Bone marrow mesenchymal stem cell exosome and application thereof

Abstract

The invention discloses a bone marrow mesenchymal stem cell exosome and application thereof. The mesenchymal stem cell exosome disclosed by the invention is prepared by treating mesenchymal stem cells through serum containing tamoxifen-resistant traditional Chinese medicine compound active ingredients and a breast cancer drug-resistant cell exosome. The mesenchymal stem cell exosome can be used for treating endocrine drug-resistant breast cancer, especially tamoxifen drug-resistant breast cancer, and the curative effect of the mesenchymal stem cell exosome is superior to that of a traditional Chinese medicine compound administration mode.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and more specifically, to bone marrow mesenchymal stem cell exosomes and applications thereof. Background Art

[0002] Breast cancer is the most common malignant tumor in women, with hormone-dependent breast cancer accounting for the highest proportion, approximately 70%. Due to the strong dependence of breast tumorigenesis on the estrogen-ER axis, estrogen inhibitors and ER antagonists are the main treatments for HR+ breast cancer. Tamoxifen (TAM) remains one of the most effective drugs for the treatment of HR+ breast cancer. It has been used clinically for over 30 years and can reduce the annual mortality rate of breast cancer by 31%. However, approximately 30% to 50% of patients experience recurrence and metastasis due to tamoxifen resistance. Patients' survival time, quality of life, and mental health status have significantly declined, and the burden of disease treatment on families and society has increased sharply. New treatments are urgently needed.

[0003] Research has revealed that breast cancer stem cells (BCSCs), with their self-renewal capacity and multipotential differentiation, play a pivotal role in tamoxifen resistance, acting as the driving cell population for the acquisition of TAM resistance in HR+ breast cancer. Resistance signals can enhance BCSC proliferation, disrupting the balance between breast cancer stem cells and non-stem cells in the tumor microenvironment, leading to tamoxifen resistance. However, the dynamic nature of breast cancer stem cells makes them challenging to target. Exosomes (EXOs), cell-secreted vesicles approximately 50-150 nm in diameter, serve as carriers of intercellular information and regulate the dynamics of breast cancer stem cells within the tumor microenvironment. Mesenchymal stem cells (MSCs), a major component of the tumor microenvironment, are most abundant in bone marrow-derived mesenchymal stem cells (BMMSCs). They are recruited to tumor tissues and participate in reshaping the breast tumor microenvironment. They possess low immunogenicity, high stability, and efficient transport, making them suitable for delivering various genes or anticancer drugs. Exosomes released by BMMSCs can interact with exosomes derived from breast cancer cells, carrying stem cell-associated β-catenin, Wnt5, and Notch signaling proteins to mediate the induction of epithelial-mesenchymal transition and stem cell-like pathways. By transporting stem cell-associated transcription factors SOX2 and SOX9, they disrupt the homeostasis of breast cancer stem cells in the tumor microenvironment, thereby inducing tamoxifen resistance. Therefore, the discovery of new therapeutic approaches to reverse tamoxifen resistance using the BMMSCs / EXO-BCSCs platform holds great research potential. Summary of the Invention

[0004] One object of the present invention is to provide bone marrow mesenchymal stem cell exosomes prepared by treating drug-containing serum and exosomes of breast cancer resistant cells.

[0005] Another object of the present invention is to provide the use of the above-mentioned bone marrow mesenchymal stem cell exosomes in the preparation of drugs for treating endocrine-resistant breast cancer, especially in the preparation of tamoxifen-resistant breast cancer drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides bone marrow mesenchymal stem cell exosomes, wherein the bone marrow mesenchymal stem cell exosomes are prepared by treating bone marrow mesenchymal stem cells with drug-containing serum and exosomes of breast cancer resistant cells;

[0008] The drug-containing serum is the serum obtained by administering the Chinese herbal medicine extract to experimental animals through oral administration;

[0009] The traditional Chinese medicine extract is prepared from the following raw materials in parts by weight: 15-45 parts of astragalus, 9-15 parts of scutellaria barbata, 5-12 parts of epimedium, 9-15 parts of drynaria, 9-15 parts of arisaema, 12-15 parts of lithospermum erythrorhizon, 9-12 parts of gleditsia thorn, and 15-45 parts of artemisia annua.

[0010] Furthermore, the Chinese medicine extract is prepared from the following raw materials in parts by weight: 30 parts of Astragalus, 15 parts of Scutellaria barbata, 9 parts of Epimedium, 12 parts of Drynaria fortunei, 12 parts of Arisaema cinerea, 12 parts of Lithospermum officinale, 9 parts of Gleditsia sinensis, and 18 parts of Artemisia annua.

[0011] The Chinese medicine formula of the present invention, through the rational combination of various Chinese herbs, can effectively treat refractory endocrine-resistant breast cancer. Astragalus membranaceus is the main herb, which uses warming to replenish qi and blood for those with insufficient circulation and sweetness to replenish essence for those with insufficient essence. Endocrine-resistant breast cancer often metastasizes to the bone. Epimedium is pungent, bitter, and slightly warm, and enters the foot Shaoyin kidney and foot Jueyin liver meridians, nourishing tendons and bones, nourishing essence and blood. Together with Drynaria fortunei, it warms and nourishes the liver and kidneys, and can eliminate scrofula. Gleditsia sinensis and Arisaema cinerea resolve phlegm and dissipate stagnation, regulating liver qi. Scutellaria barbata and Lithospermum officinale clear heat and cool blood, detoxify and resolve blood stasis. The auxiliary herb Artemisia annua obtains the spring wood Shaoyang qi the earliest, and treats diseases of the Jueyin liver meridian, Shaoyin kidney meridian, and blood, stopping restlessness and night sweats, and detoxifying and dissipating stagnation. The entire formula works together to nourish the liver and kidneys, regulate Chong and Ren meridians, and detoxify and dissipate stagnation. This study uses active ingredients from traditional Chinese medicine and exosomes secreted by drug-resistant breast cancer cells to treat bone marrow mesenchymal stem cells. This method enhances the ability of these stem cell-derived exosomes to target drug-resistant breast cancer cells and carries anti-resistance signals. Its efficacy in treating tamoxifen-resistant breast cancer is superior to that of traditional Chinese medicine compound treatments.

[0012] Furthermore, the Chinese medicine extract can be prepared by methods known in the art. The present invention illustratively provides a preparation method, comprising: taking Astragalus, Scutellaria barbata, Epimedium, Drynaria fortunei, Arisaema consanguineum, Lithospermum officinale, Gleditsia sinensis and Artemisia annua, adding 5-8 times the total weight of the raw materials in water, soaking for 25-30 minutes, boiling over high heat, then simmering over low heat for 30-90 minutes, filtering, and concentrating the filtrate to obtain the extract.

[0013] Furthermore, the drug-containing serum can be prepared by methods known in the art. The present invention illustratively provides a preparation method comprising: administering the Chinese herbal extract to experimental animals, preferably rats, by gavage 2-3 times a day for 3 consecutive days, collecting arterial blood, and obtaining the drug-containing serum through centrifugation, filtration, and inactivation.

[0014] Furthermore, the breast cancer resistant cell exosomes are LCC9 cell exosomes (human breast cancer anti-estrogen resistant cell line exosomes).

[0015] Furthermore, the LCC9 cell exosomes were prepared by ultracentrifugation. Specifically: LCC9 cells were cultured in LCC9 cell culture medium, and when the cells grew to 80% fusion, the cell supernatant was collected; the cell supernatant was transferred to LCC9 cell exosome-free serum culture medium and continued to be cultured for 48 hours, and the cell supernatant was collected; then, the cells were centrifuged at 4°C, 2000g, for 30 minutes, the precipitate was discarded to remove dead cells, and the supernatant was collected; then, the cells were centrifuged at 4°C, 10000g, for 30 minutes, the precipitate was discarded to remove larger vesicles, the supernatant was collected again, and the cells were filtered with a 0.22μm sterile filter; the filtered supernatant was transferred to an ultracentrifuge tube, and the cells were ultracentrifuged at 4°C, 100000g for 70 minutes, the supernatant was removed, and the precipitate on the tube wall was dissolved with pre-cooled PBS to obtain LCC9 exosomes;

[0016] The LCC9 cell culture medium contains: 87.9% DMEM high glucose medium, 10% fetal bovine serum, 1% 1 mg / mL bovine insulin solution, 1% penicillin-streptomycin solution, and 0.1% 1 mmol / L 4-hydroxytamoxifen solution;

[0017] The LCC9 cell exosome-free serum culture medium contains: 87.9% DMEM high-glucose culture medium, 10% exosome-free serum, 1% 1 mg / mL bovine insulin solution, 1% penicillin-streptomycin double antibody solution, and 0.1% 1 mmol / L 4-hydroxytamoxifen solution.

[0018] Furthermore, the bone marrow mesenchymal stem cell exosomes are prepared by the following method:

[0019] (1) Bone marrow mesenchymal stem cells were obtained, and bone marrow mesenchymal stem cell conditioned medium prepared with drug-containing serum and exosomes from breast cancer resistant cells was used under normoxic conditions to culture the bone marrow mesenchymal stem cells until the fusion rate reached 90%, and the cells were collected and resuspended to obtain bone marrow mesenchymal stem cells with enhanced differentiation ability. The cells were then recultured using bone marrow mesenchymal stem cell culture medium without exosomes for 48 hours. When the cell fusion rate reached 90%, the cell supernatant was collected;

[0020] The bone marrow mesenchymal stem cell conditioned medium contains: 88.9% bone marrow mesenchymal stem cell complete medium, 10% drug-containing serum, 1% penicillin-streptomycin double antibody solution, 0.1% glutamine solution, and 30 μg of breast cancer resistant cell exosomes;

[0021] The exosome-free bone marrow mesenchymal stem cell culture medium contains: 88.9% bone marrow mesenchymal stem cell complete culture medium, 10% exosome-free serum, 1% penicillin-streptomycin double antibody solution, and 0.1% glutamine solution;

[0022] (2) Preparation of bone marrow mesenchymal stem cell exosomes by ultracentrifugation: Take the cell supernatant collected in step (1), centrifuge at 4°C, 500g for 5 minutes, remove the cells, and collect the supernatant; then centrifuge at 4°C, 2000g for 30 minutes to remove the cell debris, and continue to collect the supernatant; centrifuge at 4°C, 10000g for 45 minutes to remove the larger vesicles, filter the supernatant with a 0.45μm sterile filter, and transfer the filtered supernatant to an ultracentrifuge tube; ultracentrifuge at 4°C, 100000g for 70 minutes, and remove the supernatant; resuspend with pre-cooled PBS, ultracentrifuge at 4°C, 100000g for 70 minutes, remove the supernatant, and use pre-cooled PBS to dissolve the tube wall precipitate to obtain bone marrow mesenchymal stem cell exosomes;

[0023] Furthermore, the bone marrow mesenchymal stem cells are human bone marrow mesenchymal stem cells of passage P3-P5.

[0024] In a second aspect, the present invention provides the use of the above-mentioned bone marrow mesenchymal stem cell exosomes in the preparation of a drug for treating endocrine-resistant breast cancer, especially in the preparation of a drug for treating tamoxifen-resistant breast cancer.

[0025] The beneficial effects of the present invention are as follows:

[0026] 1. The present invention provides bone marrow mesenchymal stem cell exosomes prepared by a specific method. The preparation method is simple to operate and has low requirements for instruments and equipment. Ordinary clean benches and high-speed centrifuges can meet the requirements, which is conducive to large-scale production and preparation. Bone marrow mesenchymal stem cells are passaged to P3-P5 for intervention to ensure the purity and quality of the cell source. At the same time, the bone marrow mesenchymal stem cell conditioned culture system is supplemented with "TAM resistant cell line LCC9 cell exosomes" and "traditional Chinese medicine compound drug-containing serum", which can not only provide the nutrients required for bone marrow mesenchymal stem cell culture, but also simulate the tumor microenvironment of drug-resistant people taking traditional Chinese medicine.

[0027] 2. The Chinese medicine formula provided by the present invention uses Astragalus as the "main drug" to nourish qi and blood; Epimedium and Drynaria warm and nourish the liver and kidneys; Gleditsia sinensis and Arisaema resolve phlegm and resolve stagnation; Scutellaria barbata and Lithospermum officinale clear heat and cool blood, detoxify and resolve blood stasis. These six herbs serve as assistant drugs to regulate Chong and Ren meridians and nourish the liver and kidneys; the adjuvant Artemisia annua treats diseases of the Jueyin Liver Meridian, Shaoyin Kidney Meridian, and blood, relieves restlessness and night sweats, and detoxifies and resolves stagnation. The present invention utilizes Chinese medicine components, serum, and exosomes secreted by drug-resistant cell lines to "induce" the anti-drug resistance ability of bone marrow mesenchymal stem cells, with efficacy superior to that of traditional Chinese medicine compound administration methods.

[0028] 3. Traditional Chinese medicine plays a role in the treatment of breast cancer through multiple pathways and multiple targets. The dissolution efficiency and bioavailability of active ingredients in traditional dosage forms still have room for optimization, and the efficacy of the drugs is limited, making it difficult to meet clinical needs. The exosome-based drug delivery system provides a new strategy for the delivery of active molecules of traditional Chinese medicine. This technology can significantly improve the enrichment of traditional Chinese medicine in the lesion site and overcome the constraints of biological barriers on drug absorption. In the present invention, bone marrow mesenchymal stem cell exosomes (BMMSCs / EXO-Resistant loaded with SGYS, BER-SGYS) treated with LCC9 cell exosomes (LCC9 / EXO) and traditional Chinese medicine compound drug-containing serum are used. They can be efficiently enriched in the tumor site, thereby changing the breast cancer tumor microenvironment, reducing tumor heterogeneity, and reversing tamoxifen resistance.

[0029] 4. The bone marrow mesenchymal stem cell exosomes prepared by the present invention are used to treat endocrine-resistant breast cancer. The exosomes secreted by breast cancer-resistant cells can enhance the ability of bone marrow mesenchymal stem cell-derived exosomes to target breast cancer-resistant cells, and synergize with the drug components taken by the patient to treat endocrine-resistant breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The flowchart of the preparation of bone marrow mesenchymal stem cell exosomes of the present invention is shown.

[0031] Figure 2The CCK8 assay is shown to detect the sensitivity of MCF-7 cells, LCC9 cells, and MCF-7 cells treated with BMMSCs exosomes to TAM.

[0032] Figure 3 Representative images and bar graphs of spheroidization experiments are shown for the MCF-7+PBS, LCC9+PBS, and MCF-7+BER-SGYS groups.

[0033] Figure 4 Representative images and bar graphs of Transwell experiments are shown for the MCF-7+PBS, LCC9+PBS, and MCF-7+BER-SGYS groups.

[0034] Figure 5 The graphs show the tumor growth curves of the control group, TAM group, SGYS+TAM group, and BER-SGYS+TAM group.

[0035] Figure 6 Shown are the immunohistochemical staining images of tumor tissues in the control group, TAM group, SGYS+TAM group, and BER-SGYS+TAM group. DETAILED DESCRIPTION

[0036] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0037] Example 1 In vitro test

[0038] 1 Materials and Methods

[0039] 1.1 Experimental cells

[0040] The human breast cancer MCF-7 cell line was purchased from the National Biomedical Laboratory Cell Bank and was authenticated by short tandem repeat (STR).

[0041] Human bone marrow mesenchymal stem cells were purchased from Wuhan Punosai Biotechnology Co., Ltd. (catalog number CM-H166).

[0042] 1.2 Main reagents and consumables

[0043] Duplex medium (Gibco, catalog number 11965-092); fetal bovine serum (FBS) (Gibco, catalog number 16000-044); exosome-free serum (SBI, catalog number EXO FBS-50A-1); 1× PBS buffer (pH 7.2-7.4) (Solarbio, catalog number P1020); bovine insulin (Shanghai Yuanye, catalog number S12033); penicillin-streptomycin (double antibody) (Gibco, catalog number 15140122); 4-hydroxytamoxifen (4-OH TAM) (Sigma, catalog number H7904); bone marrow mesenchymal stem cell complete medium (catalog number: HUXMA-90011); TRIzol (Thermo Fisher, catalog number 15596026); RNase-Free Water (Beyotime, Catalog No. R0022); Trypsin-EDTA (0.05%) (Gibco, Catalog No. 25300054); Cryotubes (Corning, specification: 2 mL); Centrifuge tubes (Corning, specification: 15 mL / 50 mL);

[0044] Culture dish (with holes) (Corning, size 25 cm 2 / 75m 2 ); EP tube (Axygen, specification is 1.5 mL); disposable pipette (Costar, specification is 10 mL).

[0045] 1.3 Main instruments

[0046] MCO-18AC CO2 cell culture incubator (Sanyo, Japan); 26616 ultrahigh-speed centrifuge (ThermoFisher, USA); Centrifuge 5424R conventional centrifuge (Eppendorf, Germany); ECLIPSE Ts2 conventional optical microscope and TS10 fluorescence microscope (Nikon, Japan); ELx808 fully automatic enzyme-labeled detector (Biotek, USA).

[0047] 1.4 Cell culture

[0048] LCC9 cell culture medium contains: 87.9% DMEM high glucose medium, 10% fetal bovine serum, 1% 1 mg / mL bovine insulin solution, 1% penicillin-streptomycin solution, and 0.1% 1 mmol / L 4-hydroxytamoxifen solution. The cells are cultured in a 37°C, 5% CO2 incubator. Medium replacement or subculture is performed according to the cell growth status.

[0049] The culture medium of MCF-7 cells contains: 88% DMEM high glucose medium, 10% fetal bovine serum, 1% 1 mg / mL bovine insulin solution, and 1% penicillin-streptomycin solution. The cells are cultured in a 37°C, 5% CO2 incubator. The medium is changed or the cells are passaged according to the growth status.

[0050] 1.5 As Figure 1 As shown, bone marrow mesenchymal stem cell exosomes were prepared

[0051] (1) Preparation of Chinese herbal medicine extract: 30 g of Astragalus membranaceus, 15 g of Scutellaria barbata, 9 g of Epimedium, 12 g of Drynaria fortunei, 12 g of Arisaema cinerea, 12 g of Lithospermum officinale, 9 g of Gleditsia sinensis, and 18 g of Artemisia annua. Take the above Chinese herbs, add 5-8 times the total weight of the raw materials in water, soak for 25-30 min, boil over high heat, then simmer for 60 min, filter, and concentrate the filtrate to obtain the Chinese herbal medicine extract.

[0052] (2) Preparation of drug-containing serum: 2 mL of the Chinese herbal extract prepared in step (1) was gavaged, and the high-dose group (4 times the clinical equivalent dose) was used to prepare the drug solution. The daily raw drug dosage for 200 g rats was 117 g / 60 kg × 0.2 kg × 6.71 × 4 = 10.4676 g, and the daily gavage volume was 4 mL. The Chinese herbal extract was gavaged at a dose of 2.6169 g / mL to the rats. The frequency of gavage treatment was 2 times a day for 3 consecutive days. After the 5th gavage, the rats were fasted for 10 hours. One hour after the last administration, pentobarbital (40 mg / kg) was injected intraperitoneally. When the rats had no resistance to gastric stimulation, the surgery was started. The rats were placed in a supine position, the abdominal fur was treated with alcohol gauze, the abdominal cavity was opened, the abdominal aorta was fully exposed, and arterial blood was slowly drawn and connected to a blood collection tube. After standing at room temperature for 2 hours, centrifuge at 3000 rpm for 15 minutes at 4°C, take the supernatant, place it in a 50 mL centrifuge tube, filter it, and extinguish it in a water bath at 56°C for 30 minutes to obtain the Chinese medicine compound drug-containing serum, which is divided into 2 mL cryovials, sealed and marked, and stored in a -80°C refrigerator for later use.

[0053] (3) Preparation of LCC9 cell exosomes: LCC9 cells were placed in LCC9 cell culture medium (LCC9 cell culture medium contains: 87.9% DMEM high glucose medium, 10% fetal bovine serum, 1% 1 mg / mL bovine insulin solution, 1% penicillin-streptomycin solution, 0.1% 1 mmol / L 4-hydroxytamoxifen solution), cultured in an incubator, and when the cells grew to 80% confluence, the cell supernatant was collected; the cell supernatant was transferred to LCC9 cell exosome-free serum culture medium (LCC9 cell exosome-free serum culture medium contains: 87.9% DMEM high glucose medium, 10% exosome-free serum, 1% 1 mg / mL bovine insulin solution, 1% Penicillin-streptomycin dual antibody solution, 0.1% 1mmol / L 4-hydroxytamoxifen solution), continued to culture for 48 hours, and the cell supernatant was collected; then centrifuged at 4°C, 2000g for 30 minutes, the precipitate was discarded to remove dead cells, and the supernatant was collected; then centrifuged at 4°C, 10000g for 30 minutes, the precipitate was discarded to remove larger vesicles, the supernatant was collected again, and filtered with a 0.22μm sterile filter; the filtered supernatant was transferred to an ultracentrifuge tube, and ultracentrifuged at 4°C, 100000g for 70 minutes in an ultracentrifuge, the supernatant was removed, and the tube wall precipitate was dissolved with pre-cooled PBS to obtain LCC9 cell exosomes (abbreviated as LCC9 / EXO).

[0054] (4) Preparation of exosomes from human bone marrow mesenchymal stem cells (BMMSCs):

[0055] Human bone marrow mesenchymal stem cells of passage P3-P5 were taken and conditioned medium of bone marrow mesenchymal stem cells prepared with drug-containing serum and LCC9 cell exosomes under normoxic conditions (the bone marrow mesenchymal stem cell conditioned medium contains: 88.9% bone marrow mesenchymal stem cell complete medium, 10% drug-containing serum, 1% penicillin-streptomycin double antibody solution, 0.1% glutamine solution, 30 μg LCC9 cell exosomes, wherein the concentration of LCC9 cell exosomes is 1.21E+10 Particles / mL, and the particle size of LCC9 cell exosomes is 83.6 nm), culturing the bone marrow mesenchymal stem cells to a fusion rate of 90%, collecting and resuspending the cells to obtain bone marrow mesenchymal stem cells with enhanced differentiation ability, and then using an exosome-free bone marrow mesenchymal stem cell culture medium (the exosome-free bone marrow mesenchymal stem cell culture medium contains: 88.9% bone marrow mesenchymal stem cell complete medium, 10% exosome-free serum, 1% penicillin-streptomycin double antibody solution, and 0.1% glutamine solution), re-culturing for 48 hours, and collecting the cell supernatant when the cell fusion rate reaches 90%;

[0056] After collecting the cell supernatant, centrifuge at 4°C, 500g for 5 minutes, remove the cells, and collect the supernatant; then centrifuge at 4°C, 2000g for 30 minutes to remove cell debris, and continue to collect the supernatant; centrifuge at 4°C, 10,000g for 45 minutes to remove larger vesicles, filter the supernatant with a 0.45μm sterile filter, and transfer the filtered supernatant to an ultracentrifuge tube; ultracentrifuge at 4°C, 100,000g for 70 minutes, remove the supernatant; resuspend with 10 mL of pre-cold 1× PBS, ultracentrifuge at 4°C, 100,000g for 70 minutes, remove the supernatant, and use 300μL of pre-cold sterile PBS to dissolve the tube wall precipitate to obtain bone marrow mesenchymal stem cell exosomes.

[0057] The prepared BMMSCs-derived exosomes (BMMSCs / EXO-Resistant loaded with SGYS, BER-SGYS) are exosomes carrying drug resistance and traditional Chinese medicine information after being treated with LCC9 / EXO and traditional Chinese medicine compound drug-containing serum.

[0058] 1.6 CCK8 Experiment

[0059] Three groups were set up in total, namely MCF-7+PBS group, LCC9+PBS group and MCF-7+BMMSCs exosomes (BER-SGYS) group. In the MCF-7+PBS group, MCF-7 cells were treated with 1×PBS buffer for 48 hours; in the LCC9+PBS group, LCC9 cells were treated with 1×PBS buffer for 48 hours; in the MCF-7+BMMSCs exosomes group, MCF-7 cells were treated with 5μg / mL BMMSCs / EXO-Resistant loaded with SGYS (BER-SGYS) for 48 hours. After 48 hours, the three groups were treated with 0, 5, 10, 15, 20, 30, and 50μM TAM culture medium for 48 hours, with 6 replicates in each group and 8×10 cells seeded in each well. 3 Each well was then incubated with 100 μl of cell culture medium, with complete culture medium added to each well. The wells were then placed in a 37°C, 5% CO2 incubator. 10 μl of CCK8 reagent was added to each well, and the cells were then placed in the incubator. After 1 hour, the OD values of each well were measured at 450 nm using an immunoassay microplate reader, and an IC50 value was plotted. The calculation formula was: Cell proliferation rate (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0060] 1.7 Cell spheroidization assay

[0061] Three groups were assigned: the MCF-7 + PBS group, the LCC9 + PBS group, and the MCF-7 + BMMSCs exosome (BER-SGYS) group. The MCF-7 + PBS group treated MCF-7 cells with 1× PBS buffer for 48 hours; the LCC9 + PBS group treated LCC9 cells with 1× PBS buffer for 48 hours; and the MCF-7 + BMMSCs exosome group treated MCF-7 cells with 5 μg / mL of BMMSCs / EXO-Resistant loaded with SGYS (BER-SGYS) for 48 hours. After 48 hours, each of the three groups was treated with 50 μM TAM-containing medium for another 48 hours. The cell sphere formation medium consisted of 20 ng / mL epidermal growth factor, 10 ng / mL fibroblast growth factor, 5 μg / mL insulin, and DMEM / F12 medium. The cells after intervention in the above three groups were collected, centrifuged at 1000 rpm for 5 minutes, resuspended in PBS and counted, and 5×10 cells were seeded per well in a 24-well ultra-low adsorption culture plate. 3 Set up three replicate wells per group and add the prepared cell spheroid formation medium. Observe the cells daily and change the medium every three days. After 14 days, observe under a microscope, photograph, and count the cells.

[0062] 1.8 Transwell assay

[0063] There were three groups in total, namely the MCF-7+PBS group, the LCC9+PBS group and the MCF-7+BMMSCs exosomes (BER-SGYS) group. In the MCF-7+PBS group, 1×PBS buffer was used to intervene in MCF-7 cells for 48 hours; in the LCC9+PBS group, 1×PBS buffer was used to intervene in LCC9 cells for 48 hours; in the MCF-7+BMMSCs exosomes group, 5μg / mL of BMMSCs / EXO-Resistant loaded with SGYS (BER-SGYS) was added to intervene in MCF-7 cells for 48 hours. After 48 hours, the three groups were intervened with 50μM TAM culture medium for 48 hours. After 48 hours of intervention, the cells of the three groups were digested and centrifuged at 1000rpm for 5 minutes. The cells were blown with serum-free culture medium and counted, and the cell concentration was adjusted to 2×10 5 / mL. 100 μL of cell suspension was added to the upper chamber, and 600 μL of culture medium containing 10% fetal bovine serum was added to the lower chamber. The Transwell chamber was gently lowered onto the culture medium. Repeat three times per group and incubate in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The chamber was removed and the upper chamber liquid was aspirated with a pipette. The upper chamber was then transferred to a well containing 800 μL of methanol. After fixation for 30 minutes, the chamber was transferred to a well containing 800 μL of Giemsa dye and stained for 15 minutes. The chamber was rinsed with PBS, the upper chamber liquid was aspirated, and the cells were fixed with 4% paraformaldehyde for 10 minutes. The chamber was then inverted to dry, and crystal violet stain was added for 15 minutes. The upper chamber was rinsed with PBS, and the upper chamber was gently wiped with a cotton swab. After drying, the cells were photographed and counted.

[0064] 1.9 Statistical Methods

[0065] All data obtained in this experiment are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 27.0 software, and figures were generated using GraphPad Prism 9.4.0. Two-sample comparisons were performed using the independent-sample t-test, and multiple-sample comparisons were performed using one-way analysis of variance and multiple comparison tests. Differences were considered statistically significant when P < 0.05.

[0066] 2 Experimental results

[0067] Previous studies have demonstrated that drug-resistant LCC9 / EXO cells can transfer TAM resistance and stem cell-like properties to sensitive MCF-7 cells. LCC9 / EXO-treated MCF-7 cells exhibit TAM resistance consistent with LCC9 cells. Exosomes released by LCC9 / EXO-treated BMMSCs (BMMSCs / EXO-Resistant, BER) carry TAM resistance information and can induce drug resistance in MCF-7 cells. This study used this model.

[0068] 2.1CCK8 verification of BMMSCs exosomes to reverse TAM resistance

[0069] The results showed (see Figure 2), the sensitivity of the three groups to TAM was ranked as LCC9+PBS group (IC50=39.30)>>MCF-7+PBS group (IC50=8.03)>MCF-7+BMMSCs exosome group (BER-SGYS) (IC50=7.71) (P<0.05). The larger the IC50 value, the worse the sensitivity to TAM. The results suggest that the prepared BMMSCs-derived exosomes (BMMSCs / EXO-Resistant loaded with SGYS, BER-SGYS) can inhibit the proliferation of MCF-7 cells carrying drug resistance signals by intervening in exosome crosstalk, and can synergize with TAM to enhance drug efficacy, restore the drug sensitivity level of MCF-7 cells carrying drug resistance signals, thereby reversing TAM resistance.

[0070] 2.2 Cell spheroidization experiments showed that prepared BMMSCs exosomes can reduce the stemness of breast cancer cells

[0071] The results showed that the cell sphere-forming ability of the three groups was LCC9+PBS group (number of cell spheres: 72.33±4.51) > MCF-7+BER-SGYS (number of cell spheres: 10±2.65) > MCF-7+PBS (number of cell spheres: 5±1.73), and there was a statistical difference between the groups (P<0.05). Figure 3 The results indicate that the prepared BMMSCs-derived exosomes (BMMSCs / EXO-Resistant loaded with SGYS, BER-SGYS) reduce the stem cell-like characteristics of MCF-7 cells carrying drug resistance signals, make the tumor sphere-forming ability of MCF-7 cells that receive TAM drug resistance signals close to that of sensitive MCF-7 cells, and restore sensitivity to TAM.

[0072] 2.3 Transwell assay showed that prepared BMMSCs exosomes can reduce the invasive ability of breast cancer cells

[0073] The results showed (see Figure 4 ), the number of cell migration in the three groups was significantly higher in the LCC9+PBS group (304.67±12.42) than in the MCF-7+BER-SGYS group (182.33±27.1) and higher in the MCF-7+PBS group (110±3.6), with statistically significant differences among the groups (P<0.001). These results suggest that the prepared BMMSCs-derived exosomes (BMMSCs / EXO-Resistant loaded with SGYS, BER-SGYS) can reduce the invasive ability of MCF-7 tumors carrying drug resistance signals, thereby reversing TAM resistance.

[0074] Example 2 In vivo experiment

[0075] 1 Materials and Methods

[0076] 1.1 Experimental Animals

[0077] Female NSG mice (SPF grade, lacking mature T / B / NK cells, a highly immunodeficient animal model), 24 mice, with a weight controlled at 16±2 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., batch number: No.110324241100242824, license number: SCXK (Beijing) 2019-0010, and have passed the experimental animal ethics approval of Guang'anmen Hospital, China Academy of Chinese Medical Sciences, approval number: IACUC-GAMY-2022-018.

[0078] 1.2 Main reagents and drugs

[0079] Matrigel (Corning, Catalog No. 356234); Estradiol cypionate (MCE, Catalog No. HY-B1100); Anti-Ki67 (Affinity, Catalog No. AF0198); Paraffin (Leica, Germany, Catalog No. 39601095); Antigen retrieval solution (Beijing Zhongke Wanbang Biotechnology Co., Ltd.); Tamoxifen citrate tablets (Yangtze River Pharmaceutical Group Co., Ltd.).

[0080] The Chinese medicine formula of the present invention, i.e., the herbal formula for soothing the liver and tonifying the kidney (SGYS), is provided by the pharmacy of Guang'anmen Hospital of China Academy of Chinese Medical Sciences and is composed of 30g of Astragalus, 15g of Scutellaria barbata, 9g of Epimedium, 12g of Drynaria fortunei, 12g of Arisaema cinerea, 12g of Lithospermum officinale, 9g of Gleditsia sinensis, and 18g of Artemisia annua.

[0081] The preparation method of bone marrow mesenchymal stem cell exosomes (BER-SGYS) of the present invention is shown in Example 1.

[0082] 1.3 Main instruments

[0083] Dehydrator (JT-12S, Junjie Electronics Co., Ltd., Wuhan); embedding machine (JB-P7, Junjie Electronics Co., Ltd., Wuhan); pathology slicer (Leica, Germany, RM2235); grinder (KZ-Ⅱ, Seville, Wuhan); slice baking machine (DB-B2, Guohua Electric Appliance Co., Ltd., Changzhou).

[0084] 1.4 Animal Modeling and Tumor Preparation

[0085] 24 NSG mice were fed adaptively for 7 days. Estradiolcypionate (1 mg / ml) was injected subcutaneously 3 days before inoculation of MCF-7 cells and then injected once a week according to body weight. A subcutaneous xenograft tumor model was used, and MCF-7 cell suspension (approximately 1×10 7100 μL of cell suspension (100 μg / ml) was mixed with Matrigel at a 1:1 volume ratio and injected into the right hind limb of mice. Each mouse was injected with 200 μL of cell suspension. The mice were observed every 2-3 days, and tumor diameter and body weight were measured. The formula for calculating mouse tumor volume (mm³) is (longest diameter (mm) × width (mm) × shortest diameter (mm)) / 2. To minimize the influence of individual variability on the final results, the mice were randomly divided into four groups according to body weight after tumor formation: control group, TAM group, SGYS+TAM group, and BER-SGYS+TAM group, with 6 mice in each group.

[0086] a. Control group: Oral administration of 0.2 mL of normal saline once a day; injection of 100 μL of PBS into the tumor site twice a week, for a total of 6 times in 3 weeks.

[0087] b. The TAM and SGYS+TAM groups received drugs orally by gavage. The SGYS+TAM group received 0.2 mL of the TCM compound solution by gavage once daily. The equivalent human clinical dose of the TCM compound was calculated as 1 times the human-to-mouse drug dose conversion factor of 9.1. For an adult weighing 60 kg and a 20 g average weight mouse, the daily TCM compound raw drug dosage was 117 g / 60 kg × 0.02 kg × 9.1 × 1 = 0.3549 g. The daily gavage volume was 0.2 mL, and the raw drug concentration was 1.7745 g / mL. For the TAM and SGYS+TAM groups, tamoxifen was ground and diluted with normal saline. The commonly used clinical dose is 10 mg twice daily. Therefore, the mouse dose was converted to 20 mg / 60 kg × 0.02 kg × 9.1 = 0.0606 mg. The daily gavage volume was 0.2 mL twice daily, and the TAM concentration was 0.152 mg / mL. Both groups were injected with 100 μL of PBS at the tumor site twice a week, for a total of 6 times in 3 weeks.

[0088] c. In the BER-SGYS+TAM group, exosomes were injected into the tumor site and tamoxifen was taken orally at a dose of 30 μg / time (100 μL), twice a week for a total of 6 times in 3 weeks. Tamoxifen was ground and administered orally at a concentration of 0.2 mL, 0.152 mg / mL, twice daily.

[0089] The mice were observed every 2-3 days, and the tumor diameter and body weight were measured. After 21 days of intervention, the mice were killed by cervical dislocation, and the subcutaneous tumor tissue was removed. After washing with PBS, the remaining tumor tissue was photographed and recorded. The remaining tumor tissue was stored in a -80℃ refrigerator for subsequent pathological tissue testing.

[0090] 1.5 Immunohistochemistry

[0091] Tumor tissues fixed in 4% paraformaldehyde were removed from the four groups, rinsed three times with PBS, then three times with distilled water, and then transferred to a 30-fold volume of EDTA decalcification solution for decalcification. After decalcification, the tissues were rinsed three times with distilled water. Tumor tissues were dehydrated in the order of 75% ethanol for 2 hours, 85% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, 100% ethanol A for 1 hour, and 100% ethanol B for 1 hour. Tumor tissues were cleared in the order of xylene A for 40 minutes, xylene B for 30 minutes, and xylene C for 20 minutes. Tumor tissues were paraffin-impregnated in the order of 62°C paraffin A for 1 hour, 65°C paraffin B for 1 hour, and 65°C paraffin C for 1 hour.

[0092] Paraffin molds were heated to 60°C, and after embedding the tissue overnight, sections were sliced using a microtome to 4 μm, placed on microscope slides, and dried in an oven set at 62°C for 30 minutes. The sections were then treated sequentially with xylene A for 10 minutes, xylene B for 10 minutes, xylene C for 10 minutes, 100% ethanol for 10 minutes, 95% ethanol for 10 minutes, and 80% ethanol for 10 minutes. The sections were then rinsed repeatedly with distilled water. The sections were then added to EDTA 8.0 fixative and fixed in a microwave oven using the following settings: medium for 8 minutes, off for 8 minutes, medium-low for 7 minutes, and allowed to cool naturally. The sections were then placed in PBS and washed three times for 5 minutes each on a shaker. The slides were then incubated in 3% hydrogen peroxide at room temperature for 30 minutes, and then in PBS and washed three times for 5 minutes each on a shaker. The slides were then spun dry, and the tissue was marked. 3% BSA was added and incubated at room temperature for 30 minutes. The primary antibody was then applied to the marked area and incubated overnight in a humidified chamber at 4°C.

[0093] Remove the slides and rinse them three times with PBS for 5 minutes each. After drying, apply a secondary antibody to the original marked area. Incubate at 37°C for 30 minutes, then rinse three times with PBS for 5 minutes each. Dry the slides and add DAB colorimetric solution to the marked area. A light yellow color indicates positive staining. Rinse with distilled water to terminate color development. Counterstain the slides in the following order: hematoxylin for 3 minutes, rinse, differentiate with 1% hydrochloric acid and alcohol, rinse, rinse with ammonia, rinse, and place in 75% alcohol for 6 minutes, 85% alcohol for 6 minutes, 100% alcohol A for 6 minutes, 100% alcohol B for 6 minutes, and xylene A for 5 minutes. Dehydrate until transparent, air-dry, and mount. Observe the slides under a microscope.

[0094] 1.6 Statistical methods

[0095] SPSS 23.0 statistical software was used for statistical analysis. GraphPad Prism 8 software was used to process the experimental data. Unpaired T-test was used to compare the differences between two groups, and one-way ANOVA was used to compare the differences between multiple groups.

[0096] 2 Experimental results

[0097] 2.1 Growth curve results showed that the BER-SGYS+TAM group could reverse the TAM resistance process

[0098] Three weeks after inoculation of MCF-7 cells, all 24 mice developed tumors, with a tumor formation rate of 100%. They were randomly divided into 7 groups (control group, TAM group, SGYS+TAM group, BER-SGYS+TAM group) according to body weight, with 6 mice in each group. The experimental results showed (see Figure 5 ), after 12 days of intervention, the tumors in the TAM, SGYS+TAM, and BER-SGYS+TAM groups grew slowly compared with those in the control group. The tumor volume in the BER-SGYS+TAM group shrank from the 16th day after intervention. The tumor volume at the time of sampling (mm 3 ) ranked as control group (397.59±24.65) > TAM group (327.3±21.53) > SGYS+TAM group (201.67±20.53) > BER-SGYS+TAM group (110.23±32.67) (n=6 per group, P<0.01). The BER-SGYS+TAM group was the drug-resistant group, and the results showed that this group regained sensitivity to TAM, suggesting that BER-SGYS can reverse TAM resistance. Compared with the traditional SGYS+TAM gavage group, the BER-SGYS+TAM group injected at the tumor site had a better effect in inhibiting tumor growth, suggesting that the new drug delivery method of BER-SGYS+TAM is superior to the traditional drug delivery method.

[0099] 2.2 Immunohistochemical detection of Ki-67 expression in tumor tissues of tumor-bearing mice in each group

[0100] In tumor tissue, Ki-67 is a nuclear protein closely associated with cell proliferation. Its expression level is an important indicator for assessing tumor growth rate, invasiveness, and prognosis. It reflects tumor division activity. A higher Ki67 positivity rate indicates more active tumor cell proliferation and generally indicates a higher degree of malignancy. Figure 6The results showed that the positive rate of Ki-67 was high in the control group, while the positive rate was low in the SGYS+TAM group, TAM group, and BER-SGYS+TAM group. The positive rate of the TAM group was higher than that of the SGYS+TAM group and higher than that of the BER-SGYS+TAM group. This suggests that exosomes derived from BMMSCs (BMMSCs / EXO-Resistant, BER) treated only with LCC9 / EXO can transmit drug resistance information, further promoting drug resistance and progression of breast cancer. However, the exosomes derived from BMMSCs (BMMSCs / EXO-Resistant loaded with SGYS, BER-SGYS) prepared by the present invention can restore the sensitivity of tumors to TAM, thereby reversing tamoxifen resistance and delaying the occurrence and development of tumors. The efficacy is better than that of the traditional gavage group, suggesting that this new drug delivery method can more efficiently deliver drugs to the tumor site.

[0101] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A bone marrow mesenchymal stem cell exosome, characterized in that: The bone marrow mesenchymal stem cell exosomes are prepared by treating bone marrow mesenchymal stem cells with drug-containing serum and exosomes of breast cancer resistant cells; The drug-containing serum is the serum obtained by administering the Chinese herbal medicine extract to experimental animals through oral administration; The traditional Chinese medicine extract is prepared from the following raw materials in parts by weight: 15-45 parts of astragalus, 9-15 parts of scutellaria barbata, 5-12 parts of epimedium, 9-15 parts of drynaria, 9-15 parts of arisaema, 12-15 parts of lithospermum erythrorhizon, 9-12 parts of gleditsia thorn, and 15-45 parts of artemisia annua.

2. The bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that The Chinese medicine extract is prepared from the following raw materials in parts by weight: 30 parts of astragalus, 15 parts of scutellaria barbata, 9 parts of epimedium, 12 parts of drynaria, 12 parts of arisaema, 12 parts of lithospermum officinale, 9 parts of gleditsia thorn, and 18 parts of artemisia annua.

3. The bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that The Chinese medicine extract is prepared by the following method: taking astragalus, Scutellaria barbata, epimedium, drynaria, Arisaema consanguineum, lithospermum officinale, Gleditsia sinensis and Artemisia annua, adding water 5-8 times the total weight of the raw materials, soaking for 25-30 minutes, boiling over high heat, then decocting over low heat for 30-90 minutes, filtering, and concentrating the filtrate to obtain the extract.

4. The bone marrow mesenchymal stem cell exosomes according to claim 1, wherein The drug-containing serum is prepared by the following method: the Chinese herbal medicine extract is administered orally to experimental animals 2-3 times a day for 3 consecutive days, arterial blood is collected, and the drug-containing serum is obtained by centrifugation, filtration, and inactivation.

5. The bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that The breast cancer resistant cell exosomes are LCC9 cell exosomes.

6. The bone marrow mesenchymal stem cell exosomes according to claim 5, characterized in that The LCC9 cell exosomes were prepared by ultracentrifugation.

7. The bone marrow mesenchymal stem cell exosomes according to claim 6, characterized in that The LCC9 cell exosomes are prepared by the following method: LCC9 cells are cultured in an LCC9 cell culture medium, and when the cells grow to 80% confluence, the cell supernatant is collected; the cell supernatant is transferred to an LCC9 cell exosome serum-free culture medium and cultured for 48 hours, and the cell supernatant is collected; then, the cell supernatant is centrifuged at 4°C, 2000g, for 30 minutes, the precipitate is discarded to remove dead cells, and the supernatant is collected; then, the cell supernatant is centrifuged at 4°C, 10,000g, for 30 minutes, the precipitate is discarded to remove larger vesicles, the supernatant is collected again, and the cell exosomes are filtered through a 0.22 μm sterile filter; the filtered supernatant is transferred to an ultracentrifuge tube, and the cell supernatant is removed by ultracentrifugation at 4°C, 100,000g, for 70 minutes, the supernatant is removed, and the precipitate on the tube wall is dissolved with pre-cooled PBS to obtain LCC9 cell exosomes; The LCC9 cell culture medium contains: 87.9% DMEM high glucose medium, 10% fetal bovine serum, 1% 1 mg / mL bovine insulin solution, 1% penicillin-streptomycin solution, and 0.1% 1 mmol / L 4-hydroxytamoxifen solution; The LCC9 cell exosome-free serum culture medium contains: 87.9% DMEM high-glucose culture medium, 10% exosome-free serum, 1% 1 mg / mL bovine insulin solution, 1% penicillin-streptomycin double antibody solution, and 0.1% 1 mmol / L 4-hydroxytamoxifen solution.

8. The bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that The bone marrow mesenchymal stem cell exosomes are prepared by the following method: (1) Bone marrow mesenchymal stem cells were obtained, and bone marrow mesenchymal stem cell conditioned medium prepared with drug-containing serum and exosomes from breast cancer resistant cells was used under normoxic conditions to culture the bone marrow mesenchymal stem cells until the fusion rate reached 90%, and the cells were collected and resuspended to obtain bone marrow mesenchymal stem cells with enhanced differentiation ability. The cells were then recultured using bone marrow mesenchymal stem cell culture medium without exosomes for 48 hours. When the cell fusion rate reached 90%, the cell supernatant was collected; The bone marrow mesenchymal stem cell conditioned medium contains: 88.9% bone marrow mesenchymal stem cell complete medium, 10% drug-containing serum, 1% penicillin-streptomycin double antibody solution, 0.1% glutamine solution, and 30 μg of breast cancer resistant cell exosomes; The exosome-free bone marrow mesenchymal stem cell culture medium contains: 88.9% bone marrow mesenchymal stem cell complete culture medium, 10% exosome-free serum, 1% penicillin-streptomycin double antibody solution, and 0.1% glutamine solution; (2) Preparation of bone marrow mesenchymal stem cell exosomes by ultracentrifugation: Take the cell supernatant collected in step (1), centrifuge at 4°C, 500g for 5 minutes, remove the cells, and collect the supernatant; then centrifuge at 4°C, 2000g for 30 minutes to remove the cell debris, and continue to collect the supernatant; centrifuge at 4°C, 10,000g for 45 minutes to remove the larger vesicles, filter the supernatant with a 0.45 μm sterile filter, and transfer the filtered supernatant to an ultracentrifuge tube; ultracentrifuge at 4°C, 100,000g for 70 minutes, and remove the supernatant; After resuspending with pre-chilled PBS, ultracentrifuge at 4°C, 100,000 g for 70 min, remove the supernatant, and dissolve the precipitate on the tube wall with pre-chilled PBS to obtain bone marrow mesenchymal stem cell exosomes; Preferably, the bone marrow mesenchymal stem cells are human bone marrow mesenchymal stem cells of passage P3-P5.

9. Use of the bone marrow mesenchymal stem cell exosomes according to any one of claims 1 to 8 in the preparation of a drug for treating endocrine-resistant breast cancer.

10. Use of the bone marrow mesenchymal stem cell exosomes according to any one of claims 1 to 8 in the preparation of a drug for treating tamoxifen-resistant breast cancer.

Images