CN112237623B - Pseudomonas aeruginosa type III secreted protein pcrV and application of macrophage induced to polarization - Google Patents

Pseudomonas aeruginosa type III secreted protein pcrV and application of macrophage induced to polarization Download PDF

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CN112237623B
CN112237623B CN202011189669.1A CN202011189669A CN112237623B CN 112237623 B CN112237623 B CN 112237623B CN 202011189669 A CN202011189669 A CN 202011189669A CN 112237623 B CN112237623 B CN 112237623B
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lung cancer
tumor
protein
pcrv
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CN112237623A (en
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余华
张克斌
何小梅
熊浚智
邱静
代黔
李园园
张乐
李巧巧
王兴敏
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Second Affiliated Hospital Army Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention belongs to the field of medicines, and particularly relates to application of pseudomonas aeruginosa type III secreted protein pcrV and macrophages induced to be polarized, wherein the application can promote apoptosis of lung cancer cells of mice and increase phagocytosis of the lung cancer cells, and has important market prospect for immunotherapy applied to tumors.

Description

Pseudomonas aeruginosa type III secreted protein pcrV and application of macrophage induced to polarization
Technical Field
The invention belongs to the field of medicines, and particularly relates to a pseudomonas aeruginosa type III secreted protein pcrV and application of induced polarization macrophages thereof.
Background
Lung cancer is one of the most common malignant tumors of the respiratory system, has extremely high morbidity and mortality, is extremely poor in prognosis, and has a five-year survival time of less than 18 percent. At present, lung cancer treatment mainly comprises surgical excision, radiotherapy and chemotherapy. However, lung cancer cells have poor sensitivity to chemoradiotherapy, high invasion and high metastasis characteristics, and often result in unsatisfactory therapeutic effects. Therefore, a new thought and strategy for lung cancer treatment are further searched, and important theoretical support can be provided for clinical lung cancer treatment.
Macrophages are important effector cells that regulate the immune response of the body, and infiltrate extensively in tumor tissue. However, tumor-associated macrophages (TAMs) exist predominantly in the M2 polarization state. It has been reported that M2 polarized TAMs promote tumor growth, angiogenesis and metastasis by producing various factors such as transforming growth factor- β (transforming growth factor- β, tgfβ), vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and matrix metalloproteinase (matrix metalloproteinase, MMP). In addition, M2 TAMs can also maintain the immunosuppressive state of tumor microenvironment (tumor microenvironment, TME) by producing various immunosuppressive factors such as interleukin 4 (IL 4 ) and IL10, thereby promoting the occurrence and development of tumors. Since macrophages have high heterogeneity and plasticity, M2-polarized TAMs can exhibit anti-tumor M1-polarization-related biological activity that is different from their pro-tumor effects upon stimulation by bacterial lipopolysaccharide LPS and pro-inflammatory factors such as interferon ifnγ. M1 polarized TAMs promote the killing of tumor cells mainly by producing a large amount of tumor necrosis factors, nitric oxide, active oxygen and other cytotoxic factors or by enhancing phagocytosis and other approaches. At present, research proves that the induction of transformation of TAMs from M2 type to M1 type has obvious effects on inhibiting growth, angiogenesis, invasion and metastasis of multiple tumors such as multiple myeloma, breast cancer, ovarian cancer, hepatocellular carcinoma and the like. However, induction of M1 polarization of TAMs may also promote expression of some of the pro-inflammatory or tumor-associated factors, thereby attenuating M1 macrophage-mediated anti-tumor effects and potentially promoting tumor progression and development. Therefore, by inducing polarization of TAMs M1 and exerting its maximum anti-tumor effect, minimizing the potential risk of tumor promotion caused by polarization of M1 is a key to using macrophages for anti-tumor immunotherapy.
At present, research has proved that the inactivated or attenuated tubercle bacillus, salmonella, listeria, clostridium and the like can obviously inhibit the growth and metastasis of tumors when being used for tumor treatment. However, bacterial therapies remain at risk for clinical studies due to their significant toxic side effects. It was found that the use of a microbial related structural component or secreted protein can promote macrophage M1 polarization by activating macrophage related signal pathway, suggesting that induction of TAMs to M1 polarization by microbial related structural component or secreted protein may play a role in anti-tumor.
Disclosure of Invention
The invention aims to provide pseudomonas aeruginosa type III secreted protein pcrV and application of the pseudomonas aeruginosa type III secreted protein pcrV in inducing polarized macrophages, which can promote apoptosis of lung cancer cells of mice and increase phagocytosis of the lung cancer cells. In addition, the treatment of the lung cancer cells of the mice by the PCRV protein does not obviously increase the growth of tumor cells and the expression of metastasis related molecules, thereby avoiding the potential risk that the PCRV can promote the growth and metastasis of the tumors when being used for tumor treatment. Therefore, the method has important application prospect in the immunotherapy of tumors.
The technical scheme of the invention is as follows:
application of Pseudomonas aeruginosa type III secretion system secretion type protein pcrV protein in preparing medicine for treating lung cancer.
The application of the pseudomonas aeruginosa type III secretion system secretion type protein pcrV protein in preparing medicines for treating lung cancer by inducing macrophage polarization.
The macrophage is M1 type.
Application of P.aeruginosa type III secretion system secretion type protein pcrV protein induced polarization M2BMs in preparing medicines for treating lung cancer is provided.
The medicament is used for phagocytizing Lewis lung cancer cells causing disease onset.
The medicament is used for promoting apoptosis of lung cancer cells in the application.
The lung cancer cell is Lewis lung cancer cell.
Applicants' experiments indicate that treatment of M2 type macrophages (TAM-like phenotype cells) with the P.aeruginosa type III secretion system secreted protein pcrV promotes polarization of the cells towards M1 type. The M2 type macrophage treated by the PCRV protein is co-cultured with the lung cancer cells of the mice, so that the apoptosis of the lung cancer cells of the mice can be promoted, and the phagocytosis of the lung cancer cells can be increased. In addition, the tumor growth of mice can be inhibited and the apoptosis of tumor tissues can be promoted by adopting the PCRV protein or macrophages induced by the PCRV protein for carrying out the intratumoral injection of Lewis lung cancer subcutaneous tumor-bearing mice, which shows that the PCRV protein has obvious biological activity in resisting lung cancer.
Compared with the traditional bacterial therapy, the application of the invention regulates the polarization state of macrophages in TME by adopting bacterial proteins, further plays an anti-tumor effect, is more beneficial to activating the anti-tumor immune response of the organism, and greatly reduces the toxic and side effects of bacteria. In addition, the bacterial protein can be obtained in a large quantity through an escherichia coli expression system, bacterial endotoxin can be removed through the existing mature experimental technology, and the use cost and risk of the protein are greatly reduced. The application of the composition in the immunotherapy of tumors has important potential application value.
Drawings
FIG. 1 shows that M2BMs polarized by the pcrV protein promotes apoptosis in mouse LLC cells, wherein A: detecting LLC apoptosis by flow cytometry; b: apoptosis statistics;
FIG. 2 shows the shear situation of LLC cell Caspase 3 after the co-culture of M2 BMs-LLC cells by Western blot detection;
FIG. 3 shows enhanced phagocytosis of mouse LLC cells by the PCRV protein polarized M2BMs, wherein A: flow cytometry detects phagocytosis of LLC cells by macrophages; b: a cytophagocytic statistical map;
fig. 4 shows that PcrV protein can inhibit tumor growth in Lewis lung cancer subcutaneous tumor-bearing mice, wherein a: determining the tumor volume of the mice; b: mouse tumor weight;
FIG. 5 shows that the PCRV protein polarized macrophage can inhibit the growth of Lewis lung cancer subcutaneous tumor-bearing mice, wherein A: determining the tumor volume of the mice; b: mouse tumor weight;
FIG. 6 shows TUNEL staining for detecting apoptosis in tumor tissue of mice.
FIG. 7 shows the detection of cell growth and transfer related genes and transfer ability after treatment of mouse LLC lung cancer cells with PCRV protein, wherein A: LLC cell growth and transfer related gene expression level detection; b: detecting the cell transfer capacity;
Detailed Description
Materials and reagents
Animal and cell
Male C57BL/6 mice of 6-8 weeks old Beijing Bai Osai Gene Biotechnology Co., ltd;
mouse Lewis lung cancer cell (LLC cell) cell bank of Chinese national academy of sciences.
(II) principal reagents and formulation
10% FBS/DMEM formulation: 90ml of DMEM was added to 10ml of FBS and 1ml of the double antibody to green streptomycin. PBS buffer formulation (0.01 mol/L, pH 7.4): naCl 8.00g,KCl 0.20g,KH 2 PO 4 0.20g，NaH 2 PO 4 ·H 2 O 1.56g，1000ml ddH 2 O is dissolved, sterilized at high temperature and high pressure and stored at 4 ℃.
(III) Main instrumentation
2. Examples
Example 1 tumor-associated M2 macrophage (M2 BMs) induction
(1) C57BL/6 mice were anesthetized with 1% sodium pentobarbital, the femur and tibia were harvested without bacteriostasis, and the muscles were stripped. Bone ends were cut off, and bone marrow cells were flushed out by aspiration of medium with a 10ml syringe.
(2) The cell suspension was gently blown off and adjusted to 1X 10 using 10% FBS/DMEM (containing 50ng/ml mouse M-CSF) 6 Each ml, plate, 37 ℃, 5% CO 2 Culturing for 3 days.
(3) Full exchange with 10% FBS/DMEM (50 ng/ml mouse M-CSF), 37℃and 5% CO 2 The culture was continued for 3 days, i.e., induced differentiation of mature BMDMs cells (BMs cells).
(4) Mouse IL4 at 37℃and 5% CO was used at 20ng/ml 2 BMs cells were treated for 24h to obtain tumor-associated M2 type macrophages (M2 BMs).
EXAMPLE 2 Co-cultivation of M2BMs cells and lung cancer cells
(1) The M2BMs were digested with 5mM EDTA at 37℃for 10min. Cells were adjusted to 1X 10 with 10% FBS/DMEM (containing 20ng/ml mouse M-CSF) 6 Each ml was plated in 6-well plates at 37℃in 5% CO 2 The cells were cultured for 24h.
(2) The supernatant was discarded and M2BMs cells were treated with 10% FBS/DMEM medium containing 10. Mu.g/ml pcrV (containing 20ng/ml mouse M-CSF) for 24h.
(3) On the same day of M2BMs treatment with pcrV protein, 80% confluence of fine lung cancer of mouse LLC was obtainedCells were gently blown down and the concentration was adjusted to 1X 10 6 And each ml. Taking 1×10 6 The LLC cells were seeded in a Transwell chamber (0.4 μm pore size) upper chamber at 37℃with 5% CO 2 The cells were cultured for 24h.
(4) Transferring cells containing LLC cells into 6-well plate of M2BMs cells treated with pcrV at 37deg.C with 5% CO 2 The cells were cultured for 24h.
EXAMPLE 3 Effect of M2BMs cells on apoptosis of lung cancer cells
Taking the LLC cells after co-culture, washing for 2 times by using PBS, obtaining cell sediment, re-suspending the cells by using 100 mu l of apoptosis detection buffer solution, adding 5 mu l of Annexin V-PE and 5 mu l of 7-AAD dye solution, incubating for 30 minutes at room temperature in a dark place, diluting the apoptosis detection buffer solution to 300 mu l, and detecting the apoptosis by using a flow cytometer.
Example 4 detection of cleavage event of the apoptosis-related protein Caspase-3 of lung cancer
(1) And adding 200 mu l of Western blot/IP cell lysate into the LLC cells subjected to the co-culture, and performing lysis at 4 ℃ for 30min.
(2) The cells were centrifuged at 12000rpm at 4℃for 5min, and the cell lysate was collected.
(3) The protein concentration was determined using the BCA protein assay kit, and specific methods were performed with reference to BCA protein assay kit instructions.
(4) Referring to the "molecular cloning laboratory Manual" (third edition, scientific Press 2002[ Mei ] J. Sambrook, D.W, lassel, huang Peitang, et al) a 5% strength protein laminating gel and a 12% separating gel were prepared, and 50. Mu.g of protein was sampled per well. Electrophoresis: laminating adhesive 80V; the gel was separated by 120V and the electrophoresis time was about 2 hours.
(5) And transferring the protein on the gel onto a PVDF film by adopting a wet transfer method, wherein the film transferring condition is 100V and 90min.
(6) 5% skimmed milk powder/TBST was used and shaken at room temperature for 1h to block unbound protein sites on the membrane.
(7) The membranes were incubated with rabbit anti-sheared Caspase-3 antibody or rabbit anti-GAPDH reference antibody overnight at 4℃and washed 4 times with TBST.
(8) The membranes were incubated with HRP-labeled goat anti-rabbit antibodies for 1h at room temperature and washed 4 times with tbst.
(9) ECL chemiluminescent liquid emits light to detect protein expression.
EXAMPLE 5 detection of phagocytosis of lung cancer cells by M2BMs cells
(1) M2BMs were digested with 5mM EDTA at 37℃for 10min. Cells were adjusted to 1X 10 with 10% FBS/DMEM (containing 20ng/ml mouse M-CSF) 6 Each ml was plated in 6-well plates at 37℃in 5% CO 2 The cells were cultured for 24h.
(2) The supernatant was discarded and M2BMs cells were treated with 10% FBS/DMEM medium containing 10. Mu.g/ml pcrV (containing 20ng/ml mouse M-CSF) for 24h.
(3) Taking mouse LLC lung cancer cells with 80% confluence, gently blowing, and adjusting concentration to 1×10 6 And each ml. LLC cells were labeled by staining with 5. Mu.M CFSE at room temperature for 30min.
(4) LLC cells were grown at 1:5 proportion of the mixture into M2BMs cells at 37 ℃ and 5% CO 2 Culturing for 1, 2 and 3 hours.
(5) The cells were digested with 5mM EDTA at 37℃for 10min. Mu.l of APC-labeled anti-mouse F4/80 antibody was added to the cell suspension, and incubated at room temperature for 20min.
(6) Cells were washed 2 times with PBS and CFSE was detected by flow cytometry + F4/80 + Cell populations to define phagocytosis of lung cancer cells by M2BMs cells.
EXAMPLE 6 Lewis lung carcinoma subcutaneous tumor-bearing mouse model construction and PcrV protein treatment
(1) Mouse LLC lung cancer cells were cultured in 10% FBS/DMEM to 80% confluence, and after gentle blowing, PBS was adjusted to 1×10 7 And each ml.
(2) Male C57BL/6 mice were anesthetized briefly with isoflurane, shaved on the back, and injected with 100 μl of LLC lung cancer cell suspension at 6-8 weeks of age. The tumor size of the mice was observed and measured every 3 days, and the tumor volume of the mice was calculated as follows:
tumor volume of mice (mm) 3 ) =tumor long diameter (mm) ×tumor short diameter (mm) 2 /2
(3) To the tumor volume of about 60mm 3 About, mice were divided into two groups, and PBS or PcrV protein (0.5 mg +.kg), 2 total injections.
(4) After 4 days of the second injection, mice were sacrificed and tumor tissues were harvested and weighed.
EXAMPLE 7 Lewis lung carcinoma subcutaneous tumor-bearing mice in vivo macrophage purge and PCrV polarized macrophage reinfusion
(1) Male C57BL/6 mice, 6-8 weeks old, were injected intraperitoneally with 200 μl of macrophage scavenger (Clodronate liposomes) at a concentration of 5mg/ml in vivo; the injection was repeated once after 7 days.
(2) On the day of the second injection of macrophage scavenger, mice were briefly anesthetized with isoflurane, shaved on the back, and 100 μl of 1×10 containing formulation was injected on the back 6 LLC cells.
(3) Cells were obtained by the BMs cell culture method described above, using 10. Mu.g/ml of pcrV protein at 37℃and 5% CO 2 BMs cells were treated for 24h.
(4) After the current day and 7 days of in vivo macrophage scavenger injection, control or PcrV-treated BMs cells were taken at 2×10 tumor tissues per mouse 6 Individual cells were injected intratumorally.
(5) Mice were sacrificed 7 days after the second cell injection, and mouse tumor tissue was harvested and weighed.
Example 8 preparation of mouse tumor tissue fixation, paraffin embedding and section
(1) Fixing: the mouse tumor tissue was fixed in 10% neutral formalin for 48h.
(2) Flushing: the fixed tissue was rinsed with tap water for 10min and cut into small pieces.
(3) Dehydrating: step-by-step dehydration is carried out by adopting 70%, 80%, 90%, 95% and 100% gradient alcohol for 10-30min in each stage.
(4) And (3) transparency: the transparent treatment is carried out by adopting dimethylbenzene, and the process is repeated for 1 time each time for 20-30min.
(5) Wax dipping: pouring the dissolved paraffin into a paraffin dipping cup, and placing the paraffin into transparent tissue blocks, and replacing paraffin liquid for two times each time for 20-30min.
(6) Embedding: the melted paraffin was poured into the embedder and placed into the waxed tissue. The embedding device is moved into the water surface, slowly immersed in water, and taken out after the paraffin is completely coagulated.
(7) Slicing: the slice thickness was 5. Mu.m.
(8) Spreading and sticking sheet: placing the cut wax strips into a water bath kettle with the temperature of 40-50 ℃, taking the slide glass flattened tissue slices subjected to silanization treatment, and drying the slide glass at the temperature of 45-50 ℃.
(9) Dewaxing: the tissue pieces were placed in dye vats I and II containing xylene to dissolve paraffin 3min each time.
(10) And (3) water: the tissue slices were washed with distilled water after 100%, 95%, 90%, 80% and 70% alcohol treatment at each stage.
EXAMPLE 9 TUNEL staining method for detecting apoptosis in tumor tissue in mice
Taking the dewaxed mouse tumor tissue slice, and detecting tumor tissue apoptosis by using TUNEL tissue staining kit, wherein the specific method is carried out according to the reagent instruction book. The TUNEL-dyed tissue slice is dripped with a cell nucleus dye solution (DAPI dye solution), is dyed for 10min at room temperature in a dark place, is washed for 3 times by PBS, and is sealed by an anti-fluorescence quenching sealing tablet. And observing the apoptosis condition of the tissue by a laser confocal microscope.
Example 10 fluorescent quantitative PCR method for detecting Gene expression in mice Lung cancer cells
(1) Mouse LLC lung cancer cells were cultured in 10% FBS/DMEM,37℃and 5% CO 2 Culturing to 80% confluence.
(2) LLC cells were treated with different concentrations of PcrV protein (0, 5, 10 and 20. Mu.g/ml) and incubated at 37℃with 5% CO 2 Culturing for 24h.
(3) The cells were collected and lysed by RNAiso and total RNA was extracted by reference to the RNA kit instructions.
(4) The total RNA of the cells is reverse transcribed by using a reverse transcription kit to obtain cDNA, and the specific method is carried out according to the instruction of the kit.
(5) The expression of mouse LLC lung cancer cell gene is detected by fluorescent quantitative PCR, and the method is carried out according to the instruction book of the kit.
EXAMPLE 11 detection of the metastatic Capacity of mouse lung cancer cells
(1) Mouse LLC lung cancer cells were cultured in 10% FBS +.DMEM，37℃、5％CO 2 Culturing to 80% confluence.
(2) LLC cells were gently blown down with 10% FBS/DMEM medium and adjusted to a concentration of 1X 10 5 And each ml. 1ml of the cell suspension was inoculated into the upper chamber of a Transwell cell (8 μm pore size) and 2ml of 10% FBS/DMEM medium was added to the lower chamber.
(3) Adding PcrV protein (10 μg/ml) into the culture medium, LLC cells at 37deg.C and 5% CO 2 Culturing for 48h.
(4) The Transwell chamber was washed with PBS buffer and the cells were scraped off using a cotton swab.
(5) The Transwell chamber was placed in a 0.5% crystal violet dye for 5min at room temperature.
(6) The Transwell cells were washed with PBS buffer and photographed using an optical microscope.
3. Experimental results
EXAMPLE 12 PcrV protein polarized M2BMs promote apoptosis in mouse LLC cells
The co-culture of M2BMs cells or M2BMs cells polarized by pcrV protein (see Front Microbiol,2020;11:1971;Type III Secretion Protein,PcrV,Impairs Pseudomonas aeruginosa Biofilm Formation by Increasing M1 Macrophage-Mediated Anti-bacterial Activities) with mouse LLC lung cancer cells was adopted, and apoptosis was detected by flow cytometry, which showed that the co-culture of pcrV protein polarized M2BMs cells with mouse LLC lung cancer cells significantly increased apoptosis of LLC cells compared to the co-culture group of M2 BMs+LLC cells (see FIG. 1), indicating that pcrV protein can promote macrophage-induced apoptosis of lung cancer cells.
Example 13 polarization of the PcrV protein M2BMs promote cleavage and activation of the mouse LLC apoptosis-related protein caspase 3
Caspase-3 (Caspase-3) is a key effector molecule involved in apoptosis. Normally, it exists as Pro-caspase 3. When cells are stimulated for apoptosis, caspase-3 is sheared and activated and induces apoptosis through a series of reactions. Therefore, we further examined Caspase-3 cleavage of LLC cells by Western blot when M2BMs were co-cultured with LLC cells. From the results, the cleavage of LLC apoptosis-related protein Caspase-3 was significantly promoted by the PCRV protein polarized M2BMs cells compared with the M2BMs+LLC cell co-culture group (see FIG. 2), further demonstrating that the PCRV protein can promote macrophage-induced apoptosis of lung cancer cells.
EXAMPLE 14 enhancement of phagocytosis of mouse LLC cells by PCRV protein-polarized M2BMs
Macrophages are important immune cells with phagocytic functions, and phagocytosis of tumor cells is a key element in the development of anti-tumor immunity. Co-culturing M2BMs with mouse lung cancer LLC cells and detecting phagocytosis of LLC cells by M2BMs macrophages by a flow cytometer revealed that the phagocytosis of LLC cells by M2BMs polarized by pcrV protein was significantly enhanced compared to the group of M2BMs cells not polarized by pcrV protein (see FIG. 3), indicating that pcrV protein can promote phagocytosis of mouse lung cancer cells by macrophages.
Example 15PcrV protein inhibits growth of tumor in Lewis lung carcinoma subcutaneous tumor-bearing mice
Mice were used to subcutaneously support tumors using Lewis lung cancer cells (LLC cells) and intratumorally injected with the PCRV protein, and as a result, the volume and weight of tumors in the intratumorally injected mice with the PCRV protein were significantly lower than those in the PBS-injected group (see FIG. 4). In addition, lung cancer tissue apoptosis induced by intratumoral injection of PcrV protein was also found to be significantly higher in mice than in PBS intratumoral injection group (see fig. 6) by detecting tumor tissue apoptosis. The result shows that the PCRV protein can obviously inhibit the growth of lung cancer of mice.
EXAMPLE 16 macrophage polarized by pcrV protein inhibits tumor growth in Lewis lung carcinoma subcutaneously tumor-bearing mice
The volume and weight of tumor in mice injected intratumorally with PcrV/BMs cells was found to be significantly lower than in the intratumoral BMs injection group as a result of the in vivo macrophage scavenger Clodronate liposomes to scavenge the tissue macrophages of mice and subsequent subcutaneous tumor-bearing by LLC cells and intratumoral injection of BMs macrophages polarized with PcrV protein. In addition, tumor tissue apoptosis was also found to be significantly higher in the PcrV/BMs intratumoral injection group than in the BMs intratumoral injection group by detecting tumor tissue apoptosis (see fig. 6), indicating that PcrV-induced macrophage polarization can significantly inhibit lung cancer growth in mice.
EXAMPLE 17 PcrV protein treatment of LLC Lung cancer cells does not significantly increase tumor cell growth, metastasis associated molecular expression and cell transfer Capacity
Treatment of mouse LLC lung cancer cells with different concentrations of PcrV protein and detection of gene expression levels by fluorescent quantitative PCR revealed that PcrV protein did not significantly increase tumor cell growth and metastasis associated molecular gene expression compared to untreated LLC cells (see fig. 7A). By examining the transfer capacity of the PcrV protein treated LLC cells, it was found that PcrV protein did not significantly increase the transfer capacity of LLC cells compared to normal LLC cells (see fig. 7B).

Claims (5)

1. Application of Pseudomonas aeruginosa type III secretion system secretion type protein pcrV protein in preparing medicine for treating lung cancer.
2. Application of P.aeruginosa type III secretion system secretion type protein pcrV protein induced polarization M2BMs in preparing medicines for treating lung cancer is provided.
3. The use according to claim 2, characterized in that: the medicament is used for phagocytizing Lewis lung cancer cells.
4. Use according to any one of claims 1-2, characterized in that: the medicine is used for promoting apoptosis of lung cancer cells.
5. The use according to claim 4, characterized in that: the lung cancer cell is Lewis lung cancer cell.
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细菌感染在促进肺癌发生与发展及治疗中的作用研究进展;李有军等;中华医院感染学杂志;第28卷(第17期);2716-2720 *

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