Preparation of dendritic cell vaccine for pancreatic cancer and use thereof in treatment of pancreatic cancer

Abstract

The present application relates to the technical field of biomedicine, and relates to the preparation of a dendritic cell vaccine for pancreatic cancer and the use thereof in the preparation of a drug for treating pancreatic cancer. The preparation method comprises the preparation of Panc-1 and AsPC-1 lysates, the preparation of immature dendritic cells, and the preparation of a DC vaccine loaded with the Panc-1 and AsPC-1 lysates.

Description

Preparation of a dendritic cell vaccine for pancreatic cancer and its application in the treatment of pancreatic cancer. Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the preparation of a dendritic cell vaccine for pancreatic cancer and its application in the treatment of pancreatic cancer. Background Technology

[0002] Pancreatic cancer is a highly malignant tumor with a five-year survival rate of less than 7%. It is predicted that by 2030, pancreatic cancer will become the second leading cause of death. Most patients already have local invasion and distant metastasis at the time of initial diagnosis, which is a major factor contributing to the high mortality rate of pancreatic cancer. Currently, surgical resection is the most effective treatment for pancreatic cancer. However, due to the tumor's high invasiveness and metastatic potential, even after surgery, more than 76% of recurrent patients will experience distant metastasis. In addition, pancreatic cancer is relatively insensitive to chemotherapy and radiotherapy.

[0003] In recent years, immunotherapy has been proven to be another important anti-tumor method besides surgery and chemotherapy. Immunotherapy is a treatment method that fights tumors by activating the body's immune system. It can kill tumor cells and control tumor development by stimulating and enhancing the patient's immunity. Currently, immunotherapy for pancreatic cancer mainly includes immune checkpoint inhibitors, adoptive T-cell therapy, oncolytic viruses, and specific immunomodulators. However, clinical trial results show that none of the above immunotherapies have significantly prolonged patients' overall survival or progression-free survival. In comparison, DC vaccines have shown relatively better therapeutic effects in clinical studies of pancreatic cancer, and can prolong patients' overall survival. As the most important antigen-presenting cells in the body, DC vaccines can process and present tumor antigens to the surface of T cells, thereby generating tumor-specific cytotoxic T lymphocytes.

[0004] In existing technologies, there are several reports of using DC vaccines to treat pancreatic cancer. DC vaccines aim to force the presentation of tumor antigens to the patient's immune system. This strategy typically involves using DCs prepared from patient-derived mononuclear cells, loading them with identified tumor antigens or protein lysates, activating them in vitro with cytokines and adjuvants to prepare a DC vaccine, and then reinfusing it into the patient to achieve anti-tumor and antiviral effects. Relevant targets for pancreatic cancer treatment include CLDN18, WT1, MUC1, CEACAM5, and MSLN. To gain a deeper understanding of the differential expression of tumor-associated antigens in pancreatic cancer, we compared the gene transcription levels of CLDN18, WT1, MUC1, CEACAM5, and MSLN in pancreatic cancer tissue and adjacent normal tissue. As shown in Figure 1, the expression of these five genes in pancreatic cancer tissue is significantly higher than in adjacent normal tissue, and the results of database bioinformatics analysis are consistent with literature reports.

[0005] The antigens loaded on dendritic cells (DCs) mainly include tumor-associated antigenic peptides (TAPS) and whole-cell lysates of pancreatic cancer. The main TPS are WT1 and MUC1, while the pancreatic cancer cell lysates include pancreatic cancer cell lysates, lysates of patient pancreatic cancer stem cells, and tumor cells expressing α-Gal epitopes. Studies have shown that using DCs loaded with WT1 or WT1 / MUC1 can significantly prolong the overall survival and progression-free survival of pancreatic cancer patients after surgery. Hanada et al. used WT1-loaded DCs to treat six patients after pancreatic cancer resection, achieving a progression-free survival of 19.9 months and an overall survival of 59 months. Nagai, K et al. used WT1 / MUC1-loaded DCs to treat ten patients after pancreatic cancer resection, achieving a progression-free survival of 17.7 months and an overall survival of 46.4 months. However, two single peptides are relatively limited and cannot cover most of the tumor antigens in pancreatic cancer. Immune escape is one of the key mechanisms leading to tumor development, and antigenic peptides as vaccine antibodies are limited by the patient's MHC type, meaning that not all patients can benefit from them.

[0006] Furthermore, we used the CCLE database to search for the tumor-associated antigens CLDN18 and WT1 of pancreatic cancer patient tumor cell lines, Panc-1 and AsPC-1, which were stably passaged in vitro. ， Gene expression, including MUC1, CEACAM5, and MSLN, was observed in both cell lines, as shown in Figure 2. Using this combination of cell lines as an antibiotic for DC loading resulted in a higher immune response.

[0007] Besides in vitro experiments, pancreatic cancer cells also contain a large number of tumor-associated antigens and specific antigens. Current research has attempted to use modified whole pancreatic cancer cells as vaccine antigens, such as GVAX, a type of pancreatic cancer cell irradiated with gamma rays and transduced with the GM-CSF gene. GVAX can secrete GM-CSF, attracting and activating dendritic cells (DCs), thereby inducing antigen-specific T cell responses. Although this vaccine has shown good safety and tolerability, it has not yet demonstrated superior efficacy compared to existing standard treatments.

[0008] Furthermore, autologous dendritic cells (DCs) were loaded with allogeneic tumor cell lysates, which induced tumor-specific T cell responses in pancreatic cancer patients. This was achieved using HLA-A2. +Using pancreatic cancer cell Panc-1 lysate to load dendritic cells (DCs) can generate a specific T-cell immune response against pancreatic cancer. However, due to the heterogeneity of pancreatic cancer, a single Panc-1 lysate is unlikely to cover a wide range of tumor antigens, and it needs to be used in combination with other tumor lysates or tumor antigen peptides, which may produce a stronger immune response. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing a pancreatic cancer dendritic cell vaccine and its application in the treatment of pancreatic cancer. The specific method is as follows:

[0010] The preparation of a pancreatic cancer dendritic cell vaccine includes the following steps:

[0011] 1) Take equal amounts of Panc-1 and AsPC-1 cells and place them into cryovials, and repeatedly freeze and thaw the cells at low temperature and room temperature.

[0012] 2) The cells were sonicated and centrifuged separately. The supernatant was collected and filtered sterilely. The filtrate was the cell lysate. The protein concentration was detected by the BCA method and the protein concentration was adjusted accordingly.

[0013] 3) Place CD14 + Cell culture was transferred to six-well plates, 2 x 10⁶ cells per well. 5 -2x10 6 One cell, with a volume of 1 mL, was added to a six-well plate with recombinant GM-CSF and recombinant IL-4.

[0014] 4) Place the six-well plate on a clean bench and gently shake it three times in each direction to disperse the cells evenly. Incubate at 37°C and 5% CO2 for 3 days.

[0015] 5) Add recombinant GM-CSF (final concentration 400-4000 IU / mL) and recombinant IL-4 (final concentration 200-1000 IU / mL) to the six-well plate;

[0016] 6) Place the cells in an incubator at 37°C and 5% CO2 for 2 days to obtain immature dendritic cells; add appropriate amounts of Panc-1 and AsPC-1 (final concentrations of 50 μg / mL to 3000 μg / mL) cell lysates and mix with the immature dendritic cells for 6 hours.

[0017] 7) Then add 2 μL of TNF-α (final concentration 400-4000 IU / mL), 2 μL of LPS (final concentration 1-5 μg / mL) and 1 μL of Poly(I:C) (final concentration 0.5-5 μg / mL) to stimulate dendritic cells for 24 hours to induce DC cell maturation. The matured cells are the DC vaccine.

[0018] The DC vaccine prepared using the above technical solution not only stimulates T cells to produce more IFN-γ, but also induces the production of stronger tumor-killing T cells, providing a new approach for the development of DC vaccines for pancreatic cancer. Furthermore, using Panc-1 and AsPC-1 lysates as antigens for the DC vaccine solves the problem of obtaining autologous tumor antigens. Moreover, using Panc-1 and AsPC-1 lysates as antigens for the DC vaccine, this mixed lysate contains a rich and diverse range of tumor antigens, which can prevent tumor immune escape.

[0019] Furthermore, the low temperature mentioned in step 1) is -60℃ to -200℃.

[0020] Furthermore, the ambient temperature mentioned in step 1) is 34℃~38℃.

[0021] Furthermore, the number of freeze-thaw cycles described in step 1) is 3 to 6.

[0022] Furthermore, the ultrasonic fragmentation time described in step 2) is 1 to 10 minutes.

[0023] Furthermore, the centrifugation speed mentioned in step 2) is 1000 to 3000 rpm.

[0024] Furthermore, the centrifugation time described in step 2) is 5 to 20 minutes.

[0025] Furthermore, in step 6), appropriate amounts of the Panc-1 and AsPC-1 cell lysates prepared in step 1 are added respectively. These lysates can be Panc-1 or Panc-1 and AsPC-1 cell lysates.

[0026] Furthermore, in step 6), the final concentration of the prepared Panc-1 and AsPC-1 cell lysis mixture protein added is 50 μg / mL to 3000 μg / mL.

[0027] The application of a pancreatic cancer dendritic cell vaccine as described in any of the preceding items in the treatment of pancreatic cancer.

[0028] In summary, this application includes at least one of the following beneficial technical effects regarding the preparation of a pancreatic cancer dendritic cell vaccine and its application in the treatment of pancreatic cancer:

[0029] In application, the prepared DC vaccine not only stimulates T cells to produce more IFN-γ, but also induces the production of stronger tumor-killing T cells, providing a new approach for the development of DC vaccines for pancreatic cancer. Furthermore, using Panc-1 and AsPC-1 lysates as antigens for the DC vaccine solves the problem of obtaining autologous tumor antigens. Moreover, using Panc-1 and AsPC-1 lysates as antigens for the DC vaccine, this mixed lysate contains a rich and diverse range of tumor antigens, which can prevent tumor immune escape. Attached Figure Description

[0030] Figure 1 shows a comparison of the expression of five genes in cancerous and adjacent tissues of pancreatic cancer;

[0031] Figure 2 shows the expression levels of the five genes in the Panc-1 and AsPC-1 cell lines;

[0032] Figure 3 shows the CD3 mainly provided in this application. + The level of INFγ produced by T cells;

[0033] Figure 4 shows the CD3 mainly provided in this application. + T cell killing activity against pancreatic cancer cells (Panc-1). Detailed Implementation

[0034] To facilitate a clear understanding of the technical means, creative features, objectives, and effects of this invention, the invention will be further described below in conjunction with specific embodiments.

[0035] The present application will be further described in detail below with reference to Figures 3-4.

[0036] This application discloses the preparation of a dendritic cell vaccine for pancreatic cancer and its application in the treatment of pancreatic cancer.

[0037] Example 1

[0038] The preparation of a pancreatic cancer dendritic cell vaccine includes the following steps:

[0039] 1) Take equal amounts of Panc-1 and AsPC-1 cells and place them into cryovials. Repeatedly freeze and thaw these cells at low and room temperatures. The low temperature is -60℃ to -200℃, preferably -180℃. The room temperature is 34℃ to 38℃, preferably 37℃. The number of freeze-thaw cycles is 3 to 6, preferably 5.

[0040] 2) The cells are sonicated and centrifuged separately. The supernatant is collected and filtered sterilely. The filtrate is the cell lysate. The protein concentration is determined using the BCA method, and the protein concentration is adjusted accordingly. The sonication time is 1–10 minutes, preferably 5 minutes. The centrifugation speed is 1000–3000 rpm, preferably 1500 rpm. The centrifugation time is 5–20 minutes, preferably 10 minutes.

[0041] 3) Place CD14 + Cell culture was transferred to six-well plates, 2 x 10⁶ cells per well. 5 -2x10 6 One 1 mL sample of cells was added to a six-well plate containing recombinant GM-CSF and recombinant IL-4. GM-CSF is a granulocyte-macrophage colony-stimulating factor, a cytokine secreted by various cell types (such as T cells, macrophages, and endothelial cells), which plays an important regulatory role in the production and function of blood cells. IL-4 is an important cytokine belonging to the interleukin family. It is secreted by activated T helper cells (mainly Th2 cells), mast cells, basophils, and certain types of dendritic cells.

[0042] The final concentration of recombinant GM-CSF is 400–4000 IU / mL, preferably 2000 IU / mL. The final concentration of recombinant IL-4 is 200–1000 IU / mL, preferably 800 IU / mL.

[0043] 4) Place the six-well plate on a clean bench and gently shake it three times in each direction to disperse the cells evenly. Incubate at 37°C and 5% CO2 for 3 days.

[0044] 5) Add recombinant GM-CSF and recombinant IL-4 to the six-well plate.

[0045] 6) Place the cells in an incubator at 37°C and 5% CO2 for 2 days to obtain immature dendritic cells; add appropriate amounts of Panc-1 and AsPC-1 cell lysates and mix them with the immature dendritic cells for 6 hours.

[0046] Add an appropriate amount of prepared Panc-1 and AsPC-1 cell lysates. These lysates can be either Panc-1 or a mixture of Panc-1 and AsPC-1 cell lysates. Preferably, the lysates are a mixture of Panc-1 and AsPC-1 cell lysates. The final concentration of the protein in the Panc-1 and AsPC-1 cell lysate mixture is 50–3000 μg / mL, preferably 1500 μg / mL.

[0047] 7) Then, add TNF-α (final concentration 400-4000 IU / mL), LPS (final concentration 1-5 μg / mL) and Poly(I:C) (final concentration 0.5-5 μg / mL) to stimulate dendritic cells for 24 hours to induce DC cell maturation. The matured cells are the DC vaccine.

[0048] Preferably, the dosage of TNF-α is 2 μL with a final concentration of 2000 IU / mL, the dosage of LPS is 1 μL with a final concentration of 3 μg / mL, and the dosage of Poly(I:C) is 1 μL with a final concentration of 3 μg / mL.

[0049] LPS is a complex molecule found in the cell walls of Gram-negative bacteria and is an important endotoxin. LPS is significant in immunology and microbiology, primarily because it strongly activates the immune system and triggers an inflammatory response. Preferably, the final concentration of LPS is 2 μg / mL.

[0050] Poly(I:C) is a long double-stranded RNA molecule composed of hypoxanthine and cytidine. This structure mimics the double-stranded RNA of certain viruses, thus effectively activating the immune system and being used to study immune responses. Preferably, the final concentration of Poly(I:C) is 1 μg / mL.

[0051] Isolation of peripheral blood mononuclear cells:

[0052] Step 1: Add 4.5 mL of [unspecified ingredient] to each of the two centrifuge tubes. Paque Plus solution;

[0053] Step 2: Use a pipette to draw blood from the pancreatic cancer patient and slowly inject it along the wall of the centrifuge tube. The upper layer of Paque Plus solution, 10 mL per tube. Centrifuge at 800g for 20 min at room temperature; Paque Plus solution is a density gradient medium commonly used for cell isolation. It is frequently used in laboratory techniques such as cell isolation and blood sample processing, particularly in immunology and cell biology research.

[0054] Step 3: Remove the centrifuge tube. The sample is divided into four layers, from top to bottom: plasma, mononuclear cells, Ficoll solution, red blood cells, and granulocytes.

[0055] Step 4: Carefully aspirate the mononuclear cell layer and transfer it to a 15 mL centrifuge tube. Add PBS / 1% FBS solution to bring the volume to 14 mL and mix thoroughly. Centrifuge at 800 g for 5 min at room temperature. In the prior art, PBS / 1% FBS solution is a commonly used cell processing and experimental buffer designed to maintain the physiological state and activity of cells and reduce possible cell damage during experiments.

[0056] Step 5: Discard the supernatant, gently tap the bottom of the tube to loosen the cells, and resuspend the cells in 14 mL of PBS / 1% FBS solution. Centrifuge at 700g for 5 min at room temperature.

[0057] Step 6: Discard the supernatant and gently tap the bottom of the tube to loosen the cells. Resuspend the cells in 14 mL of RPMI / 10% FBS solution. Centrifuge at 400 g for 5 min at room temperature. In the prior art, RPMI / 10% FBS solution is used to resuspend the cells. Here, "RPMI" refers to RPMI 1640 medium, and "10% FBS" refers to the medium supplemented with 10% fetal bovine serum.

[0058] Step 7: Remove the supernatant and gently tap the bottom of the tube to loosen the cells. Resuspend the cells in 10 mL of RPMI / 10% FBS solution, mix well by pipetting, and count the cells.

[0059] Step 8: Centrifuge at 700g for 5 min at room temperature, discard the supernatant, and add an appropriate amount of PBS / 1% FBS for subsequent experiments. Isolate and obtain CD14. + Mononuclear cells: EasySep was used in this embodiment. TM CD14 positive selection kit.

[0060] Step 9: Transfer the mononuclear cell population isolated from peripheral blood to a 5 mL flow cytometer tube;

[0061] Step 10: Add an appropriate amount of selection cocktail solution to a final concentration of 100 μL / mL, mix thoroughly by pipetting, and incubate at room temperature for 10 min; In the prior art, selection cocktail solution is a solution used to select or enrich specific cell populations.

[0062] Step 11: Prepare CD14 anode magnetic beads and use RapidSphere. TM The solution is vortexed for 30 seconds to ensure uniform dispersion of the magnetic beads; in existing technologies, RapidSphere... TM A solution is a commercially available reagent or solution for cell separation and enrichment. It is a density gradient separation medium designed to provide rapid and efficient cell separation solutions.

[0063] Step 12: Add an appropriate amount of RapidSphere to the flow cytometer.TM The solution was brought to a final concentration of 100 μL / mL, thoroughly mixed by pipetting, and incubated at room temperature for 3 min.

[0064] Step 13: Add an appropriate amount of PBS / 2% FBS with 1mM EDTA solution to the flow cytometer until the total volume is 2.5mL, and mix thoroughly by pipetting. In the prior art, PBS / 2% FBS with 1mM EDTA solution is a commonly used buffer solution in cell biology research, mainly used for cell processing and separation.

[0065] Step 14: Insert the flow meter vertically into EasySep TM magnet, incubate at room temperature for 3 minutes; in existing technologies, EasySep TM A magnet is a device used for cell separation, designed to efficiently and easily separate and enrich specific types of cells.

[0066] Step 15: Invert the magnet and collect the cell fluid flowing from the flow cytometry tube into a 15mL centrifuge tube. Keep the magnet inverted for 3 seconds. Do not shake or suck up the liquid on the tube wall.

[0067] Step 16: Position the magnet upright and remove the flow cytometer.

[0068] Step 17: Repeat steps 15 and 16 twice.

[0069] Step 18: Add 2 mL of RPMI / 10% FBS to a flow cytometry tube to resuspend the cells, stain with Trypan Blue, and count the cells.

[0070] Induction of immature dendritic cells:

[0071] Step 1: Place CD14 + Cell lysate was transferred to six-well plates, 2 × 10⁶ cells per well. 5 -2×10 6 One cell, 1 mL in volume, is added to a six-well plate with recombinant GM-CSF and recombinant IL-4. The final concentration of recombinant GM-CSF is 400-4000 IU / mL, preferably 2000 IU / mL. The final concentration of recombinant IL-4 is 200-1000 IU / mL, preferably 800 IU / mL.

[0072] Step 2: Place the six-well plate on a clean bench and gently shake it three times in each direction to disperse the cells evenly. Incubate at 37°C and 5% CO2 for 3 days.

[0073] Step 3: Add recombinant GM-CSF and recombinant IL-4 to the six-well plate. The final concentration of recombinant GM-CSF is 400-4000 IU / mL, preferably 2000 IU / mL. The final concentration of recombinant IL-4 is 200-1000 IU / mL, preferably 800 IU / mL.

[0074] Step 4: Place the cells in an incubator at 37°C and 5% CO2 for 2 days to obtain immature dendritic cells.

[0075] Preparation of cell lysates:

[0076] Step 1: Resuspend Panc-1 and AsPC-1 separately in RPMI 1640 at a density of 5 × 10⁻⁶. 6 / mL, with a volume of 1mL.

[0077] Step 2: Freeze the cells in liquid nitrogen for 20 seconds, and then immediately thaw the cells completely in a 37°C water bath.

[0078] Step 3: Repeat step 2 5 times.

[0079] Step 4: Centrifuge the cells that have undergone repeated freeze-thaw cycles, collect the supernatant, and test the protein concentration to prepare for loading DC cells.

[0080] Induction of mature dendritic cells:

[0081] Immature DC cells were divided into four groups: unloaded antigen group, Panc-1 lysate loaded group, AsPC-1 lysate loaded group, and Panc-1 and AsPC-1 lysate loaded group.

[0082] Different groups of antigens were added to the corresponding immature dendritic cells, and the final concentration of Panc-1 lysate histone alone was 20-200 μg / mL. Preferably, the final concentration of Panc-1 lysate histone alone was 100 μg / mL.

[0083] The final concentration of each individually loaded AsPC-1 lysate histone is 20-200 μg / mL, preferably 120 μg / mL. The final concentration of the mixed protein loaded with Panc-1 and AsPC-1 lysates is 20-200 μg / mL each, preferably 90 μg / mL each.

[0084] After culturing for 6 hours, dendritic cells were stimulated for 24 hours with Poly(I:C) (0.5-5 μg / mL) containing TNF-α (final concentration 400-4000 IU / mL), LPS (final concentration 1-5 μg / mL), and LPS, to induce DC cell maturation. These mature cells are the DC vaccine. Preferably, the final concentration of TNF-α is 2000 IU / mL, the final concentration of LPS is 3 μg / mL, and the final concentration of Poly(I:C) is 2 μg / mL.

[0085] Example 2

[0086] Functional verification of DC cells

[0087] Preparation of T cells:

[0088] Step 1: PBMC cell counting, centrifuge at 300g for 5 min, discard the supernatant, and resuspend the cells in PBS / 1% FBS to a concentration of 1×10⁻⁶ cells / mL. 8 / mL, transfer to a 5mL sterile flow cytometer tube. The volume added to the flow cytometer tube at one time should be between 0.1-2mL.

[0089] Step 2: Following the instructions of the magnetic sorting kit, add an appropriate volume of Selection Cocktail, mix thoroughly, and incubate at room temperature for 5 minutes. In existing technologies, Selection Cocktail can promote cell growth.

[0090] Step 3: Dextran RapidSpheres TM Vortex for 30 seconds, then add the corresponding volume of Dextran RapidSpheres according to the sorting kit instructions. TM Mix thoroughly and incubate at room temperature for 3 minutes. In the prior art, Dextran RapidSpheres... TM These are magnetic beads used in magnetic separation technology. These beads are typically coated with dextran and have a magnetic core. This design allows them to be used in a variety of biological separation and purification applications.

[0091] Step 4: Add PBS / 2% FBS with 1mM EDTA solution to the flow cytometry tube until the total volume is 2.5mL. Mix thoroughly and then insert the flow cytometry tube vertically into EasySep. TM Magnet, incubate at room temperature for 3 minutes. EasySep TM Magnet is a magnetic rack device used for cell sorting, which separates specific types of cells from a mixed cell population using magnetic separation technology.

[0092] Step 5: Invert the magnet and collect the cell effluent from the flow cytometry tube into a 15mL centrifuge tube. Keep the magnet inverted for 3 seconds, without shaking or sucking up the liquid on the tube wall; turn the magnet upright and remove the flow cytometry tube.

[0093] Step 6: Repeat steps 4 and 5 twice.

[0094] Step 7: Add 2 mL of RPMI / 10% FBS to the flow cytometry tube to resuspend the cells for counting and subsequent experimental procedures.

[0095] DC in vitro activation of CD3 + T cells:

[0096] The experiment was divided into four groups: negative control group (DCs without antigen loading), experimental group 1 (DCs loaded with Panc-1 lysate), experimental group 2 (DCs loaded with Panc-1 lysate), and experimental group 3 (DCs loaded with Panc-1 and AsPC-1 lysate), with three replicates per group. The DC density was adjusted to 2 × 10⁻⁶ cells / well. 5 / mL, adjust autologous CD3 + T lymphocyte density reached 2 × 10⁻⁶ 6 / mL.

[0097] First put CD3 + T cells were seeded in 96-well plates, and then DCs were added at a ratio of 1:10 (DC:T) at D0, D7, D14, and D21, respectively, along with CD3+. + In T cells, IL-2 (10-100 units / mL) is added simultaneously. IL-2 is a cytokine secreted by T cells and plays an important regulatory role in the immune system. Preferably, the active unit of IL-2 is 80 units / mL. IL-2 and IL-7 (1-10 ng / mL), preferably IL-7 at 6 ng / mL, are also added. Forty-eight hours after the second stimulation, the cell culture supernatant is collected, and the level of INF-γ in the supernatant is detected by ELISA. After four DC stimulations, CD3+ is collected. + T cells were used to detect their killing activity against pancreatic cancer cells (Panc-1).

[0098] Referring to Figure 1, the ELISA method detects CD3. + The level of INFγ produced by T:

[0099] Add the cell culture supernatant from the above steps to each well (100 μl / well), seal the wells with sealing tape, and incubate at 37°C for 90 minutes, washing 4 times. Add 100 μl of horseradish peroxidase-labeled anti-cytokine antibody. Seal the plate and incubate at room temperature for 60 minutes. Wash 4 times and blot dry. Add the enzyme conjugate working solution (100 μl / well), seal the wells with sealing tape, and incubate at 37°C for 30 minutes, washing 4 times. Add 100 μl / well of chromogenic solution and incubate at 37°C for 15 minutes in the dark. Add 100 μl / well of stop solution, mix well, and immediately measure the OD. 450 Values. The concentrations of cytokines are calculated using the standard curve formula.

[0100] Refer to Figure 2, CD3 + T's killing activity against pancreatic cancer cells (Panc-1):

[0101] The CD3 groups obtained above + T cells were added to a 96-well plate, with 8 × 10⁸ cells per well. 5 4×10 5 1×10 5 Each group has three sets of holes. Add 1×10⁻⁶ ppm of the solution to each of the above holes. 4 Panc-1 with CSFE markers makes CD3 + The T cell to Panc-1 ratios were 80:1, 40:1, and 10:1. CSFE is a fluorescent dye used to label the cytoplasm of live cells. After 10 hours of culture, cells from each well were collected and stained with Annexin V. Annexin V is a protein widely used in cell biology and biomedical research, especially in the study of apoptosis (programmed cell death). It is a calcium-dependent phospholipid-binding protein mainly used to detect the eversion of phosphatidylserine (PS) on the cell membrane. The activity of Panc-1 cells was detected by flow cytometry, and the Panc-1 specific killing percentage (%) was calculated.

[0102] Experimental results show that the prepared dendritic cell vaccine can activate T cells, induce T cells to produce a large amount of INFγ, and specifically kill pancreatic cancer cells (Panc-1).

[0103] The above are all preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A preparation of a pancreatic cancer dendritic cell vaccine, characterized in that, Includes the following steps: 1) Take equal amounts of Panc-1 and AsPC-1 cells and place them into cryovials, and repeatedly freeze and thaw the cells at low temperature and room temperature. 2) The cells were sonicated and centrifuged separately. The supernatant was collected and filtered sterilely. The filtrate was the cell lysate. The protein concentration was detected by the BCA method and the protein concentration was adjusted accordingly. 3) CD14 + Cell suspension was transferred to a six-well plate at 2 x 10 5 -2 x 10 6 cells per well in a volume of 1 mL, and recombinant GM-CSF and recombinant IL-4 were added to the six-well plate; 4) Place the six-well plate on a clean bench and gently shake it three times in each direction to disperse the cells evenly. Incubate at 37°C and 5% CO2 for 3 days. 5) Add recombinant GM-CSF (final concentration 400-4000 IU / mL) and recombinant IL-4 (final concentration 200-1000 IU / mL) to the six-well plate; 6) Place the cells in an incubator at 37°C and 5% CO2 for 2 days to obtain immature dendritic cells; add appropriate amounts of Panc-1 and AsPC-1 (final concentrations of 50 μg / mL to 3000 μg / mL) cell lysates and mix with the immature dendritic cells for 6 hours. 7) Then add 2 μL of TNF-α (final concentration 400-4000 IU / mL), 2 μL of LPS (final concentration 1-5 μg / mL) and 1 μL of Poly(I:C) (final concentration 0.5-5 μg / mL) to stimulate dendritic cells for 24 hours to induce DC cell maturation. The matured cells are the DC vaccine.

2. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The low temperature mentioned in step 1) is -60℃ to -200℃.

3. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The ambient temperature mentioned in step 1) is 34℃~38℃.

4. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1 and its application in the treatment of pancreatic cancer, characterized in that: The number of freeze-thaw cycles mentioned in step 1) is 3 to 6.

5. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1 and its application in the treatment of pancreatic cancer, characterized in that: The ultrasonic fragmentation time mentioned in step 2) is 1 to 10 minutes.

6. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The centrifugation speed mentioned in step 2) is 1000-3000 rpm.

7. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The centrifugation time mentioned in step 2) is 5 to 20 minutes.

8. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: In step 6), appropriate amounts of the Panc-1 and AsPC-1 cell lysates prepared in step 1 are added respectively. The lysates can be Panc-1 or Panc-1 and AsPC-1 cell lysates.

9. The preparation of a pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The final concentration of the prepared Panc-1 and AsPC-1 cell lysis mixture protein added in step 6) is 50 μg / mL to 3000 μg / mL.

10. The use of a pancreatic cancer dendritic cell vaccine according to any one of claims 1-9 in the treatment of pancreatic cancer.

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