Preparation method of traditional Chinese medicine polysaccharide assisted pancreatic cancer DC vaccine

Abstract

The invention discloses a preparation method of a traditional Chinese medicine polysaccharide assisted pancreatic cancer DC vaccine. The preparation method comprises the following steps: extracting mouse bone marrow dendritic cells; the preparation method comprises the following steps: cracking and centrifuging mouse bone marrow dendritic cells through a red blood cell lysis solution, cleaning a culture medium, and culturing in a special culture medium to obtain immature dendritic cells; collecting the PANC2 pancreatic cancer cells subjected to amplification culture, counting, putting into a cryopreservation tube, and preparing a tumor antigen by a low-temperature and normal-temperature repeated freezing and thawing method; the preparation method comprises the following steps: co-culturing immature dendritic cells and tumor antigen for 12-24 hours, adding traditional Chinese medicine polysaccharide, continuously culturing until the dendritic cells are mature, and re-suspending the co-cultured cells in normal saline to obtain the pancreatic cancer DC vaccine. The prepared traditional Chinese medicine polysaccharide assisted pancreatic cancer DC vaccine has the advantages of high maturity, enhanced phenotypic signal molecule expression and the like, and can effectively inhibit tumor cell growth when being independently applied to a pancreatic cancer tumor-bearing mouse model.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for preparing a pancreatic cancer DC vaccine assisted by traditional Chinese medicine polysaccharides. Background Art

[0002] Pancreatic cancer is a type of malignant tumor of the digestive tract with a mortality rate closest to its incidence rate. With a five-year survival rate of approximately 10%, it is currently the seventh leading cause of cancer-related death worldwide. Traditionally, pancreatic cancer treatments have primarily included surgery, radiotherapy, chemotherapy, interventional therapy, and best supportive care. However, due to its insidious onset and invasive nature, early diagnosis is difficult, and most patients have already missed the opportunity for surgical resection by the time they present. Furthermore, radiotherapy and chemotherapy are associated with low efficacy rates and adverse reactions, necessitating an urgent need for safer and more effective treatments.

[0003] DC vaccines, or dendritic cell vaccines, are a new type of vaccine that treats diseases by inducing autoimmune responses. They are primarily composed of dendritic cells and antigens, with some also incorporating TNF-α to stimulate cells. Currently, DC vaccines have been used in basic and clinical research for a variety of cancers, including colorectal cancer, melanoma, and pancreatic cancer, with most achieving promising therapeutic effects. They offer advantages such as strong specificity, high safety, and long-lasting efficacy. However, the anti-tumor effects of pancreatic cancer DC vaccines alone are limited. This may be due to the low in vitro maturity, poor immune activity, and weak antigen phagocytosis of pancreatic cancer DCs. Furthermore, pancreatic cancer is associated with a highly immunosuppressive tumor microenvironment, which, to a certain extent, limits the efficacy of pancreatic cancer DC vaccine immunotherapy. Therefore, DC vaccine treatment for pancreatic cancer is currently often used in combination therapies.

[0004] Traditional Chinese medicine polysaccharides such as Astragalus polysaccharide (APS) have the advantages of high safety and significant immunomodulatory effects, but there have been no reports on pancreatic cancer DC vaccines adjuvanted by traditional Chinese medicine polysaccharides. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides a method for preparing a pancreatic cancer DC vaccine assisted by traditional Chinese medicine polysaccharides.

[0006] The technical solution of the present invention is: a method for preparing a pancreatic cancer DC vaccine adjuvanted by traditional Chinese medicine polysaccharides, which is carried out according to the following steps: Step 1. Isolate mouse bone marrow dendritic cells; Step 2. Mouse bone marrow dendritic cells were lysed with red blood cell lysis buffer, centrifuged, washed with culture medium, and then cultured in a dedicated culture medium to obtain immature dendritic cells; Step 3. After expansion and culture, PANC2 pancreatic cancer cells were harvested, counted, and placed in cryopreservation tubes. Tumor antigens were prepared by repeated freeze-thaw cycles at low and room temperatures. Step 4. Co-culture the immature dendritic cells with the tumor antigen for 12 to 24 hours, then add the Chinese herbal polysaccharide and continue culturing until the dendritic cells mature. Then, resuspend the co-cultured cells in physiological saline to obtain the pancreatic cancer DC vaccine.

[0007] Preferably, step 1 is to extract cells from the femur and tibia of the hind limbs of the mouse, repeatedly rinse with serum-containing RPMI1640 complete medium, collect the cell suspension and filter it using a 70 μm sterile cell sieve, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant to obtain mouse bone marrow dendritic cells.

[0008] Preferably, the step 2 is to add the extracted dendritic cells to the red blood cell lysis buffer and resuspend them, lyse them for 3 to 5 minutes, centrifuge them at 1200 rpm for 5 minutes, discard the supernatant, wash them 2 to 3 times with RPMI1640 complete medium, resuspend them in a complete medium specifically for mouse bone marrow-derived immature dendritic cells, and culture them in a 37°C, 5% CO2 incubator for 5 to 7 days to obtain immature dendritic cells.

[0009] Preferably, in step 3, the tumor antigen is prepared by repeated freezing and thawing at low temperature and room temperature, wherein the low temperature is -60 to -200°C, the room temperature is 34 to 38°C, and the number of repeated freezing and thawing is 3 to 6 times.

[0010] Preferably, in step 4, the ratio of the number of immature dendritic cells to the number of tumor antigen cells is 1:5-20, and the traditional Chinese medicine polysaccharide is astragalus polysaccharide with a final concentration of 50-300 μg / mL.

[0011] The pancreatic cancer DC vaccine adjuvanted with traditional Chinese medicine polysaccharides prepared by the present invention has the advantages of high maturity and enhanced expression of phenotypic signal molecules. It can effectively inhibit the growth of tumor cells when used alone in a pancreatic cancer-bearing mouse model. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Graphs showing the results of microscopic observation of the vaccines of the embodiment of the present invention and the control group.

[0013] Figure 2 This is a graph showing the expression results of phenotypic signal molecules in the vaccine of the embodiment of the present invention and the control group.

[0014] Figure 3 This is a graph showing the effects of the vaccines of the embodiment of the present invention and the control group on the tumor volume of pancreatic cancer-bearing mice.

[0015] Figure 4 This figure shows the effects of the example vaccines of the present invention and the control vaccine on the tumor mass in pancreatic cancer-bearing mice. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is: a method for preparing a pancreatic cancer DC vaccine adjuvanted by traditional Chinese medicine polysaccharides, which is carried out according to the following steps: Step 1. Extract mouse bone marrow dendritic cells; Specifically, 6-8 week-old C57BL / 6 male mice were sacrificed and soaked in 75% ethanol for 10 minutes. The bilateral femurs and tibias were then removed (subsequent steps were performed aseptically). The bones were then washed three times with pre-cooled sterile PBS (containing 1% penicillin / streptomycin) to completely remove any residual ethanol. The bone marrow cavity was repeatedly flushed with serum-containing RPMI1640 complete medium until the red color faded. The cell suspension was collected, filtered through a 70 μm sterile cell sieve, and centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded. Step 2. Mouse bone marrow dendritic cells were lysed with red blood cell lysis buffer, centrifuged, washed with culture medium, and then cultured in a dedicated culture medium to obtain immature dendritic cells; Specifically, the mouse bone marrow dendritic cells extracted in step 1 were resuspended in 1 mL of red blood cell lysis buffer and gently mixed for 3-5 minutes to lyse the red blood cells. The cells were then centrifuged at 1200 rpm for 5 minutes, resuspended and centrifuged in RPMI1640 complete medium, and washed three times. The cells were then resuspended in complete medium specifically for mouse bone marrow-derived immature dendritic cells and cultured in a 37°C, 5% CO2 incubator for 5 days (day 0 of the experiment) to obtain immature dendritic cells. Step 3. The PANC2 pancreatic cancer cell line purchased from Wuhan Punosai Life Science Co., Ltd. was expanded and cultured. The PANC2 pancreatic cancer cells were collected and counted and resuspended in PBS to 1×10 7 / mL, freeze and thaw 5 times in a 37°C water bath for 5 minutes, and in liquid nitrogen for 15 minutes, centrifuge at 10,000 rpm for 15 minutes, and filter the supernatant through a 0.22 μm microporous membrane to prepare tumor antigens; Step 4. Immature dendritic cells and tumor antigen cells were mixed at a ratio of 1:20 and co-cultured for 12 hours. Astragalus polysaccharide was then added to a final concentration of 100 μg / mL and cultured for another 2 days to allow the dendritic cells to mature. The co-cultured cells were then resuspended in physiological saline to obtain the pancreatic cancer DC vaccine. The astragalus polysaccharide of the embodiment of the present invention is extracted by water extraction and alcohol precipitation method, which is specifically carried out according to the following steps: (1) Decoction slices of Astragalus membranaceus are boiled in water, extracted and filtered, and the concentrated solution is added with ethanol; the supernatant is filtered out, and the precipitate is added with ethanol, and the supernatant is discarded and the precipitate is dissolved in water; (2) Add ethanol to the solution to remove the precipitate, add ethanol to the supernatant, remove the supernatant, and ultrafilter the precipitate with water; the filtrate is passed through cation and anion exchange chromatography columns, eluent filter membranes, and ultrafiltration systems for graded ultrafiltration; (3) The two-stage ultrafiltrate is frozen, re-thawed, centrifuged to remove the supernatant, the precipitate is dissolved in ethanol, centrifuged again to remove the supernatant, low-temperature dried, crushed, and mixed to obtain the product.

[0017] experiment: According to the method of the embodiment of the present invention, immature DCs were collected on the 5th day of culture in step 2, counted, and plated in a 24-well plate. 5 Cells were cultured for 12 h at a ratio of 1:20 between the number of DC cells and the number of tumor antigen cells. Recombinant TNF-α purchased from Peprotech (final concentration 20 ng / mL) was added to the DC+TNF-α group, and APS (final concentration 100 μg / mL) was added to the DC+APS group. Culture was continued for 2 days, and the cells in each group were resuspended in physiological saline to obtain the vaccine of the embodiment of the present invention and the control group.

[0018] The apparent evaluation of each vaccine group is as follows: 1) Observe the morphology of vaccine cells prepared in each group under a microscope. The results are as follows: Figure 1 The results showed that compared with the control, the number of DC cells increased after sensitization with PANC 2 pancreatic cancer tumor cell antigens, and the process morphology and colony growth state were obvious. The DC maturity of the DC+APS and DC+TNF-α groups was higher, indicating that APS and TNF-α stimulation can further promote DC maturation.

[0019] 2) Flow cytometry was used to detect the expression of DC surface signaling molecules such as CD80 and CD86. The results were as follows: Figure 2 The results showed that compared with the Control group, PANC 2 pancreatic cancer tumor antigens can promote the expression of CD80 and CD86 on the surface of DCs, stimulate DC maturation, and DC+APS has significant differences ( p < 0.01, p < 0.05); Compared with the DC group stimulated with PANC 2 antigen alone, the effects of DC+APS and DC+TNF-α were more significant, and the effect of DC+APS on CD80 was the most significant ( p < 0.05).

[0020] 3) Anti-tumor effects of DC vaccines 3.1) Establishment of pancreatic cancer-bearing mice Pancreatic cancer cells PANC2 in the logarithmic growth phase were collected and the concentration was adjusted to 1.5 × 10 7The cell suspension (containing approximately 1.5 × 10 tumor cells / mL) was inoculated subcutaneously in the right hind leg of C57 mice, with 0.1 mL (containing approximately 1.5 × 10 tumor cells) per mouse. 6 indivual).

[0021] 3.2) Two weeks after tumor inoculation, mice were divided into four groups based on tumor size: model, DC, DC+APS, and DC+TNF-α. Each group received corresponding treatments. The model group received an intraperitoneal injection of normal saline (0.2 mL / mouse), while the DC, DC+APS, and DC+TNF-α groups received an intraperitoneal injection of the corresponding vaccine (1.5 × 10 5 The first treatment was performed on the day of grouping, and the second treatment was performed 5 days later, for a total of 2 treatments. Two days after the second treatment, mice were fasted for 12 hours, euthanized, and tumor tissue was obtained and tumor volume and mass were recorded.

[0022] 3.3) The result is as follows Figure 3 、 4 As shown in the figure, compared with the model group, the tumor volume and mass of mice in each DC vaccine group were reduced, among which the effects of the DC+APS group and the DC+TNF-α group were significantly different ( p < 0.05), with DC+APS having the most significant effect, and the tumor growth inhibition rate (tumor inhibition rate = (average tumor weight of control group - average tumor weight of experimental group) / average tumor weight of control group × 100%) > 55%, suggesting that APS can enhance the anti-tumor effect of DC vaccine.

Claims

1. A method for preparing a pancreatic cancer DC vaccine assisted by traditional Chinese medicine polysaccharides, characterized in that Follow these steps: Step 1. Isolate mouse bone marrow dendritic cells; Step 2. Mouse bone marrow dendritic cells were lysed with red blood cell lysis buffer, centrifuged, washed with culture medium, and then cultured in a dedicated culture medium to obtain immature dendritic cells; Step 3. Collect the expanded PANC2 pancreatic cancer cells, count them, and place them in cryopreservation tubes. Prepare tumor antigens by repeated freeze-thaw cycles at low and room temperatures. Step 4. Co-culture the immature dendritic cells with the tumor antigen for 12 to 24 hours, then add the Chinese herbal polysaccharide and continue culturing until the dendritic cells mature. Then, resuspend the co-cultured cells in physiological saline to obtain the pancreatic cancer DC vaccine.

2. The method for preparing the pancreatic cancer DC vaccine assisted by traditional Chinese medicine polysaccharides according to claim 1, characterized in that: The step 1 is to extract cells from the femur and tibia of the posterior limbs of the mouse, repeatedly rinse with serum-containing RPMI1640 complete culture medium, collect the cell suspension and filter it through a 70 μm sterile cell sieve, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant to obtain mouse bone marrow dendritic cells.

3. The method for preparing the pancreatic cancer DC vaccine assisted by traditional Chinese medicine polysaccharides according to claim 2, characterized in that: The step 2 is to add the extracted dendritic cells to the red blood cell lysis buffer and resuspend them, lyse them for 3-5 minutes, centrifuge them at 1200 rpm for 5 minutes, discard the supernatant, wash them 2-3 times with RPMI1640 complete medium, resuspend them in a complete medium specifically for mouse bone marrow-derived immature dendritic cells, and culture them in a 37°C, 5% CO2 incubator for 5-7 days to obtain immature dendritic cells.

4. The method for preparing the pancreatic cancer DC vaccine assisted by traditional Chinese medicine polysaccharides according to claim 3, characterized in that: In step 3, the tumor antigen is prepared by repeated freezing and thawing at low temperature and room temperature, wherein the low temperature is -60 to -200° C., the room temperature is 34 to 38° C., and the number of repeated freezing and thawing is 3 to 6 times.

5. The method for preparing the pancreatic cancer DC vaccine assisted by traditional Chinese medicine polysaccharides according to claim 4, characterized in that: In step 4, the ratio of the number of immature dendritic cells to tumor antigen cells is 1:5-20, and the traditional Chinese medicine polysaccharide is astragalus polysaccharide with a final concentration of 50-300 μg / mL.

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