Dendritic cell vaccine preparation and preparation method thereof

Abstract

The invention discloses a dendritic cell vaccine preparation and a preparation method thereof, and the method comprises the following steps: collecting fresh breast cancer tissues, and carrying out serum-free culture on the breast cancer tissues to obtain GREM1 positive triple-negative breast globular cells; performing heat shock and repeated freezing and thawing on the GREM1 positive triple-negative mammary gland globular cells to obtain cell lysate; preparing an immature dendritic cell; controlling the cell lysate to load the immature dendritic cells to obtain a load product; and carrying out induced maturation treatment on the loaded product to obtain the vaccine preparation. The dendritic cell vaccine preparation provided by the invention can trigger immune response in vivo and in vitro to kill triple-negative breast cancer cells causing relapse and metastasis, thereby providing possibility for overcoming the tolerance of triple-negative breast cancer and radically treating the relapse and metastasis of triple-negative breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biological vaccines, and in particular to a dendritic cell vaccine preparation and a preparation method thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a breast cancer tissue immunohistochemical examination result that shows negative estrogen receptor, progesterone receptor and proto-oncogene, because it is associated with high visceral metastasis rate, high risk of early recurrence and poor prognosis. Existing technology, namely conventional treatment methods such as surgery, chemotherapy and radiotherapy, can remove or kill most breast cancers in the body, but there are still some residual breast cancer cells or breast cancer cells that produce drug resistance, so that patients cannot get a long survival period and have a poor prognosis. With the development of molecular biology and cell biology, clinical cancer treatment technology has also been continuously presented and played a good effect, among which cell immunotherapy for cancer has been well developed. The clinical treatment of breast cancer with the patient's autologous immune cells has achieved good results, can effectively remove residual tumor cells in the patient's body, inhibit tumor recurrence and metastasis, and has no clinical toxic side effects. Dendritic cells are the largest antigen presenting cells in the body. They phagocytose and process breast cancer cells in the body, bind to T lymphocytes and present antigen information to T lymphocytes, and the T lymphocytes produce cytotoxic effects on tumor cells. Dendritic cell vaccines have been used clinically to treat breast cancer patients. How dendritic cells can achieve better anti-tumor effects depends entirely on the antigens specifically expressed by the tumor. Therefore, screening and finding tumor-specific antigens is a key link in the function of dendritic cells.

[0003] In summary, some breast cancer vaccines in the prior art cannot effectively and accurately eliminate breast cancer cells, and cannot effectively inhibit the recurrence and metastasis of breast cancer. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a dendritic cell vaccine preparation and a preparation method thereof to solve the problems raised in the above background technology.

[0005] A method for preparing a dendritic cell vaccine preparation, the method comprising the following steps: S1, collecting fresh breast cancer tissue, and culturing the breast cancer tissue in a serum-free manner to obtain GREM1-positive triple-negative breast sphere cells; S2, performing heat shock and repeated freezing and thawing on the GREM1-positive triple-negative mammary gland sphere cells to obtain cell lysates; S3, preparing immature dendritic cells; S4, controlling the cell lysate to load the immature dendritic cells to obtain a loaded product; S5, inducing maturation treatment on the loaded product to obtain a vaccine preparation; Wherein, step S1 is specifically as follows: Fresh breast cancer tissue was collected, soaked in 75% alcohol for 2-3 min, and washed three times with saline containing 2% double antibody, fat tissue and blood vessels were removed, and the remaining tissue was minced to obtain clean tissue; The cleaned tissue is mixed with hyaluronidase and collagenase I in DMEM-F12 digestion medium for digestion, and pipetted with a 1 ml pipette every 15-20 minutes. After digestion, it is filtered and washed with DMEM / F12 containing 10% fetal bovine serum and physiological saline in sequence, and then centrifuged to obtain target cells; The target cells were inoculated in serum-free DMEM / F12 at a concentration of 1000 cells / ml for culture. After 48 hours of culture, half of the medium was replaced until the target cells became spherical and enlarged. The cells were allowed to stand and the supernatant was aspirated. Accutase was added to the precipitate for digestion and centrifugation to obtain a single cell precipitate. The single cell pellet was resuspended in fresh culture medium and inoculated into the same 6-well plate and cultured in a 37° C., 5% CO 2 incubator to obtain GREM1-positive triple-negative breast sphere cells; The serum-free DMEM / F12 contained 10 μg / mL bFGF, 10 μg / mL EGF, 1× B-27, 1× penicillin / streptomycin double antibody, 50 units / mL heparin sodium, 0.2 μM glutamine and 10 μg / mL Y27632.

[0006] According to the method for preparing the dendritic cell vaccine preparation proposed by the present invention, the beneficial effects of the present invention are: The dendritic cell vaccine preparation obtained by the method provided by the present invention can induce an immune response in vivo and in vitro to kill triple-negative breast cancer cells that cause recurrence and metastasis, thereby providing a possibility for overcoming the tolerance of triple-negative breast cancer and radically curing the recurrence and metastasis of triple-negative breast cancer.

[0007] In addition, the method for preparing the dendritic cell vaccine preparation provided by the present invention may also have the following additional technical features: Preferably, the ratio of the hyaluronidase to the collagenase I is 1:2-2.5.

[0008] Preferably, step S2 is specifically as follows: The GREM1-positive triple-negative mammary gland sphere cells were resuspended with 5 mL of RPMI 1640 culture medium and dispensed into 1.5 ml EP tubes, sealed and placed in a 42° C. water bath for heat shock for 2 h, then placed in liquid nitrogen for 10 min, taken out and placed at room temperature for 10 min, and repeatedly frozen and thawed 3 times to obtain cell lysates.

[0009] Preferably, step S3 is specifically as follows: Peripheral blood was collected and centrifuged through Ficoll-Hypaque density gradient to obtain mononuclear cells; The mononuclear cells were resuspended in RPMI 1640 medium and added to 6-well plates to adhere; After the 6-well plate was placed in a 37°C, 5% CO2 incubator for 90 min, the adherent cells were added to complete RPMI 1640 medium for induction culture; After the third day of culture, fresh medium was replaced and culture was continued for 5 days to obtain immature dendritic cells.

[0010] Preferably, the complete RPMI 1640 culture medium includes 5% autologous serum, 1× glutamine, and 100-2000 IU / mL recombinant human FMS-like tyrosine kinase 3 ligand.

[0011] Preferably, step S5 includes: Inducin was added to the loaded product for induction for 24 hours, followed by centrifugation to obtain a mature loaded product, the mature loaded product was washed three times with normal saline and resuspended in normal saline, and then human albumin was added at a mass volume ratio of 2% to obtain a vaccine preparation.

[0012] Preferably, the inducing factors are tumor necrosis factor α and lipopolysaccharide or recombinant human interferon γ.

[0013] On the other hand, the present invention further provides a dendritic cell vaccine preparation, which is prepared by the above-mentioned method for preparing the dendritic cell vaccine preparation.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 A graph showing the relative quantitative expression results of GREM1 gene in triple-negative breast cancer tissues by real-time fluorescence quantitative PCR.

[0017] Figure 2 A diagram showing mammosphere outgrowth cells cultured from triple-negative breast cancer tissue.

[0018] Figure 3 Flow cytogram showing breast cancer stem cell markers detected by flow cytometry in mammosphere cells.

[0019] Figure 4 The figure shows the results of GREM1 gene PCR detection and protein western blot of the obtained mammary gland sphere cells.

[0020] Figure 5 The figure shows the flow cytometric analysis of lymphocyte phenotype activated by DC vaccine loaded with triple-negative mammosphere cell lysate.

[0021] Figure 6 Microscopic observation of dendritic cells loaded with GREM1-positive triple-negative mammary sphere cell lysate and phenotypic flow cytometric analysis of the DC vaccine prepared.

[0022] Figure 7 A graph showing the cytotoxic activity of CTL responses against triple-negative breast cancer sphere cells generated by dendritic cells loaded with GREM1-positive triple-negative breast sphere cell lysate.

[0023] The embodiments of the present invention will be further described below with reference to the accompanying drawings. DETAILED DESCRIPTION

[0024] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the examples. Several embodiments of the present invention are given in the examples. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0025] Example 1 In Example 1 of the present invention, a method for preparing a dendritic cell vaccine preparation is provided. Before the preparation of the vaccine preparation, it is necessary to perform quantitative polymerase chain reaction detection on the target gene of human breast cancer tissue. The specific detection process is as follows: Take 30-50mg breast cancer tissue frozen at -80℃, grind it in liquid nitrogen, add 1mL of tissue lysis buffer, shake and mix thoroughly, tissue lysis buffer: chloroform = 1mL: 200μL, add chloroform according to this ratio, cover and shake for 15 seconds, centrifuge after 2 minutes, speed 12000rpm, centrifuge for 10 minutes, the sample is divided into three layers: red layer organic phase, middle layer and colorless water phase, discard the middle layer and colorless water phase, draw the upper layer into the centrifuge tube, and then proceed to the next step. Add 1 volume of 70% ethanol (prepared with RNase-free water) to the tube, blow and beat evenly with a pipette, add the mixed solution to the adsorption column, centrifuge at 12000rpm / 10 minutes, and discard the waste liquid in the collection tube. Then add 700μL of buffer RW1 to the adsorption column, then centrifuge at 12000rpm / 15 seconds, and discard the waste liquid. Put the adsorption column back into the 2 mL collection tube. Add buffer RW2 (500μL) to the adsorption column, then centrifuge at 12000rpm / 15 seconds and discard the waste liquid. Put the adsorption column back into the 2mL collection tube. Repeat the above steps. Let the adsorption column stand at room temperature for 1-2 minutes, and then proceed to the next step after it is completely dried. Add 1.5 mL of RNase-free water to the adsorption column and place it in a centrifuge tube. Add RNase-free Water (30-50μL) to the middle part of the adsorption column, let it stand at room temperature for 1 minute, then centrifuge at 12000rpm / 15 seconds, and finally collect the extracted RNA solution. Place the RNA solution in a -80℃ refrigerator for later use; Use a spectrophotometer to detect the concentration and purity of the RNA solution, adjust the zero value with RNase-free water that dissolves RNA, and drop 1μL of RNase-free deionized water or RNA sample on the surface of the measuring base. When the droplets form a liquid column on the upper and lower bases, the measurement is completed, and the system will generate various parameters related to RNA concentration and quality; cDNA reverse transcription was performed using the HiScript III first-strand cDNA synthesis kit (+gDNA wiper). The specific steps are as follows: prepare a reaction mixture on ice, i.e. 2 μL of 5×gDNA wiper mixture, 1 μg of total RNA, and 10 μL of RNase-free deionized water. Mix well and incubate at 42°C for 2 minutes. First-strand cDNA synthesis: prepare a reaction mixture, i.e. 10 μL of the previous step reaction solution, 2 μL of 10×RT mixture, 2 μL of HiScript III enzyme mixture, 2 μL of Oligo(dT) 20VN, 2 μL of random primers, and 20 μL of RNase-free deionized water. Mix well and perform reverse transcription. After shaking well, incubate at 37°C for 15 minutes, then incubate at 85°C for 5 seconds. Store the reverse-transcribed cDNA in a -20°C refrigerator for later use. Real-time quantitative PCR sample detection: All cDNA samples were configured with real-time quantitative PCR reaction system. Specific system configuration: 2×AceQ Universal SYBR qPCR Master Mix10uL, forward strand primer 0.4μL, reverse strand primer 0.4 μL, RNase-free deionized water to 18μL, mix the solution, and centrifuge at 5000 rpm for a short time. Add 18μL of the above mixed solution to the corresponding wells of the 96-PCR plate, and then add 2μL of the corresponding cDNA. Seal with sealing film, centrifuge briefly to mix, then put the prepared PCR plate on ice, and then set the PCR program. PCR reaction: Put the prepared 96-PCR plate on the real-time quantitative PCR instrument for PCR reaction. The PCR reaction program is: 95℃ (10min); 45 PCR cycles (95℃, 15s; 60℃ 60s). After the amplification reaction is completed, analyze the product specificity according to (60℃-99℃) to establish the melting curve of the PCR product. The GREM 1 gene was subjected to real-time quantitative PCR reaction, and the internal reference (GAPDH) was used for real-time quantitative PCR reaction. Each sample was tested in 3 wells, and the obtained data were analyzed for relative quantitative expression of genes. Statistical analysis was then performed based on the relative quantitative expression. The specific results are as follows: Figure 1 shown. Figure 1 The relative quantitative expression level of GREM1 gene by real-time fluorescence quantitative PCR was analyzed statistically. There was a statistically significant difference in the gene expression of GREM1 (*P<0.05, ** represents P<0.01, *** represents P<0.001). Figure 1 As shown in the data, the quantitative expression of GREM1 gene in triple-negative breast tissue was higher than that in the adjacent cancer group (P<0.001), and the relative quantitative expression of GREM1 gene in non-triple-negative breast tissue was higher than that in adjacent cancer tissue (P<0.01), indicating that GREM1 gene was specifically expressed in triple-negative breast cancer tissue.

[0026] It should be noted that in this embodiment, the cell culture plates, cell culture dishes, culture bottles, and cell culture bags used are, for example, 75 cm 2 Cell culture flask, 175cm 2 Cell culture equipment (containers) such as cell culture flasks and 250 mL cell culture bags can be used in the present invention, preferably cell culture plates. The present invention has no special restrictions on the freezing medium, but preferably, for example, 50% calf serum, 40% cell culture medium and 10% dimethyl sulfoxide, wherein the cell culture medium is more preferably DC cell culture medium.

[0027] Serum or plasma can be added to the culture medium for culture. The amount of them added to the culture medium is not particularly limited, such as greater than 0% to 10% by volume, and the amount of serum or plasma can be changed according to different culture stages, preferably 5% (volume ratio). For example, the concentration of serum or plasma can be reduced in stages for use. In addition, the source of serum or plasma can be either self (meaning the same source as the cultured cells) or non-self (meaning different from the source of the cultured cells). From the perspective of safety, serum or plasma of self-origin is preferred. In addition, separated and purified serum components such as human serum albumin can also be added.

[0028] The present invention is implemented using various components and culture media. The culture conditions used in the present invention are not particularly limited, and the conditions used in conventional cell culture can be used. For example, the cells can be cultured at 37°C, 5% CO2, etc. The following operations can also be performed: adding fresh culture media at appropriate intervals to dilute the cell culture solution, or replacing the culture media, or replacing the cell culture equipment, etc.

[0029] The preparation method of a dendritic cell vaccine preparation provided in this embodiment 1 comprises the following steps: S1, collecting fresh breast cancer tissue, and culturing the breast cancer tissue in a serum-free manner to obtain GREM1-positive triple-negative breast sphere cells; Wherein, the step S1 comprises: S11. Collect fresh breast cancer tissue, soak it in 75% alcohol for 2-3 minutes, and wash it three times with saline containing 2% double antibody, remove fat tissue and blood vessels, and mince the remaining tissue to obtain clean tissue; Specifically, the fresh breast cancer tissue collected was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University and the patient signed an informed consent. The specific ethics approval number was (2023)CDYFYYLK(08-022). After fineness with physiological saline, 1 gram of the washed tissue was cut into small pieces and completely chopped with sterile ophthalmic scissors to obtain the cleaned tissue.

[0030] S12, mixing the cleaned tissue with hyaluronidase and collagenase I in DMEM-F12 digestion medium for digestion, pipetting with a 1 ml pipette every 15-20 minutes, filtering after digestion and washing with DMEM / F12 containing 10% fetal bovine serum and physiological saline in sequence, and then centrifuging to obtain target cells; Specifically, the ratio of hyaluronidase to collagenase I is 1:2-2.5. 100 units of hyaluronidase and 200-250 units of collagenase I are added to each ml of DMEM-F12 digestion medium. Digestion is carried out at 37°C for 3-4 hours, and pipetting is performed with a 1 ml pipette every 15-20 minutes. After digestion, the single cell suspension is filtered through a 70 μm nylon mesh, washed with DMEM / F12 / 10% FBS, and then washed with physiological saline, and centrifuged at 1200 pm for 10 minutes to obtain the target cells.

[0031] S13, inoculating the target cells at a number of 1000 cells / ml in serum-free DMEM / F12 for culture, changing half of the medium after 48 hours of culture until the target cells become spherical and enlarged, letting the medium stand and absorbing the supernatant, adding accutase enzyme to the precipitate for digestion and centrifugation to obtain a single cell precipitate; The serum-free DMEM / F12 includes 10 μg / mL bFGF, 10 μg / mL EGF, 1× B-27, 1× penicillin / streptomycin double antibody, 50 units / mL heparin sodium, 0.2 μM glutamine and 10 μg / mL Y27632. After culturing for 48 hours, half of the medium is replaced until the target cells become spherical and enlarged. After standing for 5 minutes, the supernatant is aspirated, and 1-2 ml of accutase enzyme is added to the lower sediment for digestion. Digestion is carried out in an incubator at 37°C for 3-5 minutes to form discrete single cells, which are then centrifuged at 1200 rpm for 5 minutes to obtain a single cell precipitate.

[0032] S14, resuspending the single cell precipitate with fresh culture medium and inoculating the cells into the same 6-well plate and continuing to culture in a 37° C., 5% CO 2 incubator to obtain GREM1-positive triple-negative breast sphere cells; After obtaining a single cell pellet, resuspend it with fresh culture medium and inoculate it into a 6-well plate. The fresh culture medium here is fresh serum-free DMEM / F12. Pipette the culture medium containing the suspended beads, centrifuge at 800rpm for 2min, remove the supernatant, add fresh culture medium to resuspend, and inoculate them into the same 6-well plate together, and place it in a 37℃, 5% CO2 incubator for continued culture. Figure 2 As shown in AD, Figure 2 AD in the figure respectively represent the mammosphere growth cell images of triple-negative breast cancer tissue after different culture time. On the third day of culture, obvious cell sphering can be seen. As the culture time increases, the mammosphere gradually becomes larger. When there is a small black spot in the center of the mammosphere, digestion and subculture are performed to prepare GREM1-positive triple-negative mammosphere cells. After obtaining GREM1-positive triple-negative mammary gland sphere cells, some of the GREM1-positive triple-negative mammary gland sphere cells were frozen according to the cell counting results, and the freezing medium was added to the freezing tube, which was placed in a programmed cooling box and placed in a -80°C refrigerator for 24 hours, and then stored in a liquid nitrogen tank for future use.

[0033] S2, performing heat shock and repeated freezing and thawing on the GREM1-positive triple-negative mammary gland sphere cells to obtain cell lysates; Wherein, step S2 is specifically as follows: The GREM1-positive triple-negative mammary gland sphere cells were resuspended with 5 mL of RPMI 1640 culture medium and dispensed into 1.5 ml EP tubes, sealed and placed in a 42° C. water bath for heat shock for 2 h, then placed in liquid nitrogen for 10 min, taken out and placed at room temperature for 10 min, and repeatedly frozen and thawed 3 times to obtain cell lysates.

[0034] Furthermore, after preparing GREM1-positive triple-negative breast sphere cells, cell testing is required, and the testing process is as follows: Take 0.2×10 6 GREM1-positive triple-negative mammary gland sphere cells were stained with CD24-FITC and CD44-PE, or CD133-PE, isotype control mouse IgG1-FITC and mouse IgG1-PE (Biolegend), incubated in a 4°C refrigerator for 30 min, washed once with 1 mL 1×PBS, centrifuged at 2500 rpm for 3 min, discarded the supernatant and washed again with 1 mL 1×PBS, the cell pellet was resuspended in 500 μL flow cytometry buffer, and detected on a flow cytometer (FACS AriaII, BD, USA). Figure 3 Flow cytometry was used to detect the markers of breast cancer stem cells. The test results showed that the cells grown into mammospheres were CD44 + / CD24 - 96.7%, CD133 + It was 75.6%, which was consistent with the specific phenotype of breast cancer stem cells; At the same time, if Figure 4 As shown in A, Figure 4 A in the figure indicates the statistical analysis result of the relative quantitative expression level of GREM1 gene detected by real-time fluorescence quantitative PCR. There is a statistical difference in the gene expression of GREM1 (*P<0.05, ** represents P<0.01, *** represents P<0.001). Figure 4 As shown in A, the quantitative expression of GREM1 gene in mammosphere cells was higher than that in non-triple-negative breast adherent cells and normal breast cells, indicating that cells growing into mammospheres specifically express the GREM1 gene.

[0035] After that, take 2×10 6 GREM1-positive triple-negative mammary gland sphere cells were used to extract protein, namely: adding the prepared protein lysis buffer, lysing at 4°C on a shaker for 10 minutes; scraping the lysed protein with a cell scraper, transferring it to a 1.5mL EP tube, centrifuging at 4°C, 13000rpm, for 10 minutes, and gently aspirating the supernatant into a new EP tube; the protein concentration was determined by the BCA method. Preparation of SDS-PAGE: Prepare the required protein gel concentration according to the molecular weight of the target protein; Loading: Install the electrophoresis device, add 1× electrophoresis buffer pre-cooled at 4°C to the electrophoresis tank, pull out the comb, blow out the remaining gel in the well with a gun, load the sample, and fill the volume difference of each well with 1× loading; Electrophoresis: 80V constant voltage for about 30 minutes, wait for the sample to enter the separation gel, change to 100V, and stop when the sample is 1 cm away from the bottom of the separation gel; Transfer: Take out the gel plate, carefully pry open the plate, cut off the concentrated gel part, soak the separation gel and nitrocellulose membrane in the pre-cooled 1× transfer buffer, and balance on a shaker; Assemble the transfer system according to the "sandwich" structure (blackboard, sponge, filter paper, separation gel, nitrocellulose membrane, filter paper, sponge, white board), and ensure that no bubbles are generated during this process; Put it into the transfer tank and pour in the pre-cooled 1× transfer Buffer, the entire transfer tank was placed in an ice box, constant voltage 100V, 80min; after the transfer, the nitrocellulose membrane was trimmed, 5% skim milk was added, and it was blocked on a shaker at room temperature for 2 hours; after the blocking, the milk was discarded, the nitrocellulose membrane was washed with deionized water, the deionized water was aspirated, mouse anti-human GREM1 monoclonal antibody and rabbit anti-human GADPH monoclonal antibody (Wuhan Tri-Tac) were added at 1:500, and incubated on a shaker at 4°C overnight; the primary antibody was recovered, and the membrane was washed 3 times with 1×TBST, each time for 10min; the washing solution was discarded, and horseradish peroxidase-labeled goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody (Wuhan Tri-Tac) was added at 1:500, and incubated on a shaker at room temperature for 2 hours; the secondary antibody was discarded, and the membrane was washed 3 times with 1×TBST, each time for 10min; the ECL color development solution was prepared, and the color development solution was added to the nitrocellulose membrane, reacted for 30s, and the image was collected, such as Figure 4 As shown in B. Figure 4 In B, it can be observed that GREM1 protein is hardly expressed in normal breast cells MCF-10A, slightly expressed in non-triple-negative breast cancer adherent cells, and highly expressed in triple-negative breast cancer sphere cells, indicating that GREM1 expression is closely related to the malignancy of breast cancer.

[0036] S3, preparing immature dendritic cells; Wherein, the step S3 comprises: S31, collect peripheral blood and centrifuge it through Ficoll-Hypaque density gradient to obtain mononuclear cells; S32, resuspend the mononuclear cells in RPMI 1640 medium and add them to a 6-well plate to adhere to the wall; S33, place the 6-well plate in a 37°C, 5% CO2 incubator for 90 min, then add complete RPMI1640 medium to the adherent cells for induction culture; The complete RPMI 1640 culture medium includes 5% autologous serum, 1× glutamine, and 100-2000 IU / mL recombinant human FMS-like tyrosine kinase 3 ligand, and the non-adherent cells are lymphocytes and need to be collected.

[0037] S34, replacing the culture medium with fresh medium after the third day of culture, and continuing the culture for 5 days to obtain immature dendritic cells; Specifically, the fresh culture medium here contains 1000 IU / mL recombinant human FMS-like tyrosine kinase 3 ligand.

[0038] S4, controlling the cell lysate to load the immature dendritic cells to obtain a loaded product; Specifically, the cell lysate and the immature dendritic cells are loaded in a 37° C., 5% CO 2 incubator for 24 hours to obtain a loaded product.

[0039] S5. Inducing maturation treatment on the loaded product to obtain a vaccine preparation.

[0040] Wherein, step S5 comprises: Inducers were added to the loaded product for induction for 24 hours, followed by centrifugation to obtain a mature loaded product, which was washed three times with normal saline and resuspended in normal saline, followed by addition of 2% human albumin at a mass volume ratio to obtain a vaccine preparation, and after induction, cell lysate loaded mature dendritic cells were obtained.

[0041] Wherein, the inducing factor is tumor necrosis factor α and lipopolysaccharide or recombinant human interferon γ; Specifically, after induction by induction factor, the cell state is as follows Figure 6 As shown in the figure, DCs cells became larger and had protrusions and dendrites under a 10x microscope ( Figure 6 Middle A), the dendritic structure of the cell can be more clearly observed under a 20x objective ( Figure 6Middle B), indicating that DCs cells have obvious dendritic morphology, and the cell lysate loaded with mature dendritic cells was stained with CD86-PE, CD80-PE, CD40-FITC, CD83-PE, CD11c-FITC and HLA-DR-PerCP (BD Company), and the isotype control was stained with mouse IgG1-FITC, mouse IgG1-PE and mouse IgG1-PerCP (Biolegend Company). The experimental method was the same as the above method, and the phenotypic changes of mature dendritic cells loaded with cell lysate were detected by flow cytometry. Figure 6 C- Figure 6 F in the figure is the phenotypic flow cytometric analysis of the DC vaccine prepared by flow cytometry, and the results show that CD11c + / HLA-DR + 93.4%, CD83 + / CD11c + 73.5%, CD86 + / CD11c + 92.1%, CD40 + / CD80 + It was 98.5%, which was consistent with the fact that the cell lysate was loaded with the specific phenotype of mature dendritic cells and highly expressed co-stimulatory molecules.

[0042] Then the level of IFN-γ secreted by cytotoxic T lymphocytes was detected, and the process was as follows: Mixed T lymphocyte activation: (1) Cell lysate was loaded with mature dendritic cells and T lymphocytes at a ratio of 1:5, i.e. 2×10 5 The cells in the vaccine preparation were 1×10 6 T lymphocytes were co-cultured in a 6-well plate (3 mL system) in a 37°C, 5% CO2 incubator.

[0043] (2) T lymphocytes were collected after 24, 48, 72, 96 and 120 hours of co-culture, and the placental blue counts were performed to analyze the proliferation of T lymphocytes.

[0044] T lymphocytes co-cultured for 120 hours were taken for flow cytometry detection, that is, 0.2×10 6 The cells were stained with CD3-FITC, CD4-PE, CD8-PerCP and IFN-γ-APC (Biolegend), and the isotype controls mouse IgG1-FITC, mouse IgG1-PE, mouse IgG1-PerCP and mouse IgG1-APC (Biolegend), respectively. The experimental method was the same as above. Figure 5The flow cytometric analysis of lymphocyte phenotypes of mature dendritic cells loaded with cell lysates was performed by flow cytometry. The control group was T lymphocytes activated by dendritic cells without loading. + 、CD8 + The percentage of double positive cells was 4.04%, CD8 + IFN-γ + The percentage of double positive cells was 0.09%; while the cell lysate loaded mature dendritic cells activated lymphocytes CD3 + 、CD8 + The percentage is 33.6%, CD8 + IFN-γ + The percentage of double-positive cells was 20.3%, and the percentage of IFN-γ produced by mature dendritic cells loaded with cell lysate exceeded that of the control group, which could induce a better cytotoxic response.

[0045] Finally, the vaccine preparation provided by the present invention was subjected to an in vitro tumor killing experiment, as follows: In a 6-well plate, RPMI 1640 containing 5% calf serum was still used as the culture medium. The above-mentioned cell lysate loaded mature dendritic cells and T cells were mixed at a ratio of 1:5 of mature dendritic cells loaded with the above-mentioned antigen epitope polypeptides: T cells, and recombinant human FMS-like tyrosine kinase 3 ligand (final concentration of 1000U / mL) was added. The cells were cultured for 3 days, during which half of the medium (RPMI 1640 culture medium containing 5% calf serum and a final concentration of 1000U / mL Flt3L) was replaced every other day to obtain CTL cells.

[0046] Corresponding breast cancer cells (MCF-7 and GREM1-positive triple-negative mammary gland sphere cells cultured in the above example) were selected as target cells. 51 Cr labeled, i.e., target cells (2×10 6 / mL) by mixing with 300 μCi 51 Cr was incubated in RPMI 1640 medium at 37°C for 2 hours. The labeled target cells were washed three times with 1× PBS and finally resuspended in RPMI 1640 (containing 10% calf serum) to 2×10 5 The concentration of 2×10 4 Each labeled target cell (0.1 mL) was added to a well of a 96-well plate.

[0047] The CTL cells (effector cells) produced above were added to the corresponding wells at effector-target ratios of 2.5:1, 5:1, 10:1, 20:1 and 40:1, respectively, and incubated at 37°C for 4 hours. After incubation, 75 μL of the supernatant was taken and counted using a γ-radiation counter. 51 The percentage of Cr release was calculated according to the following formula.

[0048]

[0049] The number of pulses per minute of spontaneous release was obtained by culturing target cells alone (without effector cells), the number of pulses per minute of maximal release was obtained by culturing target cells alone treated with a final concentration of 2% NP-40 (surfactant, Shanghai Biotechnology), and the number of pulses per minute of detection was obtained by culturing target cells with the addition of effector cells.

[0050] Figure 7 Cell lysates loaded with mature dendritic cells are shown. Figure 7 In A, as the effector-target ratio continued to increase, the CTL response to triple-negative breast cancer spheroid cells also specifically increased, among which cell lysate-loaded mature dendritic cells triggered a more powerful cytotoxic activity response to triple-negative breast cancer spheroid cells, which was significantly stronger than the CTL cells activated by unloaded dendritic cells.

[0051] Figure 7 B in the figure shows the loaded product after induction of maturation, which triggers toxic activity against breast cancer cells MCF-7. With the effector-target ratio of 10:1 and its increase, a specific CTL response was generated against breast cancer cells MCF-7, which had similar cell killing activity to DCs loaded with breast cancer cells MCF-7, and there was a significant difference between the two. However, both were higher than the killing activity of T lymphocytes without DC activation. The results show that the CTL response generated by cell lysate loaded with mature dendritic cells has more specific cell killing activity; And according to Figure 7 It can be seen that cell lysates loaded with mature dendritic cells activated cytotoxic T lymphocytes have a significant killing effect on triple-negative breast glomerular cells and breast cancer cells MCF-7. When the effect-target ratio is 40:1, the killing efficiency reaches more than 80%. Cell lysates loaded with mature dendritic cells are co-cultured with T lymphocytes to activate the proliferation of T lymphocytes, and interferon-γ enzyme-linked immunospot analysis can detect the secretion of interferon-γ by specifically activated T lymphocytes, promote the function of cellular immune response, and produce efficient and long-lasting anti-tumor immune effects, which is very beneficial to improving the efficacy of patients and prolonging their survival.

[0052] Specifically, in another embodiment of the present invention, a dendritic cell vaccine preparation is provided, which is prepared by the preparation method of the dendritic cell vaccine preparation shown in Example 1.

[0053] In summary, the dendritic cell vaccine preparation obtained by the method provided by the present invention can induce an immune response in vivo and in vitro to kill triple-negative breast cancer cells that cause recurrence and metastasis, thereby providing a possibility for overcoming the tolerance of triple-negative breast cancer and radically curing the recurrence and metastasis of triple-negative breast cancer.

[0054] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0055] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a dendritic cell vaccine preparation, characterized in that: The method comprises the following steps: S1, collecting fresh breast cancer tissue, and culturing the breast cancer tissue in a serum-free manner to obtain GREM1-positive triple-negative breast sphere cells; S2, performing heat shock and repeated freezing and thawing on the GREM1-positive triple-negative mammary gland sphere cells to obtain cell lysates; S3, preparing immature dendritic cells; S4, controlling the cell lysate to load the immature dendritic cells to obtain a loaded product; S5, inducing maturation treatment on the loaded product to obtain a vaccine preparation; Wherein, step S1 is specifically as follows: Fresh breast cancer tissue was collected, soaked in 75% alcohol for 2-3 min, and washed three times with saline containing 2% double antibody, fat tissue and blood vessels were removed, and the remaining tissue was minced to obtain clean tissue; The cleaned tissue is mixed with hyaluronidase and collagenase I in DMEM-F12 digestion medium for digestion, and pipetted with a 1 ml pipette every 15-20 minutes. After digestion, it is filtered and washed with DMEM / F12 containing 10% fetal bovine serum and physiological saline in sequence, and then centrifuged to obtain target cells; The target cells were inoculated in serum-free DMEM / F12 at a concentration of 1000 cells / ml for culture. After 48 hours of culture, half of the medium was replaced until the target cells became spherical and enlarged. The cells were allowed to stand and the supernatant was aspirated. Accutase was added to the precipitate for digestion and centrifugation to obtain a single cell precipitate. The single cell pellet was resuspended in fresh culture medium and inoculated into the same 6-well plate and cultured in a 37° C., 5% CO 2 incubator to obtain GREM1-positive triple-negative breast sphere cells; The serum-free DMEM / F12 contained 10 μg / mL bFGF, 10 μg / mL EGF, 1× B-27, 1× penicillin / streptomycin double antibody, 50 units / mL heparin sodium, 0.2 μM glutamine and 10 μg / mL Y27632.

2. The method for preparing the dendritic cell vaccine preparation according to claim 1, characterized in that: The ratio of the hyaluronidase to the collagenase I is 1:2-2.

5.

3. The method for preparing the dendritic cell vaccine preparation according to claim 1, characterized in that: Step S2 is specifically as follows: The GREM1-positive triple-negative mammary gland sphere cells were resuspended with 5 mL of RPMI 1640 culture medium and dispensed into 1.5 ml EP tubes, sealed and placed in a 42° C. water bath for heat shock for 2 h, then placed in liquid nitrogen for 10 min, taken out and placed at room temperature for 10 min, and repeatedly frozen and thawed 3 times to obtain cell lysates.

4. The method for preparing a dendritic cell vaccine preparation according to claim 1, characterized in that: Step S3 is specifically as follows: Peripheral blood was collected and centrifuged through Ficoll-Hypaque density gradient to obtain mononuclear cells; The mononuclear cells were resuspended in RPMI 1640 medium and added to 6-well plates to adhere; After the 6-well plate was placed in a 37°C, 5% CO2 incubator for 90 min, the adherent cells were added to complete RPMI 1640 medium for induction culture; After the third day of culture, fresh medium was replaced and culture was continued for 5 days to obtain immature dendritic cells.

5. The method for preparing the dendritic cell vaccine preparation according to claim 4, characterized in that: The complete RPMI1640 culture medium includes 5% autologous serum, 1× glutamine, and 100-2000 IU / mL recombinant human FMS-like tyrosine kinase 3 ligand.

6. The method for preparing the dendritic cell vaccine preparation according to claim 1, characterized in that: Step S5 includes: Inducin was added to the loaded product for induction for 24 hours, followed by centrifugation to obtain a mature loaded product, the mature loaded product was washed three times with normal saline and resuspended in normal saline, and then human albumin was added at a mass volume ratio of 2% to obtain a vaccine preparation.

7. The method for preparing the dendritic cell vaccine preparation according to claim 6, characterized in that: The inducing factors are tumor necrosis factor α and lipopolysaccharide or recombinant human interferon γ.

8. A dendritic cell vaccine preparation, characterized in that: The dendritic cell vaccine preparation is prepared by the preparation method of the dendritic cell vaccine preparation according to any one of claims 1 to 7.

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