CN113116924A - Application of cerium oxide nanoparticles in preparation of medicine for enhancing cytotoxic CD8+ T cell activity - Google Patents
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Abstract
The invention relates to an application of cerium oxide nanoparticles in preparation of a medicine for enhancing cytotoxicity CD8+ T cell activity, belonging to the technical field of biological medicineCytokine secretion and effector molecule synthesis in sex CD8+ T cells enhances cytotoxic CD8+The specific antigen killing capacity of T cells, and in animal experiments, the cerium oxide nanoparticles enhance the virus clearing capacity of organisms. The new application of the cerium oxide nanoparticles provides a new idea for treating diseases clinically by taking cytotoxic CD8+ T cells as a treatment target.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of cerium oxide nanoparticles to preparation of a medicine for enhancing activity of cytotoxic CD8+ T cells.
Background
Cytotoxic CD8+T cell (Cytotoxic CD 8)+T cells, CTLs) are important in controlling viral, tumor and intracellular bacterial infections. CTLs in the novel coronary pneumonia (SARS-coV-2) outbreak in 2020 have been shown to play an important role in resisting SARS-coV-2 infection and preventing asymptomatic and mild infected persons from progressing to moderate and severe infections. When an organism encounters an antigen, the antigen presenting cells activate the primary CD8 in secondary lymphoid organs+T cells (
CD8+T cells). Activated CD8+T cells migrate to the infected site after clonal propagation and kill pathogenic microorganisms or cancer cells by directly killing or releasing effector molecules such as IFN-gamma, IL-2, TNF-alpha, granzyme B and perforin. Therefore, how to effectively improve the activity of CTLs to effectively kill pathogenic microorganisms and tumors is very important!
At present, the following 2 methods are commonly used for improving the killing activity of CTLs:
(1) enhancing the expression of specific transcription factors within CTLs facilitates the synthesis and release of effector molecules. T-beta and Eomes can synergistically promote the expression of IFN-gamma, granzyme B and perforin in CTLs. Blimp1 promoted migration of CTLs to sites of inflammation and infection, and enhanced expression of IFN- γ and granzyme B.
(2) Adoptive T cell therapy (ACT) is largely divided into 3 types: tumor Infiltrating Lymphocytes (TILs), TCR-T cells, and CAR-T cell therapy. Currently most widely used CAR-T therapies for tumor cell therapy require that activated T cells be genetically engineered to mount CARs (tumor chimeric antigen receptors) to specifically recognize and kill tumor cells.
However, the above two methods also have many disadvantages: enhancing the expression of specific transcription factors to promote the synthesis and release of effector molecules has great technical difficulty. At present, the method is generally realized by synthesizing small molecular compounds, but the price is high, and the small molecular compounds lack targeting property. Different transcription factors play different roles in different cells, and wrong transcription factor activation is not beneficial to the stability of organisms and even generates toxic and side effects. Although ACT therapy has achieved compelling success, CAR-T therapy in particular has been FDA approved for the treatment of certain hematologic tumors. However, it is expensive, has poor curative effect on solid tumors, can only act against specific antigens, and has severe cytokine release syndrome and the like which severely restrict the application of the drug.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an application of cerium oxide nanoparticles in preparing a drug for enhancing the activity of cytotoxic CD8+ T cells, and an object of the present invention is to provide a drug for enhancing the activity of cytotoxic CD8+ T cells.
In order to achieve the purpose, the invention provides the following technical scheme:
1. use of cerium oxide nanoparticles for the preparation of a medicament for enhancing the activity of cytotoxic CD8+ T cells.
As one of the preferable technical proposal, the preparation method of the cerium oxide nano-particles is that Ce (NO)3)3·6H2Adding O and trioctylphosphine oxide into ethanol and 1-octadecene, heating to 90 deg.C under vacuum, maintaining for 15min, adding oleylamine, heating to 190 deg.C, maintaining for 15min, cooling at room temperature, and washing to colorless to obtain cerium oxide nanoparticles, wherein Ce (NO) is cerium oxide3)3·6H2The mass-volume ratio of O, trioctylphosphine oxide, ethanol, 1-octadecene and oleylamine is 0.7:1:3:5:100, g: g: mL: mL: μ L.
As one of the preferred technical solutions, the cytotoxic CD8+ T cell is a therapeutic target for diseases.
As one of the preferable technical schemes, the diseases are virus infectious diseases, tumors and intracellular bacteria infection.
As one of the preferable technical schemes, the activity of the cytotoxic CD8+ T cells is specifically enhanced, and the killing capacity of the cytotoxic CD8+ T cells is improved.
As one of the preferable technical schemes, the activity of the cytotoxic CD8+ T cell is enhanced, in particular, the secretion of cytokines and the synthesis of effector molecules in the cytotoxic CD8+ T cell are enhanced.
As one of the preferable technical schemes, the signaling pathway for enhancing the activity of the cytotoxic CD8+ T cells is that the mitochondrial membrane potential in the cytotoxic CD8+ T cells is reduced, so that the generation of active oxygen is reduced, and the NF-kB signaling pathway is activated.
2. A medicament for enhancing cytotoxic CD8+ T cell activity, the medicament comprising cerium oxide nanoparticles and one or more pharmaceutically acceptable carriers or excipients.
The invention has the beneficial effects that:
the invention proves that the cerium oxide nano-particles have no cytotoxicity to CTLs and do not influence the proliferation capacity of cells through cell experiments, and remarkably enhances the synthesis and secretion of CTLs effector molecules and cytokines such as IL-2, TNF-alpha, granzyme B and perforin; the enhanced synthesis and secretion of cytokines and effector molecules allows CTLs to be more effective in killing target cells presenting specific antigens in vitro. In an LCMV (liquid crystal display virus) infection model in a mouse, the injection of the cerium oxide nanoparticles helps the mouse to more effectively eliminate virus infection of organs (heart, liver, spleen, lung and kidney) of each tissue; and the enhancement of CTLs function by the cerium oxide nanoparticles is dependent on the activation of NF-kappa b channels. The new application of the cerium oxide nanoparticles provides a new idea for treating diseases clinically by taking cytotoxic CD8+ T cells as a treatment target.
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For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:
fig. 1 is a design and characterization of cerium oxide nanoparticles. a is a structure diagram of the cerium oxide nano-particles, and b is a scanning electron microscope detection diagram.
FIG. 2 is a graph showing the survival and proliferation potency of CTLs treated with cerium oxide nanoparticlesAnd (6) measuring. a is the survival condition of CTLs detected after the CTLs and the cerium oxide nanoparticles are co-cultured for 4 days; b is a statistical graph of the percentage of live cells; c is a representative diagram of cell division of CTLs; d is Ki67+Scale of cells; e is Ki67+Cell ratio histogram.
Fig. 3 shows the effect of cerium oxide nanoparticles on the production and release capacity of CTLs cytokines and effector molecules after treatment. a is a flow-type representative graph and a positive cell proportion statistical graph of the IFN-gamma positive cell population, and a statistical graph of average fluorescence intensity values; b is a flow representation diagram and a positive cell proportion statistical diagram of the IL-2 positive cell population; c is a flow representation diagram and a positive cell proportion statistical diagram of the TNF-alpha positive cell population; d is a flow-type representative graph and a positive cell proportion statistical graph of the granzyme B positive cell population, and a mean fluorescence intensity value statistical graph; e is a flow representation diagram of the perforin positive cell population, a positive cell proportion statistical diagram and a mean fluorescence intensity value statistical diagram.
Fig. 4 is a graph showing the effect of cerium oxide nanoparticles on the killing ability of CTLs. a is an in vitro specific killing experiment of CTLs; b is the virus titer of serum, liver, spleen and kidney of mice infected by CNPs intraperitoneal injection LCMV, and 5 days later.
FIG. 5 shows the result of the activation of NF- κ b pathway by the cerium oxide nanoparticles by scavenging ROS. a is DCFH-DA+Flow representation of cells and statistical plots of MFI values; b, c is a flow representation diagram and a proportion statistical diagram of the CTLs polarized by mitochondria; d is WB for detecting NF-kappa B channel related proteins IKK beta, p-I kappa B alpha and I kappa B alpha.
Fig. 6 is a schematic diagram of the cerium oxide nanoparticles enhancing the function of CTLs.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1
Preparation and morphology detection of cerium oxide nanoparticles
The preparation method of Cerium oxide nanoparticles (CNPs) comprises the following steps: 0.7g Ce (NO)3)3·6H2O (Acros, 99%) was mixed with 1g trioctylphosphine oxide (Sigma-Aldrich, 99%) in 3mL ethanol and 5mL 1-octadecene (Sigma-Aldrich, 90%), heated to 90 deg.C under vacuum for 15min, 100. mu.L oleylamine was added, heated to 190 deg.C for 15 min. After cooling to room temperature, washing with acetone until the supernatant is colorless, and obtaining the cerium oxide nanoparticles.
The scanning electron microscope showed that the cerium oxide nanoparticles showed a and b in fig. 1, and the CNPs were uniform nanoparticles with a diameter of about 4 nm and were spherical.
Example 2
Cerium oxide nanoparticle pair CD8+Effect of T cells
Screening purification of initial CD8 from mice+T cells, the specific method is as follows:
WT mice were sacrificed and their spleens were ground in a petri dish containing 3mL of ACK and left for 3-5 min. The reaction was terminated by adding 6mL of FACS, and transferred to a 15mL centrifuge tube after being flushed well with a pipette. Centrifuge at 1600rpm for 5min in a 4 ℃ centrifuge. The tube was removed from the supernatant, 1mL of FACS solution was added and the cells resuspended by pipetting with a pipette gun, and the cell suspension was pipetted into an EP tube. Centrifuge at 2000rpm for 5min at 4 ℃. The antibody suspension was prepared according to the 600. mu.L system, the supernatant was removed after centrifugation in an EP tube, the antibody suspension was added, and the mixture was spun on a spinner in a freezer at 4 ℃ for 30 min. The beads were prepared, aspirated into a new EP tube, placed on a magnetic rack, washed twice with PBS solution, and then resuspended in an equal amount of PBS solution. Add 100. mu.L of the prepared magnetic beads to the cell solution and spin on the rotator in a 4 ℃ freezer for 20 min. The magnetic beads enter the liquid and are linked to the FC end of the antibody, while the Fab end of the antibody binds to the cell surface marker. Placing the EP tube containing cell, antibody and magnetic bead suspension on magnetic frame for 1-2min, after the magnetic beads are completely adsorbed on the tube wall of the EP tube, extending the pipette into one side of the tube wall, carefully sucking supernatant, transferring into another new EP tube, adding 50 μ L of the prepared magnetic beads, and rotating in a rotator in a 4 deg.C freezer for 20 min. The EP tube was placed on a magnetic stand, after the liquid was clarified, the supernatant was aspirated off and transferred to another new EP tube. And (4) counting the cells obtained by screening and detecting the purity of the cells.
(1) Effect of cerium oxide nanoparticles on the survival and proliferative Capacity of CTLs
a. Detection of cytotoxicity of cerium oxide nanoparticles to CTLs
The primary CD8+ T cells obtained by screening were co-cultured with the addition of cerium oxide nanoparticles at different concentrations (0, 10. mu.M, 100. mu.M, 500. mu.M, and 1000. mu.M) while stimulating activation in vitro. After 4 days of co-culture, the proportion of surviving cells was examined by Annexin V and 7-AAD staining. The results are shown in fig. 2 a, b, with about 90% cell survival and no statistical difference between the groups, indicating that the cerium oxide nanoparticles are not toxic to CTLs.
b. Detection of the Effect of cerium oxide nanoparticles on the proliferative Capacity of CTLs
After primary CD8+ T cells were fluorescently labeled with carboxyfluorescein diacetate (CFSE), cerium oxide nanoparticles of different concentrations (0,10 μ M,100 μ M) were added while stimulating activation in vitro, and co-cultured for 4 days for flow detection. The CFSE fluorochrome binds to the cell membrane and its fluorescence intensity is halved with the binary division of the cell, so a peak in the flow chart represents one generation of proliferation. As a result, CTLs showed similar proliferation efficiency (about 3 generations) under the culture conditions of cerium oxide nanoparticles at different concentrations, as shown in FIG. 2, c. According to the same method, after adding cerium oxide nanoparticles (0,10 mu M and 100 mu M) with different concentrations for co-culture for 4 days while stimulating activation in vitro, cell proliferation marker Ki-67 protein is detected, and as a result, as shown in d and e in FIG. 2, the ratio of Ki-67 protein expressed by CTLs under different concentration conditions is similar. The above results indicate that cerium oxide nanoparticles have no effect on the proliferation of CTLs.
(2) Cerium oxide nanoparticles for improving generation and release capacity of CTLs (cytokine variants) cytokines and effector molecules
While stimulating and activating the original CD8+ T cells in vitro, adding cerium oxide nanoparticles with different concentrations (0,10 μ M,100 μ M) for co-culture, and after 4 days of co-culture, the CD8+ T cells are cultured at about 2-4 × 10 per well6The cells were added to a 96-well plate, centrifuged at 1800rpm for 2min, and the supernatant was removed. The cells were washed with freshly prepared FACS solution. The FACS solution in 200. mu.L of liquid is sucked by a discharge gun, added into a 96-well plate, uniformly blown, centrifuged at 1800rpm for 2min, the supernatant is removed, washed for 2 times, and pre-cooled corresponding surface staining antibody is added in a ratio of 1: 200. Add 100. mu.L of antibody suspension (prepared according to different experimental purposes) to each well, mix well, and incubate in a refrigerator at 4 ℃ for 30min in the dark. The 96-well plate was centrifuged at 1800rpm for 2min, and the supernatant was discarded. Each well was washed 2 times with 200. mu.L of FACS solution, and the supernatant was discarded. mu.L of the membrane-penetrating fluid (Cytofix/cytoperm solution, BD) was added to each well, and incubated at 4 ℃ in a dark environment for 30 min. The membrane penetrating fluid contains paraformaldehyde, the cell is fixed and can be killed, and then other components in the reagent can make small holes in the cell membrane of the cell, so that the flow-type antibody enters the cytoplasm and is combined with corresponding protein. The 96-well plate was removed, centrifuged at 2100rpm for 3min, and the supernatant was discarded. Cells were washed 2 times with 200. mu.L of 1 XPerm/Wash Buffer per well. The antibody suspension was prepared at a ratio of 1:50 using 1 XPerm/Wash Buffer, and 100. mu.L of the antibody suspension was added to each well and incubated at 4 ℃ in a dark environment for 30 min. The 96-well plate was removed, centrifuged at 2100rpm for 3min, the supernatant was discarded, and 200. mu.L of 1 XPerm/Wash Buffer was added to each well to Wash the cells 2 times. The appropriate amount of FACS solution was added and transferred to a flow tube, tested on the machine and analyzed by Flowjo software.
The effect of cerium oxide nanoparticles on the secretion of CTLs cytokines the results are shown in fig. 3 a, b, c, where most of the activated CTLs expressed IFN- γ and the mean fluorescence intensity values were very similar between groups (a in fig. 3); after cerium oxide nanoparticle treatment, the treated group had a higher proportion and mean fluorescence intensity of IL-2 secretion compared to the control group, and this increase was dose-related (fig. 3 b); although activated CTLs showed similar secretion frequency in the treated and control groups, the MFI value of TNF-. alpha.in the treated cells was clearly increased, indicating that the treatment with cerium oxide nanoparticles enhanced the TNF-. alpha.secretion capacity of individual CTLs (FIG. 3, c).
Effect of cerium oxide nanoparticles on CTLs effector synthesis results are shown in fig. 3 d, e, that CTL cells release cytolytic effector molecules such as perforin and Granzyme B when they encounter infected or dysfunctional cells. Under the action of perforin, Granzyme B enters the cytoplasm of the target cell and eventually leads to apoptosis of the target cell. After the cerium oxide nanoparticles were added, CTLs had higher Granzyme B secretion frequency and MFI value (d in fig. 3) compared to the control group; the secretion frequency of perforin of the treated cells was also significantly higher than that of the control group (e in FIG. 3).
(3) Cerium oxide nanoparticles for improving killing capacity of CTLs
a. Cerium oxide nanoparticles improve the in vitro killing ability of CTLs
While the primary CD8+ T cells were stimulated to activate in vitro, cerium oxide nanoparticles at various concentrations (0,10 μ M,100 μ M) were added for co-culture for 4 days. EL4 cells with GP33-41 peptide fragment stained with lower concentration CFSE staining solution as target cells for specific killing, called CFSElowAnd (4) grouping. EL4 cells without GP33-41 peptide fragment were stained with higher doses of CFSE as a control, called CFSEhighAnd (4) grouping. After two groups of cells are uniformly mixed according to the ratio of 1:1, the two groups of cells and CTLs treated by cerium oxide nanoparticles are cultured together for 4 hours according to the ratio of 1:30, and then the detection is carried out. As a result, as shown in fig. 4, although the cells of the control group were not changed after co-culturing with CTLs, the target cells were significantly decreased, and the decrease increased with the increase of the treatment dose of CNPs, indicating that the cerium oxide nanoparticles promote the killing ability of CTLs in vitro.
b. Cerium oxide nanoparticles improve the ability to clear viruses in vivo
8 weeks of C57BL/6J mice were injected with different concentrations (0, 10. mu.M, 100. mu.M) of cerium oxide nanoparticles and 2 hours later (2X 10)5PFU) LCMV viral infection. The virus titer profile in different tissues of mice was tested by RT-qRCR on day 5 post infection. The results are shown in b in FIG. 4, in liver, kidney and spleen of miceIn the viscera, the serum and the lung, the virus titer of the treatment group is obviously reduced compared with that of the control group, and the reduction is dose-dependent, and the virus in the tissues of the high-dose group is almost completely eliminated in the liver, the spleen and the serum, which indicates that the cerium oxide nanoparticles effectively improve the killing capability of the CTLs in vivo.
(4) Activation of NF-kappa b pathway by scavenging ROS by cerium oxide nanoparticles
a. The cerium oxide nanoparticles are effective in reducing the active oxygen level and mitochondrial membrane potential level of CTLs
Adding cerium oxide nanoparticles with different concentrations (0,10 mu M and 100 mu M) into the initial CD8+ T cells for co-culture while stimulating and activating in vitro, and performing an ROS staining experiment by using an ROS Detection Assay Kit of Abcam corporation in the United states after co-culture for 4 days to detect the ROS level; the mitochondrial membrane potential level of the cells was detected by Staining using the Mitochondria stabilizing Kit (JC-1) Kit from MultiSciences company.
Results as shown in a, b, c in fig. 5, the ROS levels of CTLs were significantly reduced in the treated group compared to the control group, and this reduction was dose-dependent (a in fig. 5); the mitochondrial membrane potential of P14 cells was also significantly decreased after ceria nanoparticle treatment and also exhibited a dose-dependent decrease (b, c in fig. 5). Taken together, the results indicate that the cerium oxide nanoparticles reduce the level of ROS in CTLs by reducing the mitochondrial membrane potential.
b. Activation of NF-kappa b pathway by scavenging ROS by cerium oxide nanoparticles
The initial CD8+ T cells are stimulated and activated in vitro, cerium oxide nanoparticles with different concentrations (0,10 mu M and 100 mu M) are added for co-culture for 4 days, and the cells are collected and subjected to Western blot detection. Protein electrophoresis was performed by adding to 10% SDS-PAGE gel at a protein content of 30ug per group of samples, followed by protein transfer to PVDF membrane. After the membrane transfer was complete, the PVDF membrane was blocked with 5% skim milk, followed by a 1: 1000 (. beta. -action (clone 8H10D10, Cell Signaling Technology), I.kappa.Balpha. (clone 44D4, Cell Signaling Technology), Phospho-I.kappa.Balpha. (Ser32) (clone 14D4, Cell Signaling Technology), IKK.beta. (clone D30C6, Cell Signaling Technology)) were added to assay the proteins of interest. After overnight incubation, secondary antibody was added and stained for 1 hour for detection.
The results are shown in fig. 5, d, and the treated cells showed higher expression levels of IKK β, p-I κ B α and lower expression levels of I κ B α compared to the untreated cells. In the classical NF-. kappa.b signaling pathway, I.kappa.B.alpha.proteins are in an inactive state by masking the nuclear localization sequence of NF-. kappa.b proteins, thereby leaving the NF-. kappa.b/RelA complex in the cytoplasm. Upon phosphorylation of I κ B α, the resulting p-I κ B α is subsequently degraded by the ubiquitin proteasome pathway. As I κ B α degrades, NF- κ B complex is released into the nucleus and regulates expression of target genes within the nucleus. The above results indicate that treatment with CNPs activates NF-. kappa.B signaling. FIG. 6 is a schematic diagram of enhancement of CTLs function by cerium oxide nanoparticles, showing that CNPs activate NF-kb pathway by eliminating excessive ROS in CTLs, enhancing CTLs function, enhancing the ability to eliminate viral infection, and potentially eliminating bacterial infection and tumors.
Taken together, the results demonstrate that CNPs reduce ROS production and ultimately activate NF- κ B signaling pathways by down-regulating Mitochondrial Membrane Potential (MMP) of CTL cells.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (8)
1. Use of cerium oxide nanoparticles for the preparation of a medicament for enhancing the activity of cytotoxic CD8+ T cells.
2. The use according to claim 1, wherein the cerium oxide nanoparticles are prepared by a process of Ce (NO)3)3·6H2O and trioctylphosphine oxide to ethyleneHeating alcohol and 1-octadecene to 90 deg.C under vacuum, maintaining for 15min, adding oleylamine, heating to 190 deg.C, maintaining for 15min, cooling at room temperature, and washing to colorless to obtain cerium oxide nanoparticles, wherein Ce (NO) is3)3·6H2The mass-volume ratio of O, trioctylphosphine oxide, ethanol, 1-octadecene and oleylamine is 0.7:1:3:5:100, g: g: mL: mL: μ L.
3. The use of claim 1 or 2, wherein the cytotoxic CD8+ T cells are therapeutic targets for a disease.
4. The use of claim 3, wherein the disease is a viral infectious disease, a tumor, an intracellular bacterial infection.
5. The use according to claim 1, wherein the enhancement of cytotoxic CD8+ T cell activity is in particular an increase of the killing capacity of cytotoxic CD8+ T cells.
6. Use according to claim 1, wherein the enhancement of cytotoxic CD8+ T cell activity is in particular the enhancement of cytokine secretion and effector molecule synthesis in cytotoxic CD8+ T cells.
7. The use of claim 1, wherein the signaling pathway that enhances the activity of cytotoxic CD8+ T cells is the down-regulation of mitochondrial membrane potential in cytotoxic CD8+ T cells, thereby reducing reactive oxygen species production and activating the NF- κ B signaling pathway.
8. A medicament for enhancing cytotoxic CD8+ T cell activity, said medicament comprising said cerium oxide nanoparticles and one or more pharmaceutically acceptable carriers or excipients.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104519914A (en) * | 2012-07-19 | 2015-04-15 | 米兰比可卡大学 | Nanoconstructs with pharmacological activity |
| CN113577100A (en) * | 2021-07-20 | 2021-11-02 | 中国人民解放军陆军军医大学 | Application of cerium oxide nanoparticles in preparation of medicines for treating sepsis damage caused by various causes |
-
2021
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104519914A (en) * | 2012-07-19 | 2015-04-15 | 米兰比可卡大学 | Nanoconstructs with pharmacological activity |
| CN113577100A (en) * | 2021-07-20 | 2021-11-02 | 中国人民解放军陆军军医大学 | Application of cerium oxide nanoparticles in preparation of medicines for treating sepsis damage caused by various causes |
Non-Patent Citations (4)
| Title |
|---|
| SHUPEI TANG，等: "Ceria nanoparticles promoted the cytotoxic activity of CD8+ T cells by activating NF-κB signaling", 《BIOMATERIALS SCIENCE》 * |
| 周澜: "氧化铈纳米颗粒对CD8+T细胞功能的影响及其代谢产物对骨骼系统的潜在风险研究", 《中国优秀博硕士学位论文全文数据库（硕士） 医药卫生科技辑》 * |
| 唐舒沛: "CNPs对细胞毒性CD8+T细胞的作用及其机制研究", 《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》 * |
| 徐兴良，等: "金属纳米复合催化剂合成与应用的最新进展", 《无机化学学报》 * |
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