Exosome preparation with treatment function and preparation method thereof

Abstract

The invention provides an exosome preparation with a treatment function and a preparation method thereof, and belongs to the technical field of cell biology. The exosome preparation is prepared by subjecting stem cells to first-stage alpinetin stimulation and second-stage low-intensity magnetic field stimulation, and can effectively inhibit bladder cancer cell proliferation and migration and enhance the sensitivity of bladder cancer cells to chemotherapy drug paclitaxel, so that a novel biological treatment strategy is provided for overcoming bladder cancer chemotherapy drug resistance; the important clinical transformation value is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and in particular relates to an exosome preparation with therapeutic function and a preparation method thereof. Background Art

[0002] Exosomes are a class of nanoscale membrane vesicles secreted by cells. They possess a phospholipid bilayer structure and can carry a variety of bioactive molecules, such as miRNA, mRNA, proteins, and lipids, playing a crucial role in intercellular communication. In the field of cancer treatment, exosomes derived from mesenchymal stem cells (MSC), particularly bone marrow-derived MSCs (BMSC), have begun to be used in disease treatment due to their natural homing ability and ability to regulate the microenvironment. However, the therapeutic effects of naturally occurring exosomes are generally weak, requiring high concentrations to exert their effects. This, coupled with the complexity of exosome extraction, makes them difficult to meet the needs of disease treatment.

[0003] Bladder cancer is one of the most common malignant tumors in the urinary system and currently relies mainly on surgical resection, chemotherapy and immunotherapy. Chemotherapy drugs such as paclitaxel and cisplatin are currently the first-line treatment options, but some patients gradually develop drug resistance during treatment, resulting in decreased efficacy and increased recurrence rate. Therefore, there is an urgent need to explore new adjuvant treatment strategies to reverse tumor resistance and enhance chemotherapy sensitivity. Exosome-based biotherapy is considered a promising solution due to its good tissue targeting and delivery efficiency. However, how to effectively enhance the killing effect of exosomes on tumor cells and their sensitization effect on chemotherapy drugs remains a difficulty and challenge in current research. Summary of the Invention

[0004] The purpose of the present invention is to provide an exosome preparation with therapeutic function and a preparation method thereof, so as to achieve efficient inhibition of bladder cancer and utilize the exosome preparation to enhance the chemotherapy sensitivity of bladder cancer.

[0005] To achieve the above object, the present invention provides the following technical solutions: In the first aspect, the present invention provides an exosome preparation for inhibiting bladder cancer and enhancing the sensitivity of bladder cancer chemotherapy drugs. The exosomes are produced by stem cells after a first-stage stimulation with galangin and a second-stage stimulation with a low-intensity magnetic field. The CAS number of the galangin is 36052-37-6.

[0006] Preferably, the first-stage stimulation with shanjaroline is 2.5-7.5 μM shanjaroline for 24 hours; The low-intensity magnetic field stimulation method is 3-8mT magnetic field intensity, 1Hz frequency stimulation for 2h; The stem cells are bone marrow mesenchymal stem cells.

[0007] Preferably, the method for preparing exosomes comprises the following steps: 1) Cell seeding and culture: Human bone marrow mesenchymal stem cells from passages P3 to P6 were seeded in α-MEM medium containing exosomes and depleted of fetal bovine serum and cultured to 70% to 80% confluence; 2) First-stage stimulation by galangin: The α-MEM medium was discarded, and after washing, serum-free α-MEM medium containing a final concentration of 2.5-7.5 μM shanjiangsu was added and cultured for another 24 h; 3) Two-stage low-intensity magnetic field stimulation: The serum-free α-MEM medium was removed, the cells were washed, and then replaced with serum-free α-MEM medium. The cells were exposed to a low-intensity magnetic field with a magnetic field intensity of 3 to 8 mT and a frequency of 1 Hz for 2 hours. 4) Exosome collection: After the second stage of low-intensity magnetic field stimulation, the cells were cultured under serum-free conditions for 48 hours, and the culture supernatant was collected. The cells were centrifuged at 300 × g for 10 minutes, 2,000 × g for 20 minutes, filtered through a 0.22 μm filter membrane, and ultracentrifuged at 100,000 × g for 70 minutes to precipitate the exosomes. The cells were resuspended in PBS and ultracentrifuged at 100,000 × g for 70 minutes. Finally, the exosomes were resuspended in PBS to obtain the exosome preparation.

[0008] Preferably, the inhibition of bladder cancer is the inhibition of bladder cancer cell proliferation and migration; The enhancing the sensitivity of bladder cancer chemotherapy drugs is enhancing the sensitivity of bladder cancer cells to paclitaxel; The concentration of exosomes in the exosome preparation is 12.5 μg / ml-100 μg / ml.

[0009] In a second aspect, the present invention provides use of the exosome preparation in the preparation of a drug for inhibiting bladder cancer.

[0010] Preferably, the medicament is used to inhibit the growth and migration of bladder cancer cells.

[0011] In a third aspect, the present invention provides the use of an exosome preparation in the preparation of a drug for enhancing the sensitivity of bladder cancer chemotherapy drugs.

[0012] Preferably, the drug is used to enhance the sensitivity of bladder cancer cells to paclitaxel.

[0013] In a fourth aspect, the present invention provides a method for preparing exosomes for enhancing the sensitivity of bladder cancer cells to chemotherapy drugs and inhibiting the growth and migration of bladder cancer cells, the method comprising the following steps: 1) Cell seeding and culture: Human bone marrow mesenchymal stem cells from passages P3 to P6 were seeded in α-MEM medium containing exosomes and depleted of fetal bovine serum and cultured to 70% to 80% confluence; 2) First-stage stimulation by galangin: The α-MEM medium was discarded, and after washing, serum-free α-MEM medium containing a final concentration of 2.5-7.5 μM shanjiangsu was added and cultured for another 24 h; 3) Two-stage low-intensity magnetic field stimulation: The serum-free α-MEM medium was removed, the cells were washed, and then replaced with fresh serum-free α-MEM medium. The cells were exposed to a low-intensity magnetic field with a magnetic field strength of 3 to 8 mT and a frequency of 1 Hz for 2 hours. 4) Exosome collection: After the second stage of low-intensity magnetic field stimulation, the cells were cultured in serum-free conditions for 48 h, and the culture supernatant was collected and centrifuged at 300 × g for 10 min, 2,000 × g for 20 min, filtered through a 0.22 μm filter membrane, and ultracentrifuged at 100,000 × g for 70 min to precipitate the exosomes. The cells were resuspended in PBS and ultracentrifuged at 100,000 × g for 70 min. Finally, the exosomes were resuspended in PBS to obtain exosomes.

[0014] Preferably, the CAS number of the alpinol is 36052-37-6; The chemotherapy drug is paclitaxel.

[0015] The beneficial effects of the present invention are: This invention provides an innovative dual-stimulation method for inducing the secretion of highly active exosomes from bone marrow mesenchymal stem cells (BMSCs) and its application in the treatment of bladder cancer. This method utilizes a first-stage chemical stimulation with 2.5-7.5 μM sanshinol for 24 hours, combined with a second-stage physical stimulation with a low-intensity alternating magnetic field (1 Hz) of 3-8 mT for 2 hours, significantly enhancing the anti-tumor efficacy of the exosomes. Experimental studies have demonstrated that the resulting exosomes (12.5-100 μg / mL) can dose-dependently inhibit the proliferation and migration of bladder cancer cells while significantly enhancing their chemosensitivity. This provides a novel biotherapeutic strategy for overcoming chemoresistant bladder cancer and holds significant clinical translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This figure shows the comparative results of the growth inhibitory effects of different exosomes prepared in Example 1 and Comparative Examples 1-4 on T24 cells after treatment; Figure 2 This is a graph showing the inhibitory effect of the exosomes prepared in Example 1 at different concentrations (12.5, 25, 50, and 100 μg / mL) on the proliferation of T24 bladder cancer cells; Figure 3 This figure shows the comparison of the migration inhibition effects of T24 cells after treatment with different exosomes prepared in Example 1 and Comparative Examples 1-4; Figure 4 The inhibitory effect of exosomes at different concentrations on T24 cells after 6 and 12 hours of action in Example 1; Among them, A is the inhibitory effect under 6 hours, and B is the inhibitory effect under 12 hours; Figure 5 The difference in the inhibitory effect of paclitaxel after 24 h of pretreatment with different exosomes prepared in Example 1 and Comparative Examples 1-4 at different concentrations; Wherein, A is the inhibitory effect of paclitaxel after pretreatment with different exosomes prepared in Example 1 and Comparative Examples 1-4 at 12.5 μg / mL; B is the inhibitory effect of paclitaxel after pretreatment with different exosomes prepared in Example 1 and Comparative Examples 1-4 at 25 μg / mL. DETAILED DESCRIPTION

[0017] Alpinin (CAS No.: 36052-37-6, MedChemExpress (MCE)) is a flavonoid isolated from Alpinia officinalis. Its structural formula is as follows:

[0018] It has anti-tumor, anti-inflammatory, liver-protective, cardiovascular-protective, lung-protective, antibacterial, antiviral, and neuroprotective properties. Regarding stem cells, existing literature suggests it has the ability to activate hair follicle stem cell regeneration. However, there are currently no reports on methods for enhancing the therapeutic potential of mesenchymal stem cells using shanjiangsuan.

[0019] Example 1 This embodiment provides a method for preparing exosomes with high bladder cancer therapeutic activity, comprising the following steps: Step 1: Cell seeding and culture (1) Select P6 human bone marrow mesenchymal stem cells, gently wash them with PBS, and then inoculate the cells into a sterile cell culture dish; (2) Add α-MEM complete medium containing 10% Exo-free FBS (fetal bovine serum without exosomes) and 1% penicillin / streptomycin; (3) Place the cells in a 37°C, 5% CO2 constant temperature incubator and culture them conventionally until the cell confluence reaches about 80%.

[0020] Step 2: First stage induction stimulation (1) Aspirate the complete culture medium and gently wash with PBS to remove serum components; (2) Add serum-free α-MEM medium containing a final concentration of 5 μM galangin and continue incubating the cells for 24 h to complete the first stage of induction stimulation.

[0021] Step 3: Second stage induction stimulation (1) Aspirate the culture medium and gently wash the cells with serum-free α-MEM to remove residual shanjiangsuin; (2) Serum-free α-MEM medium was added and treated with a magnetic field intensity of 5 mT and a frequency of 1 Hz for 2 h to perform the second stage of induction stimulation; Step 4: Exosome secretion and collection (1) After the magnetic field stimulation treatment, the cells were cultured at 37°C and 5% CO2 for 48 hours; (2) Carefully collect the culture supernatant and centrifuge at 300 × g at 4°C for 10 minutes to remove living cells; (3) Transfer the supernatant to a new sterile tube and centrifuge at 2,000 × g, 4°C for 20 minutes to remove cell debris and large particles. (4) The supernatant was sterile filtered using a 0.22 μm PES membrane filter to further remove particles larger than the exosome particle size; (5) Transfer the filtrate into an ultracentrifuge tube and use an ultracentrifuge at 100,000 × g, 4°C for 70 minutes to precipitate the exosomes; (6) Carefully discard the supernatant, resuspend the precipitate in sterile PBS, and use an ultracentrifuge at 100,000 × g, 4°C for 70 minutes to remove impurities and obtain exosomes; (7) Resuspend the exosomes in PBS and store at -80°C.

[0022] Example 2 This embodiment provides a method for preparing exosomes with high bladder cancer therapeutic activity, comprising the following steps: Step 1: Cell seeding and culture (1) Select P3 human bone marrow mesenchymal stem cells, gently wash them with PBS, and then inoculate the cells into a sterile cell culture dish; (2) Add α-MEM complete medium containing 10% Exo-free FBS (fetal bovine serum without exosomes) and 1% penicillin / streptomycin; (3) Place the cells in a 37°C, 5% CO2 constant temperature incubator and culture them conventionally until the cell confluence reaches about 70%.

[0023] Step 2: First stage induction stimulation (1) Aspirate the complete culture medium and gently wash with PBS to remove serum components; (2) Add serum-free α-MEM medium containing a final concentration of 7.5 μM galangin and continue incubating the cells for 24 h to complete the first stage of induction stimulation.

[0024] Step 3: Second stage induction stimulation (1) Aspirate the culture medium and gently wash the cells with serum-free α-MEM to remove residual shanjiangsuin; (2) Serum-free α-MEM medium was added and treated with a magnetic field intensity of 3 mT and a frequency of 1 Hz for 2 h to perform the second stage of induction stimulation; Step 4: Exosome secretion and collection (1) After the magnetic field stimulation treatment, the cells were cultured at 37°C and 5% CO2 for 48 hours; (2) Carefully collect the culture supernatant and centrifuge at 300 × g, 4°C for 10 minutes to remove live cells; (3) Transfer the supernatant to a new sterile tube and centrifuge at 2,000 × g, 4°C for 20 minutes to remove cell debris and large particles. (4) The supernatant was sterile filtered using a 0.22 μm PES membrane filter to further remove particles larger than the exosome particle size; (5) Transfer the filtrate into an ultracentrifuge tube and use an ultracentrifuge at 100,000 × g, 4°C for 70 minutes to precipitate the exosomes; (6) Carefully discard the supernatant, resuspend the precipitate in sterile PBS, and use an ultracentrifuge at 100,000 × g, 4°C for 70 minutes to remove impurities and obtain exosomes; (7) Resuspend the exosomes in PBS and store at -80°C.

[0025] Example 3 Step 1: Cell seeding and culture (1) Select P6 human bone marrow mesenchymal stem cells, gently wash them with PBS, and then inoculate the cells into a sterile cell culture dish; (2) Add α-MEM complete medium containing 10% Exo-free FBS (fetal bovine serum without exosomes) and 1% penicillin / streptomycin; (3) Place the cells in a 37°C, 5% CO2 constant temperature incubator and culture them conventionally until the cell confluence reaches about 70%.

[0026] Step 2: First stage induction stimulation (1) Aspirate the complete culture medium and gently wash with PBS to remove serum components; (2) Add serum-free α-MEM medium containing a final concentration of 2.5 μM galangin and continue incubating the cells for 24 h to complete the first stage of induction stimulation.

[0027] Step 3: Second stage induction stimulation (1) Aspirate the culture medium and gently wash the cells with serum-free α-MEM to remove residual shanjiangsuin; (2) Serum-free α-MEM medium was added and treated with a magnetic field strength of 8 mT and a frequency of 1 Hz for 2 h to perform the second stage of induction stimulation; Step 4: Exosome secretion and collection (1) After the magnetic field stimulation treatment, the cells were cultured at 37°C and 5% CO2 for 48 hours; (2) Carefully collect the culture supernatant and centrifuge at 300 × g, 4°C for 10 minutes to remove live cells; (3) Transfer the supernatant to a new sterile tube and centrifuge at 2,000 × g, 4°C for 20 minutes to remove cell debris and large particles. (4) The supernatant was sterile filtered using a 0.22 μm PES membrane filter to further remove particles larger than the exosome particle size; (5) Transfer the filtrate into an ultracentrifuge tube and use an ultracentrifuge at 100,000 × g, 4°C for 70 minutes to precipitate the exosomes; (6) Carefully discard the supernatant, resuspend the precipitate in sterile PBS, and use an ultracentrifuge at 100,000 × g, 4°C for 70 minutes to remove impurities and obtain exosomes; (7) Resuspend the exosomes in PBS and store at -80°C.

[0028] Comparative Example 1 Step 1: Cell inoculation and culture are the same as in Example 1; Step 2: The first stage of stimulation uses low-intensity magnetic field stimulation, and the conditions are the same as those in Example 1; Step 3: The second stage of stimulation uses drug stimulation, and the conditions are the same as those in Example 1; Step 4: Exosome secretion and collection are the same as in Example 1.

[0029] Comparative Example 2 Step 1: Cell inoculation and culture are the same as in Example 1; Step 2: The first stage of stimulation uses drug stimulation, and the conditions are the same as those in Example 1; In step 3, the second stage of stimulation was not performed, and only serum-free culture was performed for 2 h; Step 4: Exosome secretion and collection are the same as in Example 1.

[0030] Comparative Example 3 Step 1: Cell inoculation and culture are the same as in Example 1; In step 2, the first stage of stimulation was not performed, and only serum-free culture was performed for 24 h; Step 3: The second stage of stimulation uses low-intensity magnetic field stimulation, and the conditions are the same as those in Example 1; Step 4: Exosome secretion and collection are the same as in Example 1.

[0031] Comparative Example 4 Step 1: Cell inoculation and culture are the same as in Example 1; In step 2, the first stage of stimulation was not performed, and only serum-free culture was performed for 24 h; In step 3, the second stage of stimulation was not performed, and only serum-free culture was performed for 2 h; Step 4: Exosome secretion and collection are the same as in Example 1.

[0032] Experimental test 1 This experiment tested the inhibitory effect of exosomes obtained by different methods on bladder cancer growth The exosomes obtained in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 were all prepared into a concentration of 50 μg / ml using culture medium; Bladder cancer T24 cells were digested and collected by centrifugation, resuspended in DMEM medium containing 10% FBS serum, counted, and adjusted to a single cell suspension density of 20,000 cells / ml; Add 100 μl of cell suspension to each well of a 96-well plate, and add PBS to the peripheral wells to reduce evaporation interference; Group design: Control group: untreated T24 cells, 5 replicate wells; Experimental group: exosomes treated in Example 1 (50 μg / ml) + T24 cells, 5 replicate wells; Comparative group 1: exosomes treated with comparative example 1 (50 μg / ml) + T24 cells, 5 replicate wells were set; Comparative group 2: exosomes treated with comparative example 2 (50 μg / ml) + T24 cells, 5 replicate wells were set; Comparative group 3: exosomes treated with comparative example 3 (50 μg / ml) + T24 cells, 5 replicate wells were set; Comparative group 4: exosomes treated with comparative example 4 (50 μg / ml) + T24 cells, 5 replicate wells were set; The inoculated 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere; The original culture medium was discarded and fresh culture medium containing 50 μg / ml exosomes was added to each well according to the group design; After 48 h of continuous culture, 10 μl of CCK-8 reagent was added to each well, gently shaken to mix, and then returned to the incubator for 3 h. The absorbance was read using a microplate reader at a wavelength of 450 nm.

[0033] Experimental results: The results of experimental test 1 are as follows Figure 1 As shown, the OD values of the experimental groups were: control group 1.031, experimental group 0.489, comparative example group 1 0.716, comparative example group 2 0.762, comparative example group 3 0.803, and comparative example group 4 0.8606.

[0034] Data analysis shows that: The experimental group (alpinia officinalis + low-intensity magnetic field two-stage stimulation) had the best inhibitory effect (OD=0.489), which was significantly better than the other groups, confirming that the two-stage stimulation can significantly enhance the anti-tumor activity of exosomes.

[0035] Although the single stimulation groups (Comparative Examples 2 and 3) were better than the non-stimulation group (Comparative Example 4), there were significant differences between them and the experimental group (p<0.01), and the simple addition of their effects was still lower than that of the experimental group, proving that the two-stage stimulation had a synergistic effect.

[0036] Further analysis showed that although the control group 1, which first underwent low-intensity magnetic field stimulation and then galangin stimulation, had better effects than control groups 2 and 3, it was significantly different from the experimental group, indicating that the timing design of the two-stage stimulation is a necessary condition for this technical solution to achieve high therapeutic activity.

[0037] Experimental test 2 This experiment tested the inhibitory effect of different concentrations of exosomes obtained in Example 1 on bladder cancer The exosomes obtained in Example 1 were prepared in culture medium to concentrations of 12.5, 25, 50, and 100 μg / ml; Bladder cancer T24 cells were digested and collected by centrifugation, resuspended in serum-containing medium, counted, and the density was adjusted to 20,000 cells / ml of single-cell suspension; Add 100 μl of cell suspension to each well of a 96-well plate, and add PBS to the peripheral wells to reduce evaporation interference; The inoculated 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere; The original culture medium was discarded, and fresh culture medium containing 12.5, 25, 50, or 100 μg / ml exosomes was added to each well; After 48 h of continuous culture, 10 μl of CCK-8 reagent was added to each well, gently shaken to mix, and then returned to the incubator for 3 h. The absorbance was read using a microplate reader at a wavelength of 450 nm.

[0038] Experimental results The results of experimental test 2 are as follows Figure 2 As shown, the OD values of the experimental groups were: 1.025 for the control group, 0.923 for the 12.5 μg / ml group, 0.729 for the 25 μg / ml group, 0.493 for the 50 μg / ml group, and 0.397 for the 100 μg / ml group.

[0039] The results show that after 48 hours of treatment, the exosomes prepared in Example 1 at a concentration of 12.5 μg / ml can significantly inhibit the growth of bladder cancer cells, showing a clear concentration dependence.

[0040] Experimental test 3 This experiment tested the inhibitory effect of exosomes obtained by different methods on bladder cancer metastasis The exosomes obtained in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 were all prepared into a concentration of 50 μg / ml using culture medium; T24 cells were incubated with medium containing 50 μg / ml exosomes (experimental and control groups 1-4) or medium without exosomes (control group) for 24 hours. The cells were collected by digestion and resuspended in serum-free medium containing 1% FBS to adjust the density to 2×10 5 cells / ml; Add 200 μl of pretreated cell suspension to the upper chamber of the Transwell chamber, add 600 μl of culture medium containing 10% FBS to the lower chamber, and place the Transwell chamber in an incubator to continue culturing for 24 h; The chamber was removed, washed with PBS, fixed with 4% formaldehyde for 10 minutes, and stained with 0.5% crystal violet for 30 minutes; The non-migrated cells in the upper chamber were mechanically removed with a cotton swab, and 10 fields of view (400×) were randomly selected under a microscope to count the average number of penetrating cells in each group.

[0041] Experimental results: The results of experimental test 3 are as follows Figure 3As shown, the average number of penetrating cells in each group was: 162.6 in the control group, 72.6 in the experimental group, 118.4 in the comparative example 1 group, 128.7 in the comparative example 2 group, 134.1 in the comparative example 3 group, and 145.6 in the comparative example 4 group.

[0042] Data analysis shows that: Similar to the results of inhibiting bladder cancer growth, the experimental group (alpinia officinalis + low-intensity magnetic field two-stage stimulation) showed the best inhibitory effect on bladder cancer cell migration, significantly outperforming all other groups. Compared with the control group (162.6), the number of penetrating cells in the experimental group decreased by approximately 55.4%, demonstrating a strong inhibitory effect. This two-stage stimulation confirms that exosomes can significantly enhance the ability of the exosomes to inhibit bladder cancer cell migration.

[0043] Experimental test 4 This experiment tested whether the exosomes obtained in Example 1 can enhance the inhibitory ability of paclitaxel on bladder cancer A. Screening for the appropriate concentration and time for exosome treatment The exosomes obtained in Example 1 were prepared in culture medium to concentrations of 12.5, 25, and 50 μg / ml; Bladder cancer T24 cells were digested and collected by centrifugation, resuspended in serum-containing medium, counted, and the density was adjusted to 20,000 cells / ml of single-cell suspension; Add 100 μl of cell suspension to each well of a 96-well plate, and add PBS to the peripheral wells to reduce evaporation interference; The inoculated 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere; The original culture medium was aspirated and discarded, and fresh culture medium containing 12.5, 25, and 50 μg / ml exosomes was added to each well. A blank group containing culture medium without exosomes was also set up. Continue to culture for 6 h. After 12 h, add 10 μl of CCK-8 reagent to each well, shake gently to mix, and return to the incubator for incubation for 3 h; The absorbance was read at a wavelength of 450 nm using a microplate reader, and the cell viability was calculated as follows: OD450 of the experimental group (control group) - OD450 of the blank group / OD450 of the control group - OD450 of the blank group × 100%.

[0044] B. Detection of the sensitization effect of different exosomes on paclitaxel Bladder cancer T24 cells were digested and collected by centrifugation, resuspended in serum-containing medium, counted, and the density was adjusted to 20,000 cells / ml of single-cell suspension; Add 100 μl of cell suspension to each well of a 96-well plate, and add PBS to the peripheral wells to reduce evaporation interference; The inoculated 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere; The cells were pretreated for 6 h using 12.5 μg / ml of the exosomes obtained in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 and 25 μg / ml of the exosomes obtained in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4; After the treatment, the original culture medium was removed and culture medium containing 5 μM paclitaxel was added, and the treatment was continued for 24 h; Add 10 μl of CCK-8 reagent to each well, shake gently to mix, and then return to the incubator to incubate for 3 hours; The absorbance was read at a wavelength of 450 nm using a microplate reader, and the cell inhibition rate was calculated as 1-(OD450 of the experimental group-OD450 of the blank group / OD450 of the control group-OD450 of the blank group) × 100%.

[0045] The results of experimental test 4A are as follows Figure 4 As shown, from Figure 4 A shows that after 6 h of treatment, 12.5 μg / ml and 25 μg / ml of the exosomes from Example 1 had no significant inhibitory effect on bladder cancer cells T24. However, when the concentration of the exosomes from Example 1 was 50 μg / ml, a significant inhibitory effect began to be produced on bladder cancer cells T24.

[0046] from Figure 4 As shown in Figure B, when treated for 12 hours, only the concentration of 12.5 μg / ml of the exosomes in Example 1 had no inhibitory effect on bladder cancer cell T24. When the concentration was 25 μg / ml and 50 μg / ml, the bladder cancer cell T24 was significantly inhibited.

[0047] Therefore, based on the above results, in order to exclude the interference of exosomes themselves on the inhibitory effect of T24 cells, this study selected exosome concentrations of 12.5 μg / ml and 25 μg / ml for 6 hours for subsequent detection of the effect of exosomes in Example 1 on the sensitization effect of paclitaxel.

[0048] The results of experimental test 4B are as follows Figure 5 As shown, from Figure 5 As can be seen, at a low concentration of 12.5 μg / ml, the experimental group pretreated with only the exosomes from Example 1 exhibited a significant sensitization effect. Specifically, the cell inhibition rate in the experimental group was 44.39%, a significant increase compared to the 28.46% in the control group. In contrast, the cell inhibition rates in Comparative Examples 1 to 4 did not show a significant increase, indicating that the exosomes from Example 1 significantly enhanced the inhibitory effect of paclitaxel on bladder cancer cells at low concentrations.

[0049] from Figure 5 As can be seen in Figure 2, at a lower concentration of 25 μg / ml, the cell inhibition rate in the experimental group further increased to 57.45%, also significantly higher than the 29.29% in the control group. Furthermore, the cell inhibition rate in comparison group 1 was 33.68%, which, while slightly higher than the control group, was much lower than the increase in the experimental group. This indicates that the exosomes from Example 1 still had a significant sensitization effect at a concentration of 25 μg / ml, and their effect was superior to that of the other comparison groups.

[0050] Therefore, the above results show that treatment with the exosomes of Example 1 significantly sensitized bladder cancer cells to paclitaxel, enabling significant inhibition of bladder cancer cells at low concentrations. Therefore, the exosomes prepared by the present invention can be used as sensitizing drugs to achieve higher therapeutic effects at lower doses, thereby reducing drug toxicity and side effects and improving patient tolerance.

Claims

1. An exosome preparation for inhibiting bladder cancer and enhancing the sensitivity of bladder cancer chemotherapy drugs, characterized in that: The exosomes are exosome preparations produced by stem cells after a first-stage stimulation with shanpin and a second-stage stimulation with a low-intensity magnetic field. The CAS number of the shanpin is 36052-37-6.

2. The exosome preparation according to claim 1, characterized in that The first stage of stimulation with shanjaroline is to stimulate the culture with 2.5-7.5 μM shanjaroline for 24 hours; The low-intensity magnetic field stimulation method is 3-8mT magnetic field intensity, 1Hz frequency stimulation for 2h; The stem cells are bone marrow mesenchymal stem cells.

3. The exosome preparation according to claim 2, characterized in that The method for preparing exosomes comprises the following steps: 1) Cell seeding and culture: Human bone marrow mesenchymal stem cells from passages P3 to P6 were seeded in α-MEM medium containing exosomes and depleted of fetal bovine serum and cultured to 70% to 80% confluence; 2) First-stage stimulation by alpinol: The α-MEM medium was discarded, and after washing, serum-free α-MEM medium containing a final concentration of 2.5-7.5 μM shanjiangsu was added and cultured for another 24 h; 3) Two-stage low-intensity magnetic field stimulation: The serum-free α-MEM medium was removed, the cells were washed, and then replaced with fresh serum-free α-MEM medium. The cells were exposed to a low-intensity magnetic field with a magnetic field strength of 3 to 8 mT and a frequency of 1 Hz for 2 hours. 4) Exosome collection: After the second stage of low-intensity magnetic field stimulation, the cells were cultured under serum-free conditions for 48 hours, and the culture supernatant was collected. The cells were centrifuged at 300 × g for 10 minutes, 2,000 × g for 20 minutes, filtered through a 0.22 μm filter membrane, and ultracentrifuged at 100,000 × g for 70 minutes to precipitate the exosomes. The cells were resuspended in PBS and ultracentrifuged at 100,000 × g for 70 minutes. Finally, the exosomes were resuspended in PBS to obtain the exosome preparation.

4. The exosome preparation according to claim 3, characterized in that The inhibition of bladder cancer is to inhibit the proliferation and migration of bladder cancer cells; The enhancing the sensitivity of bladder cancer chemotherapy drugs is enhancing the sensitivity of bladder cancer cells to paclitaxel; The concentration of exosomes in the exosome preparation is 12.5 μg / ml-100 μg / ml.

5. Use of the exosome preparation according to any one of claims 1 to 4 in the preparation of a medicament for inhibiting bladder cancer.

6. The use according to claim 5, characterized in that The drug is used to inhibit the growth and migration of bladder cancer cells.

7. Use of the exosome preparation according to any one of claims 1 to 4 in the preparation of a drug for enhancing the sensitivity of bladder cancer chemotherapy drugs.

8. The use according to claim 7, characterized in that The drug is used to enhance the sensitivity of bladder cancer cells to paclitaxel.

9. A method for preparing exosomes for enhancing the sensitivity of bladder cancer cells to chemotherapy drugs and inhibiting the growth and migration of bladder cancer cells, characterized in that: The method comprises the following steps: 1) Cell seeding and culture: Human bone marrow mesenchymal stem cells from passages P3 to P6 were seeded in α-MEM medium containing exosomes and depleted of fetal bovine serum and cultured to 70% to 80% confluence; 2) First-stage stimulation by alpinol: The α-MEM medium was discarded, and after washing, serum-free α-MEM medium containing a final concentration of 2.5-7.5 μM shanjiangsu was added and cultured for another 24 h; 3) Two-stage low-intensity magnetic field stimulation: The serum-free α-MEM medium was removed, the cells were washed, and then replaced with fresh serum-free α-MEM medium. The cells were exposed to a low-intensity magnetic field with a magnetic field strength of 3 to 8 mT and a frequency of 1 Hz for 2 hours. 4) Exosome collection: After the second stage of low-intensity magnetic field stimulation, the cells were cultured in serum-free conditions for 48 h, and the culture supernatant was collected and centrifuged at 300 × g for 10 min, 2,000 × g for 20 min, filtered through a 0.22 μm filter membrane, and ultracentrifuged at 100,000 × g for 70 min to precipitate the exosomes. The cells were resuspended in PBS and ultracentrifuged at 100,000 × g for 70 min. Finally, the exosomes were resuspended in PBS to obtain exosomes.

10. The method according to claim 9, characterized in that The CAS number of the alpinol is 36052-37-6; The chemotherapy drug is paclitaxel.

Images