim 1, characterized in that: The stem cells are human placental mesenchymal stem cells, gp96 is encoded by the HSP90B1 gene, and the protein sequence number is: P14625.

1.

3. The engineered exosome containing gp96 antigen information according to claim 2, characterized in that: The exosomes are cup-like structures with a particle size of 30-200 nm.

4. A method for preparing engineered exosomes containing gp96 antigen information, characterized in that: The following steps are involved: S1: Isolate the tissue mass containing stem cells and culture the tissue mass containing stem cells with the gp96 antigen complex; S2: Change the medium at the set time, subculture when the cell density reaches the set value, and collect the culture supernatant of the 3rd to 5th generations; S3: Exosomes are separated from the culture supernatant to obtain engineered exosomes containing gp96 antigen information.

5. The method for preparing engineered exosomes containing gp96 antigen information according to claim 4, characterized in that: The gp96 antigen complex is a protein complex derived from human placental tissue. The gp96 antigen complex contains antigen information including ARHGAP33, ACTR5, MAML1, MUC3B, MAMDC4, LRP2, KCNT1, ITPR2, FBXO34, FLRT3, DHCR24, and C1orf115.

6. The method for preparing engineered exosomes containing gp96 antigen information according to claim 4, characterized in that: The specific steps of S1 are as follows: S101: Obtain a fresh intact placenta, wash the placenta, and then remove the amniotic membrane tissue on the placenta; S102: Clean the amniotic membrane tissue and cut it into pieces of 0.5 to 1.0 cm 2 of tissue blocks; S103: Spread the tissue block into a T75 culture flask and place it in a cell culture incubator for 1-3 hours to allow the tissue block to adhere to the wall; S104: Add 10 to 15 mL of serum-free stem cell culture medium supplemented with gp96 antigen complex and culture together with the tissue pieces.

7. The method for preparing engineered exosomes containing gp96 antigen information according to claim 6, characterized in that: The culture medium in S104 includes the following components: 450-475 mL of stem cell serum-free culture medium, 25-50 mL of platelet lysate with a volume fraction of 5-10%, 2.5-10 mL of 0.5-2% penicillin-streptomycin, and the final concentration of the gp96 antigen complex in the culture medium is 3 ug / mL to 15 ug / mL.

8. The method for preparing engineered exosomes containing gp96 antigen information according to claim 1, characterized in that: The medium was changed every 3-5 days in S2. When the density of adherent cells around the tissue block reached 80-90%, the cells were subcultured. Trypsin was used to recover the cells during subculture. The subcultured cells were divided into 1-2.5X10 4 / cm 2 The density of culture was 25cm 2 In the culture flask, the medium was changed every 3-5 days, and the cells were passaged at a ratio of 1:2 or 1:3 when the cell growth density reached 80%-90%.

9. The method for preparing engineered exosomes containing gp96 antigen information according to claim 7, characterized in that: The specific steps of S3 are as follows: S301: Centrifuge the supernatant obtained in S2 at 2-8°C, 300-500g for 5-10 minutes, and take the supernatant; S302: Centrifuge the supernatant obtained in S301 at 2000-4000 g for 10-20 minutes at 2-8°C, and take the supernatant; S303: centrifuging the supernatant obtained in S302 at 2 to 8°C and 10,000 to 13,000 g for 30 to 60 minutes, and taking the supernatant; S304: performing ultrafiltration and concentration treatment on the supernatant obtained in S303 to obtain an ultrafiltrate; S305: centrifuging the ultrafiltrate at 2 to 8°C and 5,000 to 10,000 g for 20 to 30 minutes to obtain a supernatant; S306: The supernatant obtained in S305 is filtered and sterilized to obtain a concentrated solution of engineered exosomes containing gp96 antigen information.

10. An application of engineered exosomes containing gp96 antigen information in the treatment of osteoarthritis, characterized in that: The exosomes are the exosomes according to any one of claims 1 to 3.

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