CN116178302B - CD47 protein ubiquitination modified agonist and application thereof - Google Patents
CD47 protein ubiquitination modified agonist and application thereof Download PDFInfo
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- CN116178302B CN116178302B CN202210563765.0A CN202210563765A CN116178302B CN 116178302 B CN116178302 B CN 116178302B CN 202210563765 A CN202210563765 A CN 202210563765A CN 116178302 B CN116178302 B CN 116178302B
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Abstract
The invention discloses a CD47 protein ubiquitination modified agonist and application thereof, belonging to the technical field of CD47 ubiquitination promotion. The invention uses the USP2 inhibitor ML364 to inhibit the function of the deubiquitination modification of USP2, promote the ubiquitination modification of CD47, ensure that the protein expression quantity of the CD47 is down-regulated, activate the anti-tumor immune response of immune cells in tumor tissues and strengthen the effect of anti-tumor immune treatment. The CD47 ubiquitination activator can enhance the immune response in tumor tissues, has a synergistic effect when being combined with other immune detection point antibodies such as anti-PD-1 antibodies, and can enhance the effect of single drug use so as to better treat tumors. Compared with the existing CD47 blocking agent, the CD47 protein ubiquitination modified agonist provided by the invention does not influence the quantity of red blood cells and platelets, and has relatively fewer toxic and side effects.
Description
Technical Field
The invention belongs to the technical field of CD47 ubiquitination promotion, and particularly relates to a CD47 protein ubiquitination modified agonist and application thereof.
Background
With the wide clinical application of PD-1/PD-L1 targeted drugs, immunotherapy has become another key means for treating tumors after traditional radiotherapy and chemotherapy. However, the anti-PD-1/PD-L1 drugs are only suitable for 20-30% of tumor patients clinically at present, and the drugs have the problems of drug resistance or toxic and side effects and the like. Therefore, finding new tumor immune combination therapeutic strategies and developing new tumor immune therapeutic targets have become one of the key problems to be solved in clinic.
Cluster of differentiation 47 (CD 47) is one of the members of the immunoglobulin superfamily, and this highly glycosylated membrane protein is expressed in a variety of human cells, and this molecule is composed mainly of three parts, the N-terminal IgV domain, 5 transmembrane domains, and a short C-terminal cytoplasmic tail. CD47 can bind to ligands such as signal regulatory protein α (sirpa), integrin, thrombospondin-1 (TSP-1), and vascular endothelial growth factor receptor 2 (EGFR-2), and participate in regulating various cellular activities in humans, such as cell proliferation, cell motility, cell phagocytosis, T-cell activation, and inflammatory responses; through regulating the clearance function of erythrocytes, the trimming function of neuronal synapses and the like maintain physiological homeostasis, and play an important role in pathological processes such as tissue fibrosis diseases, atherosclerosis and the like.
There have been many studies demonstrating high expression of CD47 on the surface of cancer cells of solid and hematological tumors, including lung cancer, ovarian cancer, breast cancer, glioblastoma, liver cancer, prostate cancer, acute myeloid leukemia, and non-hodgkin's lymphoma. CD47 on the surface of cancer cell membrane is combined with SIRPa ligand to send out signal of ' don't eat me ' to macrophage, inhibit phagocytosis of tumor cell by macrophage, promote tumor cell to evade immune monitoring. Thus the CD47-sirpa axis is considered as a tumor phagocytosis checkpoint signal axis, CD47 also becoming a new target for tumor immunotherapy. Blocking therapies targeting CD47-sirpa can promote immune responses in tumor tissues by activating the activity of innate and adaptive immune cells through a variety of mechanisms. Various therapeutic agents targeting CD47-SIRPa have been developed, such as a monospecific antibody targeting CD47 only, a bispecific antibody targeting PD-1, and SIRPa Fc fusion proteins, etc., and some therapeutic antibodies have entered phase ii or phase iii clinical trials. The antibodies have certain curative effects in various tumor detection, for example, after CD47 is blocked by CD47 antibodies, phagocytosis of cancer cells by macrophages can be induced, and growth of small cell lung cancer is inhibited; combination therapy of CD47 antibodies with rituximab has therapeutic effects on non-hol-based lymphomas.
However, since CD47 is also widely expressed on the surface of erythrocytes, normal erythrocytes are destroyed when the antibody is used for treatment at present, and serious toxic and side effects such as anemia, thrombocytopenia and the like appear, which limit the clinical application of therapeutic antibodies targeting CD 47-SIRPa. However, the molecular mechanisms that regulate CD47 expression in cancer cells at the protein level are currently under further investigation. Although it has been reported that the glutamine peptide cyclotransferase-like protein (QPCTL) inhibits phagocytic function of macrophages by promoting the formation of pyroglutamic acid residues at the N-terminus of the CD47 protein molecule, promoting the binding of CD47 and sirpa, no regulation of the ubiquitination modification of CD47 has been reported. Therefore, further research on the regulatory mechanism of CD47 in cancer cells and other cells, in order to seek to specifically regulate the expression of CD47 in tumor cells, and thus, development of therapeutic drugs specifically targeting CD47 of tumor cells, would be one of the important strategies to clinically improve the therapeutic effect on tumors.
Ubiquitination modification refers to the process of dynamically modifying target protein by ubiquitin molecules under the catalysis of a series of enzymes, is a main way for regulating and controlling the stability of protein in cells, participates in important life activities including apoptosis, cell cycle progress, DNA damage repair, membrane transport and the like, and plays a key role in the processes of tumor growth, inflammatory reaction and the like. Ubiquitin molecules have 7 lysine residues (K6, K11, K27, K29, K33, K48, K63) and one methionine residue (M1), and are linked by lysine residues or methionine residues to create different ubiquitin chains, which when linked to a substrate protein will determine the different fate of the substrate protein. However, ubiquitination modification and deubiquitination modification are a dynamic reversible process, in which ubiquitin E3 ligase is responsible for covalently attaching ubiquitin chains to substrate proteins, whereas deubiquitination proteases specifically remove isopeptidic linkages formed between ubiquitin molecules and target proteins, leaving ubiquitin molecules or ubiquitin chains off the target proteins, protecting the substrate proteins from degradation, repositioning or activation, etc. There are about 100 deubiquitinated proteases (DUBs) found in humans, including six structurally distinct families, of which the ubiquitin-specific protease (USP) family is the largest subfamily of deubiquitinated proteases, with about 54 members.
USP2 is one of the members of the USP family and plays an important role in various physiological pathological processes such as tumorigenesis, circadian rhythm variation, inflammatory response, etc. However, research in recent years has shown that USP2 is highly expressed in various cancers such as prostate cancer, liver cancer, bladder cancer, etc., and promotes the development of tumors by stabilizing a substrate protein. USP2 can modulate the stability of critical tumor proteins. For example, USP2 promotes palmitate synthesis by inhibiting Fatty Acid Synthase (FASN) degradation; and palmitate can inhibit apoptosis of tumor cells, so that apoptosis can be slowed down by regulating fat metabolism of tumor cells when USP2 stably exists, and further growth of the tumor cells is promoted. Thus, the use of small molecule inhibitors to inhibit the functional activity of USP2 may be a new target for the treatment of tumors. At present, a small molecule inhibitor ML364 specifically targeting USP2 has been reported to cause cell cycle arrest and degradation of cyclin D1, and inhibit DNA repair mediated by homologous recombination, thereby inhibiting tumor growth and metastasis. Document Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models (Davis MI et al) discloses that small molecule ML364 can induce cell cycle arrest by USP2 inhibition and inhibit the growth of colorectal cancer and mantle cell lymphoma cell lines. Document The deubiquitylase USP main ErbB2abundance via counteracting endocytic degradation and represents a therapeutic target in ErbB-positive breast cancer (Jinrui Zhang et al) discloses that USP2 maintains ErbB2abundance by counteracting endocytic degradation, and is a therapeutic target for ErbB2 positive breast cancer. The document USP2a Supports Metastasis by Tuning TGF-beta Signaling (Yin Zhao et al) discloses the role of USP2a in promoting tumor metastasis by promoting TGF-beta triggered Signaling. However, the functions of USP2 are diverse, and it has not been reported to date what kind of connection or influence exists between USP2 and the ubiquitination modification process of CD47, whether the connection or influence participates in the regulation of tumor immunity, and the influence of the small molecule inhibitor ML364 of USP2 on the activity of tumor immune cells.
Disclosure of Invention
The invention aims to provide a CD47 protein ubiquitination modified agonist and functions and applications thereof in tumor immunotherapy. Modulating the ubiquitination modification of CD47 by using a CD47 protein ubiquitination modification agonist, thereby altering the stability of CD47, enhancing immune responses in tumor tissue; the effect of anti-tumor immunotherapy can be enhanced when a CD47 protein ubiquitination modified agonist is used in combination with other immune checkpoint antibodies (e.g., PD-1 antibodies); meanwhile, compared with the prior CD47 blocking agent, the CD47 protein ubiquitination modified agonist has fewer toxic and side effects. Based on the research results, the invention provides a novel CD47 protein ubiquitination modified agonist and corresponding application, which are realized by the following technology.
A CD47 protein ubiquitination modified agonist comprising a small molecule inhibitor ML364, the small molecule inhibitor ML364 having a chemical structure of:
the invention also provides application of the CD47 protein ubiquitination modified agonist in preparation of tumor immunotherapy medicaments.
Preferably, the CD47 protein ubiquitination modified agonist is combined with an anti-PD-1/PD-L1 monoclonal antibody, so that the anti-tumor treatment effect can be enhanced.
Preferably, the CD47 protein ubiquitination modified agonist is used in combination with an anti-CTL 4 monoclonal antibody, so that the anti-tumor therapeutic effect can be enhanced.
Preferably, the tumor is ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblast derived CNS tumor, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma or mast cell derived tumor for use in the preparation of a medicament for tumor immunotherapy.
The invention also provides a tumor immunotherapeutic agent comprising a CD47 protein ubiquitination modified agonist as claimed in any one of claims 1 to 3.
Preferably, the tumor immunotherapy drug further comprises an anti-PD-1/PD-L1 monoclonal antibody or an anti-CTL 4 monoclonal antibody.
Preferably, the tumor immunotherapy drug further comprises a carrier applied to pharmacy.
Preferably, the tumor is an ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuroblastoma-derived CNS tumor, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma or mast cell derived tumor.
In the present invention, we have found for the first time that USP2 can increase the stability of CD47 in tumor cells by removing the polyubiquitination modification of CD47, thereby up-regulating the expression level of CD 47. However, the use of the USP2 inhibitor ML364 or the CRISPR technique to knock out the USP2 gene can promote ubiquitination modification of CD47 to degrade CD47 protein molecules, i.e. can significantly reduce CD47 expression in tumor cells; finally activating the anti-tumor immune response in tumor tissues and enhancing the effect of anti-tumor immune treatment.
Further, in the mouse tumor model, compared with the ML364 or the anti-PD-1 antibody, the combination therapy of the ML364 and the PD-1 antibody can obviously inhibit the growth of the mouse tumor and prolong the survival period of the mouse. More importantly, we have found that either ML364 alone or in combination with anti-PD-1/PD-L1 monoclonal antibodies, or anti-CTL 4 monoclonal antibodies, significantly increases CD8 in tumor tissue + The number and activity of T cells and no significant downregulating effect on the number of erythrocytes, hemoglobin and platelet in mice. Based on the similarity of the mechanism of action of anti-CTL 4 monoclonal antibodies with anti-PD-1 antibodies, it can be reasonably deduced that ML364, anti-CTL 4 monoclonal antibodies are equally possessed when combined therapy is performed.
Therefore, our study not only proves that inhibiting USP2 can down regulate the expression level of CD47 in tumor cells, but also proves that when the USP2 inhibitor and the PD-1 antibody are combined to treat tumor, the effect on tumor treatment can be remarkably improved, and the possibility is provided for clinically applying a new tumor immunotherapy scheme.
Compared with the prior art, the invention has the following advantages:
1. the function of the USP2 deubiquitination modification is inhibited by using the USP2 inhibitor ML364, the ubiquitination modification of the CD47 is promoted, the protein expression quantity of the CD47 is down-regulated, the anti-tumor immune response in tumor tissues is activated, and the effect of anti-tumor immune therapy is enhanced.
2. The CD47 ubiquitination activator can enhance the immune response in tumor tissues, and when being combined with other immune checkpoint antibodies such as anti-PD-1 antibodies, the CD47 ubiquitination activator can play a synergistic role, and can enhance the effect of single drug use so as to better treat tumors.
3. Compared with the existing CD47 blocking agent, the CD47 protein ubiquitination modified agonist provided by the invention does not influence the quantity of red blood cells and platelets, and has relatively fewer toxic and side effects.
Drawings
FIG. 1 is a graph showing that inhibitors inhibit the down-regulation of CD47 expression by deubiquitinase USP2 function;
in FIG. 1, panel A shows the detection of CD47 expression after 14h treatment of PC9 cells with 2.5. Mu.M inhibitors of the USP deubiquitinase family, respectively;
panel B shows the detection of CD47 protein expression after 16h treatment of PC9 cells with 0.75. Mu.M, 1. Mu.M, 2. Mu.M USP2 inhibitor, respectively;
panel C shows the detection of CD47 mRNA levels after 16h of treatment of PC9 cells with 0.75. Mu.M, 1. Mu.M, 2. Mu.M USP2 inhibitor, respectively;
panel D shows the detection of CD47 protein expression after 16H treatment of H1975 cells with 0.75. Mu.M, 1. Mu.M, 2. Mu.M USP2 inhibitor, respectively;
panel E shows the detection of CD47 mRNA levels after 16H of treatment of H1975 cells with 0.75. Mu.M, 1. Mu.M, 2. Mu.M USP2 inhibitor, respectively;
FIG. 2 is a schematic representation of knockdown of USP2 gene down-regulating CD47 expression;
in FIG. 2, panel A shows infection of A549 cells with lentiviruses, and protein expression of USP2 and CD47 was examined after knocking down USP2 with shRNA in A549 cells;
panel B shows infection of H1975 cells with lentivirus, and protein expression of USP2 and CD47 was detected after knocking down USP2 with shRNA in H1975 cells;
panel C shows the effect of USP2 over-expression on CD47 protein expression by transient transfection over-expression of CD47 in 293T cells and gradient over-expression of USP 2;
panel D shows transient transfection of CD47 in 293T cells and transfection of empty plasmid or wild-type USP2WT or USP 2C 276A, the effect of CD47 protein expression was examined;
panel E shows the detection of CD47 protein degradation after transient transfection of CD47 in 293T cells and transfection of empty plasmid or USP2WT, treatment of cells with Cycloheximide (CHX) at various time points;
FIG. F is a graph showing the gray scale analysis statistics of the CD47 protein expression level after treatment with CHX for different times in FIG. E;
panel G shows the transient transfection of CD47 and either USP2WT or USP 2C 276A plasmids in 293T cells, and detection of CD47 protein degradation after treatment of the cells with CHX at various time points;
FIG. H is a graph showing the gray scale analysis statistics of the CD47 protein expression level after treatment with CHX for different times in the graph G;
FIG. 3 shows that the USP2 protein expression level and the CD47 protein expression level are positively correlated in a patient sample;
in FIG. 3, panel A shows the detection of USP2 and CD47 expression in patient samples by immunohistochemistry in tumor tissue of lung adenocarcinoma patient with upper panel scale of 100 μm and lower panel scale of 50 μm;
panel B shows the correlation of USP2 and CD47 by a Person statistical analysis after detecting the expression of USP2 and CD47 in a patient sample by immunohistochemistry on tumor tissue of 83 lung adenocarcinoma patients, and scoring the protein expression level by a semi-quantitative method. P <0.0001, r=0.5323, n=83;
FIG. 4 shows that deubiquitinase USP2 can interact with CD 47;
in FIG. 4, panel A shows transient transfection of CD47 in 293T cells and empty or USP2 and other USP family members, the interaction of CD47 with USP2 and other USP family member proteins was detected by co-immunoprecipitation;
panel B shows the in vitro purification of CD47 protein, and the interaction between the CD47 protein and the USP2 protein is verified by GST pull down experiment;
panel C is a schematic diagram of the functional domain of the USP2 protein;
panel D shows transient transfection of CD47 and USP2 or truncated mutants of USP2 in 293T cells, validating the USP2 functional domain interacting with CD47 by co-immunoprecipitation;
FIG. E shows the purification of CD47 protein in vitro, and the interaction of the functional domain of USP2 with CD47 is verified by GST pull down experiment;
FIG. 5 shows ubiquitination modification of deubiquitinase USP2 to remove CD 47;
in FIG. 5, panel A shows transient transfection of CD47 and Ub molecules in 293T cells, while gradient transfection of USP2, validating the deubiquitination of CD47 by USP 2;
panel B shows transient transfection of CD47 and Ub molecules in 293T cells, while transfection of wild-type USP2 or USP2 deubiquitination function inactivating mutants, verifying the function of USP2 on CD47 deubiquitination modification;
panel C shows transient transfection of CD47, ub molecule and USP2 in 293T cells and treatment of the cells with USP2 inhibitor, verifying the effect of USP2 inhibitor on ubiquitination modification of CD 47;
FIG. 6 is the effect of USP2 inhibitors on enhancing tumor immunotherapy by activating immune responses;
in fig. 6, panel a shows the effect on tumor growth in mice by subcutaneously plating LLC cells in mice using a PD-1 antibody, a USP2 inhibitor, or a combination of a PD-1 antibody and a USP2 inhibitor.
Panel B shows the effect on mice survival of LLC cells grown subcutaneously with PD-1 antibody, USP2 inhibitor or a combination of PD-1 antibody and USP2 inhibitor.
Panel C shows analysis of immune cells in mouse tumor tissue, CD8 + Infiltration of T lymphocytes.
Panel D shows analysis of immune cells in mouse tumor tissue, CD8 + Expression of Granzyme B (GzmB) in T lymphocytes.
Panel E shows analysis of immune cells in mouse tumor tissue, CD8 + Expression of Interferon gamma (ifnγ) in T lymphocytes.
FIG. 7 is a study of toxic side effects of USP2 inhibitors;
in FIG. 7, panel A shows the effect on mouse body weight of LLC cells subcutaneously plated in mice using PD-1 antibody, USP2 inhibitor or a combination of PD-1 antibody and USP2 inhibitor;
panel B shows the effect on mouse erythrocyte numbers of LLC cells subcutaneously plated in mice using PD-1 antibodies, USP2 inhibitors or a combination of PD-1 antibodies and USP2 inhibitors; RBCs are red blood cells;
panel C shows the effect of LLC cell transplantation subcutaneously in mice on the hemoglobin content of mice using PD-1 antibodies, USP2 inhibitors or a combination of PD-1 antibodies and USP2 inhibitors; HGB is hemoglobin;
panel D shows the effect on the number of platelets in mice by subcutaneously plating LLC cells in mice using PD-1 antibodies, USP2 inhibitors or a combination of PD-1 antibodies and USP2 inhibitors; PLT is platelets.
Detailed Description
To date, there is no literature report on modulation of tumor immunity by the modulation of ubiquitination modification of CD 47. In the present invention, a deubiquitinase capable of removing the ubiquitination modification of CD47 is mainly discovered, the ubiquitination modification of CD47 is stabilized by using the inhibitor of the deubiquitination enzyme, the ubiquitination modification of CD47 protein is increased to degrade the CD47 protein, and when the protein amount of CD47 is reduced, the anti-tumor immune response of tumor tissues can be enhanced. Therefore, when such CD47 protein ubiquitination modified agonist is used in combination with other antitumor immune agonists, the effect of antitumor immune therapy can be further enhanced, such as in combination with an anti-PD-1/PD-L1 monoclonal antibody or an anti-CTL 4 monoclonal antibody, etc.
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
1. Experimental materials
1. Buffer formulation
Cell lysates (for immunoblotting and co-immunoprecipitation experiments): 1M Tris-HCl, ph= 7.5,5M NaCl,0.5%NP40;
protein running buffer: 25nM Tris,192mM glycine, 0.1% SDS;
protein transfer buffer: 25nM Tris,192mM glycine;
NETN buffer: 0.5%NP40,0.002M Tris-HCl, ph= 8.0,0.1M NaCl,0.001M EDTA;
TBST buffer: 150mM NaCl,50mM Tris,0.1%Tween 20,pH =7.6;
immunoblotting blocking solution: 5% nonfat dry milk, TBST solution (also used to formulate primary and secondary antibodies);
ubiquitination experiment cell lysis Buffer a (Buffer a): 6M guanidine hydrochloride, 0.1M Na 2 HPO 4 /NaH 2 PO 4 10mM imidazole;
ubiquitination assay Buffer TI (Buffer TI): 25mM Tris-HCl,20mM imidazole, pH=6.8;
LB medium: 1%TRYPTONE,0.5%YEAST EXTRACT,1%Nacl;
3×loading Buffer:6.7% SDS,34% glycerol, 5% DTT,1% bromophenol blue.
2. Chemical reagent
DUBs-IN-2 (HY-50737A), ML323 (HY-17543), ML364 (HY-100900), P22077 (HY-13865), spautin-1 (HY-12990), IU1 (HY-13817), LDN-57444 (HY-18637), PR-619 (HY-13814), BAY 11-7082 (HY-13453), MF-094 (HY-112438), EOAI3402143 (HY-111408) and MG132 (HY-13259) are all available from MCE corporation.
TCID (S7140) is purchased from Selleck; cyclohexide (C7698-5G) was purchased from Sigma; PEI (23966-1) was purchased from Polysciences.
Ampicillin sodium was purchased from source leaf organisms.
3. Cell strain
HEK-293T cell line, PC9, A549, H1975 human lung adenocarcinoma cell line, LLC mouse lung carcinoma cell line, all from ATCC cell banks, were cultured using complete medium (DMEM (Hyclone) cell culture medium plus 10% fetal bovine serum (Celmax), 100U penicillin and 100mg/ml streptomycin (Hyclone).
4. Plasmid(s)
The pEnCMV-CD47-3x HA plasmid was purchased from vast organism; his-Ub plasmid was purchased from origin;
Flag-USP 2WT, flag-USP 2C 276A, flag-USP4, flag-USP5, flag-USP7, flag-USP8, flag-USP13, flag-USP15, flag-USP21, flag-USP25, flag-USP30 were all constructed in pcDNA3-Flag empty plasmids (purchased from Addgene), all constructed by the present laboratory themselves;
GST-CD47 is constructed on pGEX-6p-2 empty plasmid (purchased from Addgene) and is self-constructed by the laboratory; shGFP and shRNA of shUSP2 are both constructed on pLko.1 empty plasmid (purchased from Addgene) and are self-constructed by the laboratory;
the shRNA sequence of shrp 2 is as follows.
shUSP2#1：5’-CCGCGCTTTGTTGGCTATAAT-3’；
shUSP2#2：5’-GCTCACAACATTTGTGAACTT-3’。
5. Antibody information
Anti-human CD47(63000，Cell Signaling Tecnology)；
anti-human USP2(AP2131c abcepta)；
anti-GAPDH (1E 6D 9) mouse monoclonal antibody (60004-1-Ig, proteintech);
anti-Vingulin (VIN-11-5) mouse monoclonal antibody (V4505, sigma);
Alexa -700-CD8a (53-6.7) monoclonal antibody (100730, biolegend);
PE/Cy7-Granzyme B (QA 16A 02) monoclonal antibody (372213, biolegend);
FITC-IFNgamma (XMG 1.2) monoclonal antibody (505506, biolegend);
Perp-Cy5.5-CD45 (30-F11) monoclonal antibody (103131, biolegend);
goat anti-rabbit IgG HRP (BL 003A, biosharp), secondary antibody;
goat anti-mouse IgG HRP (BL 001A, biosharp), secondary antibody;
HA antibody-conjugated magnetic beads (HY-K0201, MCE);
GST-coupled agarose gel beads (17-0756-05, GE);
Ni-NTA agarose gel beads (30230, QIAGEN).
Anti-PD-1 (29 f.1a12) treated mab (BP 0273, bio X Cell).
6. Experimental animal
C57BL/6 mice: directly purchased from Jizhikang, SPF grade, 6 weeks for animal experiments;
the mice used in the invention are all bred in SPF environment (temperature: 20-22 ℃, humidity: 50-60%), and the breeding room is lighted for 12 hours and dark for 12 hours alternately.
7. The tissue chip: purchased from Shanghai core super company.
2. Experimental method
1. Biochemical experiments
(1) Immunoblotting
(1) Collecting a protein sample: lysing the cells with EBC solution at 4deg.C for 15min; the lysed cells were then placed in a centrifuge at 4℃and centrifuged at 14000rpm for 10min, and the supernatant was assayed for protein concentration using the BCA kit. The supernatant was added with 3×loading Buffer, boiled for 5min, and then loaded with SDS-PAGE was performed.
(2) SDS-PAGE and transfer: protein electrophoresis was performed in a protein transfer buffer for 70min using a constant voltage of 128V, and after the electrophoresis was completed, protein transfer was performed in a protein transfer buffer for 2h using a constant current of 300 mA.
(3) Blocking and antibody application: after the membrane transfer is finished, the PVDF membrane is placed in 5% skimmed milk for 1h at room temperature, after the sealing is finished, the sealed PVDF membrane is placed in a corresponding one-antibody diluent, and the PVDF membrane is incubated at 4 ℃ overnight.
(4) ECL development: after the primary antibody incubation was completed, the PVDF membrane was washed three times with TBST, then the secondary antibody was incubated at room temperature for 1h, and after the secondary antibody incubation was completed, the PVDF membrane was washed three times with TBST. Then, ECL developer is developed.
(2) Co-immunoprecipitation
Protein was extracted and the protein concentration was measured in the same manner as in immunoblotting described above. In each experimental group, 1. Mu.g of cell lysis supernatant was taken, the volume was made up to 500. Mu.L with EBC solution, 8. Mu.L of anti-HA beads were added to each group, after 4h incubation at 4℃the non-specific binding proteins on the HA antibody-coupled beads were washed and removed using NETN Buffer, 3 XLoading Buffer was added to the HA antibody-coupled beads, and boiled for 5min for immunoblotting experiments.
(3) Detection of ubiquitination modification experiments
(1) Collecting a protein sample: and (3) using a part of cell samples for immunoblotting experiments, detecting the expression condition of corresponding proteins in cells, using 1mL Buffer A to lyse the cells and uniformly mixing the cell samples, using an ultrasonic breaker to fully lyse the cells, centrifuging at 14000rpm at room temperature for 10min, taking supernatant, adding Ni-NTA agarose gel into the supernatant, and incubating at room temperature for 3h.
(2) Washing the Ni-NTA agarose gel beads: after the incubation, the Ni-NTA agarose gel beads were washed twice with Buffer A to remove non-specific binding, then with Buffer A/Buffer TI=1:3 solution, and finally with Buffer TI once. The washed Ni-NTA agarose gel beads were added with 3×loading Buffer and boiled for 5min for immunoblotting experiments.
(4) In vitro purification of proteins
(1) Conversion: pGEX-6p-2 empty or pGEX-6p-2-CD47 plasmid is added into BL21 (DE 3) competent cells, after ice bath for 30min, water bath heat shock is carried out at 42 ℃ for 90s, ice bath is carried out for 5min, 1mL of non-resistant LB culture medium is added into competent cells, activation is carried out at 37 ℃ for 45-60min, centrifugation is carried out at 2000rpm for 3min at room temperature, 900 mu L of supernatant is removed, and after precipitation and residual supernatant are fully mixed, bacterial liquid is evenly coated on an ampicillin resistant solid LB plate.
(2) Bacterial amplification: single colonies grown on LB plates were inoculated into 4mL of ampicillin-resistant LB medium and amplified at 37℃for 4h. Then inoculating 4mL of bacterial liquid into 200mL of ampicillin-resistant LB culture medium, continuing to amplify for 1-2h at 37 ℃, detecting the OD value of the bacterial liquid by using a spectrophotometer, stopping amplifying when the OD value reaches 0.6, and preparing to add IPTG for induction.
(3) Induction of protein expression: the temperature of the shaking table is reduced to 16 ℃, then 0.1mM isopropyl-beta-D-thiogalactoside (IPTG) is added into the amplified bacterial liquid, and finally the bacterial liquid is placed into a shaking table with low temperature of 16 ℃ for induction overnight.
(4) Protein extraction: placing the induced bacterial liquid into a low-temperature centrifuge at 4 ℃, centrifuging at 8000rpm for 30min, and removing the supernatant; adding 20ml of ice-bath PBS buffer solution into the thalli, centrifuging at 8000rpm for 10min, and removing the supernatant; then PBS in ice bath is used for cleaning the thalli once; after the cleaning is finished, 10mL of PBS buffer solution in ice bath is added into the thalli, and a proteasome inhibitor is added into the thalli to fully resuspend the thalli; placing the bacterial liquid in an ultrasonic crusher to crush the bacterial liquid to release protein in the cytoplasm of the bacterial liquid, stopping 5s after the ultrasonic condition is 100W working for 5s, and circulating the steps for 20min, wherein the whole ultrasonic crushing process is carried out in an ice bath; after the ultrasonic disruption, placing the disrupted bacterial liquid in a low-temperature centrifuge at 4 ℃, centrifuging at 8000rpm for 30min, wherein the target protein is mainly present in the supernatant after centrifugation.
5, enrichment of target proteins: storing the supernatant after centrifugation in a new 15ml centrifuge tube, adding 500 mu L of GST agarose gel beads into the supernatant, and incubating for 4 hours at 4 ℃; after incubation, GST agarose gel beads were washed 2 times with ice-bath NETN buffer to remove non-specific binding, and then washed twice with ice-bath PBS buffer; after washing, the solution was resuspended in PBS buffer equal to the volume of GST agarose gel beads, and the proteasome inhibitor was added to the solution for storage at 4 ℃.
6, judging the concentration of the target protein: and taking 20 mu L of the target protein obtained by enrichment, adding 10 mu L of 3×loading Buffer into the protein solution, boiling for 5min, loading the sample, performing SDS-Page electrophoresis, and performing Coomassie brilliant blue staining after electrophoresis is finished. SDS-PAGE was performed on the same gel as the target protein using a known concentration of BCA protein, and the concentration of the target protein was determined by referring to the concentration of BCA protein.
(5) GST-pulldown experiment
Transfecting USP2 or a truncated mutant of USP2 with PEI in 293T cells, recovering the cells after 36 hours of transfection, determining the protein concentration, taking 2 μg of the protein solution in a new 1.5mL Ep tube, and adding the volume of the protein solution to 500 μl; 1 μg GST or GST-CD47 protein is added into 500 μl protein solution, GST agarose gel beads with target protein are washed by NETN Buffer solution after incubation for 4h at 4 ℃, finally 3×loading Buffer is added, SDS-PAGE electrophoresis is carried out, gel blocks of one sample are taken for coomassie brilliant blue staining after electrophoresis is finished, and immunoblotting experiment is continued on the other sample.
(6) Immunohistochemistry (IHC)
Tumor tissue was fixed with paraformaldehyde and embedded in paraffin, and cut into tissue sections with a thickness of 4 μm. The tissue sections were dewaxed in an oven at 60℃for 2h and sequentially soaked in xylene for 10min,100% ethanol for 5min,90% ethanol for 5min,80% ethanol5min, hydrating with 70% ethanol for 5min; antigen retrieval was then performed by heating in a microwave oven for 15min with 0.01M citric acid buffer (ph=6.0); then using 3%H 2 O 2 The tissues were incubated at room temperature for 15min to block endogenous peroxidase activity. After tissue is blocked by 10% normal goat serum for 1 hour, the primary antibody is incubated at 4 ℃ overnight in a refrigerator; the primary antibodies were anti-human CD47 (1:200, #63000,Cell Signaling Tecnology) and anti-human USP2 (1:1500,AP2131c abcepta) antibodies, respectively, diluted with 10% PBS solution. Washing the slices with PBS for 3 times, and washing for 5min each time; the biotin-labeled IgG secondary antibody was incubated at room temperature for 30min. After washing again with PBS, tissue staining was performed with a Servicebio Dection Kit peroxidase/Diamniobenzidine (DAB) mouse/rabbit (G1212-200T, servicebio) kit according to the instructions provided by the kit manufacturer, showing brown precipitate at the antigen; finally, the tissue is stained with hematoxylin solution to blue the nuclei. The stained tissue sections were scanned with a Lecia Aperio VERSA (Aperio imagescope v 12.3.2.8013) multifunctional scanner. The mean optical density value of each sample tissue was quantified as a positive intensity score using the Halo v3.0.311.314 analysis software Indica Labs-Area Quantification v 2.1.3.
2. Cell experiment
(1) Transfection of 293T cells with PEI
(1) Preparing cells: 293T cells were plated in 6cm dishes 1-2 days prior to cell transfection and transfection was initiated when cell densities reached 70-80%.
(2) Transfection: plasmid transfection amount was calculated according to specific experiments while adjusting PEI usage. In general, PEI is used in a quantity (. Mu.L)/mass of plasmid (. Mu.g) of 3:1. The specific transfection steps are as follows: taking a 1.5mL Ep tube, adding 300 mu L of Opti-MEM culture medium, firstly adding 3 mu g of plasmid into the Ep tube, uniformly mixing, then adding 9 mu L of PEI into the Ep tube, uniformly mixing, standing for 15min, dropwise adding into cell culture, slightly shaking the culture medium, uniformly mixing, putting the cells into a 37 ℃ incubator, and replacing the cells with fresh complete culture medium after 7-8 h; cells can be harvested for immunoblotting, immunoprecipitation, ubiquitination and other experiments after 36h transfection; can also be used for lentivirus packaging.
(2) Lentivirus-infected lung cancer cell line
(1) The relevant plasmids were transfected into 293T cells according to the PEI transfected cells, and the mass of the plasmids was generally: 1. Mu.g of the plasmid of interest, 1. Mu.g of pVSVG and 1. Mu.g of pD8.9.
(2) Viral supernatants were collected twice after 36h and 60h of transfection, respectively. The two collected viral supernatants were collected in the same 15ml centrifuge tube, mixed well and filtered through a 0.45 μm filter.
(3) A lung cancer cell line (A549 and H1975) having a density of 30-40% was prepared with a 6-well plate, 1.5mL of a virus solution and 0.5mL of fresh complete medium were added to each well, after culturing in an incubator for 12 hours, the lung cancer cell supernatant was removed, and the virus solution and complete medium were added to the lung cancer cell line in the previous manner once, and after culturing continued for 12 hours, the culture was changed to fresh complete medium for 24 hours.
(4) After infection was completed, cells were screened with 1. Mu.g/. Mu.L puromycin. The knockdown effect of USP2 was examined using immunoblotting and changes in CD47 expression.
3. Animal experiment
(1) LLC tumor model
(1) LLC cell preparation: cells were digested with pancreatin and washed twice with PBS, then resuspended with DMEM, the cells were thoroughly blown and counted to adjust the cell density to 5 x 10 6 /mL. In the process of preparing cells, the overlength of cell digestion time and the overgreat cell growth density are prevented, and the maximum density of the cells is controlled within 80 percent so as to maintain the optimal state of cell growth;
(2) LLC cell transplantation: 100 μl of LLC cell suspension was injected subcutaneously into 6 week old female C57BL/6 mice at the intersection of the mouse subcutaneous axillary midline and the 4 th rib; after injection is completed, tumor formation can be observed 3-5 days after injection;
(3) drug administration strategy of LLC tumor model: mice injected with LLC cells were divided into 4 groups, each: control group (normal saline), anti-PD-1 monoclonal antibody treatment group, ML364 USP2 inhibitor treatment group, and anti-PD-1 monoclonal antibody and USP2 inhibitor ML364 combination treatment group. In the model group, the dosage of the anti-PD-1 monoclonal antibody is 10mg/kg, and the anti-PD-1 monoclonal antibody is administrated by intraperitoneal injection every three days; the dosage of the USP2 inhibitor ML364 treatment group is 5mg/kg, and the administration is carried out by intraperitoneal injection every day.
(4) Tumor measurement and mice sacrifice: on day 3 after LLC cell injection, the length and width of the tumor of the mice were measured every two days using vernier calipers, using the formula: length x width 2 The volume of the tumor of the mice was calculated by x 0.5. According to ethics of experimental animals, when the tumor volume of the mice reaches 2000mm 3 Or when the tumor of the mouse is ulcerated, the mouse is considered dead, and the survival time of the mouse after LLC cell injection is recorded.
(5) Tumor infiltrating lymphocyte analysis: after 15 days of LLC cell subcutaneous injection, mice were sacrificed by cervical dislocation and tumor tissues were isolated. After shearing the tumor tissue, the tissue was ground on a 70 μm filter, the filtrate was again filtered with a 70 μm filter, and the tumor tissue single cell suspension obtained by the filtration twice was collected into a 50mL centrifuge tube while the single cell suspension volume was made up to 30mL.
10mL of Ficoll-Paque 1.084 reagent (17544602, GE) is added into each sample of tumor single cell suspension, then a horizontal centrifuge is used for centrifugation for 20min at 1025g, tumor infiltrated immune cells are enriched on the interface of Ficoll-Paque, the immune cells are carefully sucked out by a pipette, and the cell surface can be directly stained by using a corresponding flow antibody, so that the expression of CD45 and CD8 molecules can be detected. If secreted cytokines are detected, they are stimulated for 5h at 37℃with 1640 complete medium containing leukocyte activator (550583, BD) and then stained for intracellular secreted factors.
(6) And (3) collecting a blood sample of the mice: after 21 days of combined treatment of tumor-bearing mice with anti-PD-1 monoclonal antibody, ML364 or anti-PD-1 monoclonal antibody and ML364, the mice were removed from their eyeballs before being sacrificed, a portion of the blood samples were collected in anticoagulation tubes for routine blood tests, a portion of the blood was collected in 1.5ML Ep tubes, left at room temperature for 20min waiting for clotting, and then centrifuged at 4 ℃ for 20min at 3000rpm, and the supernatant serum was collected in a new Ep tube for liver function test.
3. Experimental results
1. Inhibition of USP2 in lung cancer cell lines would down-regulate CD47 protein expression
In the present invention, by treating human lung cancer cell line PC9 with inhibitors of various USP family members, it was found that CD47 protein levels could be significantly down-regulated only when cells were treated with the USP2 inhibitor ML-364 (FIG. 1, A). Thus, we treated PC9 and H1975 cells with ML-364, respectively, and found that the protein level of CD47 was gradually down-regulated (FIGS. 1, B and D) and that the mRNA level of CD47 was not significantly changed (FIGS. 1, C and E) as the inhibitor concentration was increased.
After finding that using ML364 can significantly down-regulate protein levels of CD47, we also packaged shRNA lentiviruses knockdown USP2 to infect human lung cancer cell lines a549 and H1975. In the shUSP2 cell line, protein levels of USP2 were down-regulated, along with significant down-regulation of the protein amount of CD47 (fig. 2, a and B). Since inhibition of USP2 significantly down-regulates the protein level of CD47, we over-expressed USP2 in 293T cells, and we found that with increasing USP2 protein expression, the protein level of CD47 was up-regulated (see fig. 2, c).
According to previous literature reports, the main deubiquitination enzyme activity site of USP2 is cysteine (Cys, C) at position 276, and USP2 will lose deubiquitination modification function when this amino acid is mutated to Alanine (Alanine, ala, a). Thus, using PEI to transiently transfect CD47 and USP2WT or USP 2C 276A plasmids in 293T cells, we found that the protein level of CD47 would be up-regulated only when USP2WT was overexpressed, whereas the CD47 protein would not be significantly altered when USP 2C 276A was overexpressed (FIGS. 2, C and D).
Next, we validated the effect of USP2 on CD47 protein half-life. Cycloheximide (CHX) inhibits protein biosynthesis, so we used CHX to treat cells where USP2WT protein was overexpressed, and CD47 degradation slowed down, indicating that overexpression of USP2 significantly prolonged the half-life of CD47 protein, whereas overexpression of USP 2C 276A protein did not (e.g. fig. 2, e-H). Based on the above experimental results, we found that inhibition of USP2 significantly down-regulates the protein level of CD47 without significantly affecting the mRNA level of CD47, and in addition, USP2 significantly increases the half-life of CD 47. Together, these results demonstrate that USP2 regulates CD47 protein levels primarily through post-translational modification.
In the present invention, we found that the protein level of USP2 and the protein level of CD47 in lung cancer tissues were positively correlated by semi-quantitative analysis of the staining results and statistics of the results using IHC to detect the expression levels of USP2 and CD47 in 83 lung cancer patient samples, except that inhibition of USP2 was demonstrated to down-regulate CD47 at the cellular level (fig. 3, a and B).
As can be seen, through cell level study and analysis of the expression level of USP2 protein and the expression level of CD47 protein in lung cancer tissue samples of patients, the expression level of CD47 protein is down-regulated when USP2 is inhibited.
2. Ubiquitination modification of USP2 to remove CD47
In the results prior to the present invention, after the use of a range of inhibitors of USP family members, it was found that only USP2 inhibitors were used to significantly down-regulate the protein level of CD47 and demonstrated that USP2 should regulate the protein level of CD47 primarily by post-translational modification (see fig. 1 and 2). We then first transiently transfected with PEI in 293T cells with USP family member molecules and CD47 molecules corresponding to the inhibitors used in (fig. 1, a) and verified whether there was an interaction between the protein molecules of these USP family members and the CD47 protein molecules. Consistent with previous experimental results, we found that only USP2 protein and CD47 protein had an interaction relationship, while other USP family members were unable to interact with CD47 (fig. 4, a).
Furthermore, we purified GST protein and GST-CD47 protein in vitro, and by GST-pull down experiments, it was demonstrated that USP2 protein and in vitro purified CD47 protein can also interact strongly (FIG. 4, B). To further explore which functional domain of USP2 the CD47 protein interacted with, we constructed two stretches of truncated mutant amino acids 1-266 and amino acids 267-605 of USP2 (see fig. 4, c). We co-transfected CD47 and USP2WT or USP2 truncation mutants, respectively, in 293T cells, we found that CD47 interacted primarily with the 1-266 segment domain of USP2 (FIG. 4, D). Similarly, we also demonstrated by GST-pull down experiments that in vitro purified CD47 protein interacted primarily with the 1-266 segment domain of USP2 (FIG. 4, E). After demonstrating that the CD47 protein can interact with USP2 protein, we need to further demonstrate whether USP2 can remove the ubiquitination modification of CD 47. Thus we co-transfected CD47, his-Ub and USP2WT or USP 2C 276A plasmids, respectively, in 293T cells, we found that only USP2WT could significantly remove the ubiquitinated modification of the CD47 protein, whereas the ubiquitinated modification of the CD47 protein molecule could not be removed when USP 2C 276A (see fig. 5, a, B).
To verify whether the ubiquitination modification of the USP2 to remove the CD47 protein molecule could be inhibited when ML364 was used, we co-transfected CD47, his-Ub and USP2WT plasmids in 293T cells and treated the cells simultaneously with ML364, we found that the ubiquitination of USP2 to CD47 was significantly inhibited when the cells were treated with ML364 (fig. 5, c).
According to the above experimental results, the present invention demonstrates that USP2 protein can interact with CD47 protein and can remove ubiquitination modification on CD47 protein molecule, however, the ubiquitination of USP2 on CD47 protein molecule can be significantly inhibited when USP2 inhibitor ML364 is used.
3. The USP2 inhibitor can improve the immunotherapeutic effect of the anti-PD-1 antibody
To explore whether USP2 inhibitors could be used to increase ubiquitination modification of CD47, promote CD47 protein degradation, down regulate CD47 protein levels, enhance the efficacy of anti-tumor immunotherapy. We constructed LLC tumor models and then treated mice tumors with ML364 and anti-PD-1 monoclonal antibodies. When the USP2 inhibitor and the anti-PD-1 monoclonal antibody are used in combination to treat tumors, the effect of the anti-PD-1 monoclonal antibody on treating tumors is remarkably enhanced. The main manifestation is that the growth of the tumor in mice was significantly inhibited in the combination group and the survival of tumor-bearing mice was significantly prolonged (fig. 6, a-B).
To demonstrate whether USP2 inhibitors achieve the effect of enhancing anti-PD-1 monoclonal antibodies in treating tumors by enhancing anti-tumor immune responses. We isolated immune cells from tumor tissue and used flow cytometry to analyze CD8 in tumor tissue + Infiltration of T lymphocytes and infiltrated CD8 + Activity of T lymphocytes. We have found that upon combination of a USP2 inhibitor and an anti-PD-1 monoclonal antibody, infiltrated CD8 + T lymphocytes were significantly increased and activity was significantly enhanced (fig. 6, c-E).
In summary, the present invention has found that when the inhibitor is used to inhibit the deubiquitinase activity of USP2, the ubiquitination modification of CD47 is increased, so that the degradation of CD47 protein is caused, and when the amount of CD47 protein is reduced, the tumor immune response is promoted, and finally the effect of anti-tumor immunotherapy is enhanced, so as to achieve the purpose of more effectively treating tumor.
4. USP2 inhibitor has less toxic and side effects
Although anti-tumor immune responses can be enhanced by blocking CD47, most of CD47 blocking antibodies currently used have significant side effects, such as the occurrence of anemia and thrombocytopenia, which limit the use of CD47 blocking antibodies.
According to the experimental results of the present invention, the use of USP2 inhibitors can also significantly down-regulate the protein level of CD47 by increasing the ubiquitination modification of CD47, achieving the effect of enhancing anti-tumor immunotherapy (fig. 1-6). Furthermore, we also performed weight monitoring of tumor-bearing mice treated with USP2 inhibitors and anti-PD-1 monoclonal antibodies, and we found that neither the single nor the combination had a significant effect on the body weight of the mice after treatment (fig. 7, a).
Since most CD47 blockers will reduce the number of erythrocytes during use, resulting in anemia in tumor patients, we collected blood samples from mice 21 days after treatment of tumor mice, examined the mice blood convention, and found that neither single or combination groups had significant adverse effects on erythrocytes, hemoglobin, and platelets in mice (fig. 7, b-D).
In summary, the invention discovers a novel treatment method, namely, the level of CD47 protein is regulated by regulating CD47 ubiquitination modification, so that the anti-tumor immunotherapy is enhanced, and compared with the prior CD47 blocking, the method has the advantage of small toxic and side effects on the premise of ensuring tumor therapy.
The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.
Claims (3)
1. A pharmaceutical composition for tumor immunotherapy, characterized in that the pharmaceutical composition consists of a CD47 protein ubiquitination modified agonist and a monoclonal antibody; the CD47 protein ubiquitination modified agonist is ML364, and the monoclonal antibody is an anti-PD-1/PD-L1 monoclonal antibody; the tumor is lung cancer tumor.
2. The tumor immunotherapeutic pharmaceutical composition of claim 1, wherein the mass ratio of ML364 to anti-PD-1/PD-L1 monoclonal antibody is 1:1.5.
3. Use of a pharmaceutical composition according to claim 1 or 2 for the preparation of a medicament for tumour immunotherapy.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210563765.0A CN116178302B (en) | 2022-05-23 | 2022-05-23 | CD47 protein ubiquitination modified agonist and application thereof |
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| CN111214664A (en) * | 2020-02-24 | 2020-06-02 | 大连医科大学 | Application of combination of USP2 and HSP90 inhibitor in inhibiting growth of ErbB2 positive breast cancer |
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Non-Patent Citations (4)
| Title |
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| "The deubiquitylase USP2 maintains ErbB2 abundance via counteracting endocytic degradation and represents a therapeutic target in ErbB2-positive breast cancer";Jinrui Zhang deng;Cell Death & Differentiation;第27卷(第9期);第2710-2725页 * |
| "Small Molecule Inhibition of the Ubiquitin‑specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models";Mindy I. Davis 等;JOURNAL OF BIOLOGICAL CHEMISTRY;第291卷(第47期);摘要，第24631页 * |
| "USP2 is an SKP2 deubiquitylase that stabilizes both SKP2 and its substrates";Fengwu Zhang 等;JOURNAL OF BIOLOGICAL CHEMISTRY;第297卷(第4期);摘要，第2页右栏、第5页右栏，图3、6 * |
| "去泛素化酶USP2抑制剂对卵巢癌的作用与机制研究";刘秀秀;中国优秀硕士学位论文全文数据库 医药卫生科技辑;摘要 * |
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