CN105832673A - Application of cuprous oxide nanoparticles to preparation of medicine for treating renal cancer diseases - Google Patents
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Abstract
The invention relates to application of cuprous oxide nanoparticles to preparation of a medicine for treating renal cancer diseases. Renal cancers include clear cell renal cancer, granular cell renal cancer, mixed cell renal cancer and undifferentiated cell renal cancer. The invention further provides the medicine for treating the renal cancers. Expression profile sequencing is conducted after the medicine for treating the renal cancers acts, and the medicine acts in in-vivo experiments of a cell line and in-vitro experiments of nude murine with tumor subcutaneously implanted. Functions and related signaling pathways of CONPs in renal cancer treatment are further explained. A theoretical foundation is laid for exploring a novel metastatic renal cell carcinoma treatment method.
Description
Technical field
The present invention relates to pharmaceutical technology field, specifically, be in preparation treatment about cuprous nano grain
Application in kidney disease medicament.
Background technology
Kidney accounts for the 2～3% of all malignant tumours.Although some patients can reach the purpose of radical cure by operation,
But 20～30% patient just have occurred and that DISTANT METASTASES IN when making a definite diagnosis, 30～40% there is recurrence and transfer after operation in patients.
Kidney is the most insensitive to chemicotherapy.Tumor tissues has special high-permeability and anelasticity, nano-scale
Carrier could pass through the gap of tumor vascular endothelium system, carries medicine and enters and accumulate in tumor tissues；
Secondly, nano-carrier can realize medicine intelligent conveying in vivo by functional modification, has target
To location and the function of drugrelease, reduce the toxic and side effect of medicine simultaneously；Additionally, special by nano-carrier
Some structures and character, can load insoluble drug.Nano medication, compared with conventional medicine, has particle diameter
Little, dissolubility compared with big, permeability is good, catalytic efficiency is high, surface-active is high, activated centre is many, absorption
The advantages such as power is strong.Have been reported display tumor tissues copper metabolic disorder to enliven, cuprous nano grain (CONPs)
The mechanism that the cuprous ion carried acts on the pharmacodynamics of renal carcinoma tissue and signal path is unclear.
Summary of the invention
It is an object of the invention to for deficiency of the prior art, it is provided that a kind of cuprous nano grain is in preparation
Application in treatment kidney medicine.
Another purpose of the present invention is to provide a kind of medicine treating kidney.
For achieving the above object, the present invention adopts the technical scheme that: cuprous nano grain is in preparation treatment
Application in kidney medicine.
The particle diameter of described cuprous nano grain is between 50-100nm.
Described kidney is clear cell type kidney, granular cell type kidney, mixed cell type kidney, undifferentiated
Cellular type kidney.
Described kidney is clear cell type kidney.
For realizing above-mentioned second purpose, the present invention adopts the technical scheme that:
A kind of medicine treating kidney, described medicine is by cuprous nano grain and pharmaceutically acceptable carrier
Or excipient composition.
The particle diameter of described cuprous nano grain is between 50-100nm.
Described kidney is clear cell type kidney, granular cell type kidney, mixed cell type kidney, undifferentiated
Cellular type kidney.
Described kidney is clear cell type kidney.
The invention has the advantages that: the present invention is checked order by express spectra after kidney medicine effect and medicine acts on carefully
The experiment in vivo of born of the same parents system and the experiment in vitro of nude mice by subcutaneous lotus knurl.CONPs is explained further treat in kidney
Effect and associated signal paths, for exploring the methods for the treatment of based theoretical of new metastatic renal cell carcinoma.
Accompanying drawing explanation
Fig. 1 .CONPs can show significant lethal effect to renal carcinoma cell line A498 and 786-O.
Fig. 2 .CONPs can significantly induce renal carcinoma cell line A498 and 786-O Apoptosis
Fig. 3 .CONPs can significantly inhibit migration and the invasion and attack of A498,786-O cell.
Fig. 4 .CONPs can cause A498 (A) and the cell cycle progression of 786-O (B) cell.
Fig. 5 .KEGG database-rise signal path
The signal path of Fig. 6 .KEGG database-gene-ratio figure-rise
The signal path of Fig. 7 .KEGG database-downward
The signal path of Fig. 8 .KEGG database ratio chart-downward
The signal path of Fig. 9 .GO database-rise
The signal path of Figure 10 .G0 database-rise
The signal path of Figure 11 .G0 database ratio chart-rise
The signal path that Figure 12 .G0 database is lowered
The signal path of Figure 13 .GO database multiple enrichment figure-downward
The signal path of Figure 14 .G0 database ratio chart-downward
Detailed description of the invention
The detailed description of the invention provided the present invention below elaborates.
1 experiment material
786-O cell is grown on RPMI1640 culture medium (the Gibco/BRL public affairs of 10% hyclone (FBS)
Department), A498 cell is grown on the MEM culture medium (Gibco/BRL company) of 10%FBS, 37 DEG C, 5%
Incubator in persistently cultivate.CCK-8 kit is purchased from east, Shanghai Renhua Science and Technology Ltd.,
AnnexinV/FITC apoptosis kit and cell cycle kit are limited purchased from Hangzhou connection section biotechnology share
Company.Transwell cell is purchased from Corning Incorporated.
2 methods
2.1, cell proliferation experiment
96 orifice plates, every hole adds the cell suspension of 200 μ l, 3000, every hole cell, overnight incubation, abandons
Culture medium, adds CONPs by concentration gradient, except for a control group, experimental group concentration gradient is set as 1.25,
2.5,5,10,20 μ g/ml are respectively 48, within 72 hours, discard the culture medium adding medicine, by 10:1's
The mixed liquor that the every hole of ratio adds the CCK8 solution of 10 μ l and 100 μ l culture mediums prepare, 37 DEG C of incubators are incubated
Educate 4 hours, at 450nm, measure cell absorbance with ELIASA.
2.2, cell apoptosis assay
In six orifice plates, every hole adds 100,000 cells, every hole 2ml culture medium, overnight incubation, and 72 hours real
Testing group and add CONPs according to concentration gradient in second day, concentration gradient is 1.25,2.5,5,10 μ g/ml.
48 hours experimental group at the 3rd day according to concentration gradient dosing.At up flow type analyzer on the same day, collect respectively
Cell supernatant enters centrifuge tube, PBS attached cell, and trypsinization attached cell, according to concentration gradient
Adding in corresponding supernatant, be centrifuged and abandon culture medium, resuspended being centrifuged of PBS abandons supernatant, Annexin V binding
Resuspended being centrifuged of buffer abandons supernatant, and cell concentration is few, and 200 μ l Annexin V binding buffer are resuspended
(cell concentration adds to 300-400ul), add Annexin V FITC 5 μ l, and lucifuge hatches 15
After minute, add 5 μ l PI, and upper machine testing.Experiment initial data flowjo 7.6 is analyzed.Note
Stay a pipe do not add Annexin V FITC/PI as blank.Whole process to operate on ice.
2.3, the cell cycle tests
In six centimetres of wares, the every ware of control group adds 50,000 cells, 1.25,2.5 μ g/ml groups, and every ware adds
100000 cells, 5 μ g/ml groups, every ware adds 200,000 cells, 10 μ g/ml groups, and every ware adds 30
Ten thousand cells.Every ware 3ml culture medium, incubator overnight incubation, respectively at second day and the 3rd day according to concentration
Gradient dosing, up flow type machine is analyzed on the same day.Abandoning supernatant, trypsinization is centrifugal abandons supernatant, PBS resuspended from
The heart abandons supernatant, adds 300 μ l staining buffer and 5 μ l PI, after room temperature lucifuge hatches 30 minutes
Up flow type machine is analyzed.Experiment initial data modfit software analysis.
2.4, Cell migration assay
Tumour cell Nature enemy 24 hours, abandons culture medium, trypsinization for second day, is centrifuged, uses serum-free
Culture medium is resuspended, and adjusting cell density is every ten thousand cells of 200 μ l1, puts into cell, cell in 12 orifice plates
Upper strata adds the suspension of 200 ten thousand cells of μ l1, and cell lower floor adds the 600 μ l cultivation containing 10% hyclone
Base.Noticing that cell does not the most form bubble, after 48 hours, by PBS drip washing cell levels, cotton swab is light
Dab the cell on cell upper strata, fix three hours with absolute ethyl alcohol, use violet staining 1 hour afterwards, wind
Amplify 100 times under microscope after dry, choose 10 visuals field at random and take pictures.
2.5, cell invasion is tested:
Tumour cell Nature enemy 24 hours, abandons culture medium, trypsinization for second day, is centrifuged, uses serum-free
Culture medium is resuspended, and adjusting cell density is every ten thousand cells of 200 μ l1, puts into cell, cell in 12 orifice plates
Upper strata adds matrigel, and simulated substrate film, cell upper strata adds the suspension of 200 ten thousand cells of μ l1, cell
Lower floor adds the 600 μ l culture medium containing 10% hyclone.Noticing that cell does not the most form bubble, 48 is little
Shi Hou, by PBS drip washing cell levels, the cell on cell upper strata wiped gently by cotton swab, solid with absolute ethyl alcohol
Fixed three hours, use violet staining 1 hour afterwards, amplify 100 times under microscope after air-drying, randomly select 10
Take pictures in the individual visual field.
The test of correlated expression spectrum and analysis after 2.6, CONPs effects
Renal cancer cell line 786-O, the application PMRI1640 culture medium containing 10% hyclone (FBS)
Cultivating, in 5%CO2 concentration, cultivate in 10cm culture dish under the conditions of 37 DEG C, experimental group and control group are equal
Support 3 wares, treat cell density reach 70%-80%, in good condition when, by every ware cell add 1ml
RNAiso, fully blows and beats mixing, is stored in-80 DEG C of ultra low temperature freezers.Extract total serum IgE, it is ensured that sample OD
260/280 value between 1.9-2.2, RNA total amount at 10 more than μ g, purify through mRNA, rich
Collection, application bivalent cation high-temperature heating method is by mRNA fragmentation, with short-movie section mRNA as template, should
Synthesize cDNA with random primed reverse transcription, rear cDNA is purified, end reparation, add adenine base,
Add the series of experiments such as sequence measuring joints, it is thus achieved that the cDNA template of purifying also sets up cDNA library, through library
After quality inspection is qualified, by a bunch generation step, check order in Illumina HIseq2500 platform；To survey
Sequence result carries out gene coverage analysis, gene expression analysis (unite by quantitative gene expression, gene expression profile
Meter, expression correlation analysis between sample) etc. statistics, search out the information such as group difference gene expression, go forward side by side
Row utilizes KEGG and GO to carry out sequencing result analysis.
3, result
3.1CONPs can significantly inhibit kidney cancer cell propagation
The CONPs process of variable concentrations is incubated at the kidney cancer cell 48 of 96 orifice plates and after 72 hours, uses
The detection CONPs impact on hyperplasia of CCK8 method, testing result shows, variable concentrations CONPs acts on
786-O cell 48 and after 72 hours, all has significant difference (P < 0.001) compared with control group.With
After batten part acts on A498 cell 48 hours, its OD value compared with control group except 1.25 μ g/ml groups
(P=0.014), remaining each group all has significant difference (P < 0.001) compared with control group, and effect 72 is little
Shi Hou, 1.25 μ g/ml group (P=0.012), remaining each group all has significant difference compared with control group
(P<0.001).And thus calculate CONPs process 786-O cell 48h IC50=2.615 μ g/ml,
The IC50=1.763 μ g/ml of 72h.CONPs processes the IC50=5.08 μ g/ml of the 48h of A498 cell,
The IC50=3.512 μ g/ml of 72h.(Fig. 1)
3.2CONPs can significantly induce kidney cancer cell generation apoptosis
Kidney cancer cell vigor is suppressed, it is possible to the generation of inducing cell apoptosis, and this research uses Annexin
The double dye method of V/PI have detected CONPs impact apoptotic on 786-O and A498.Respectively with 1.25 μ g/ml,
Two strain cells are processed 48 hours and 72 hours by 2.5 μ g/ml, 5 μ g/ml, the CONPs of 10 μ g/ml.
CONPs acts on 786-O cell 48 hours, early apoptosis from 6.64% increasing respectively to 5.49%, 12.6%,
45.6%, late apoptic increases respectively to 13.1%, 11.4%, 5.45% from 6.97%, and medicine effect 72 is little
Time after, early apoptosis increases respectively to 8.1%, 15.6%, 70.8% from 6.39%, and late apoptic is from 9.18%
Increase respectively to 13.4%, 17.2%, 13.4%.
The CONPs of same concentration gradient acts on A498 cell 48 hours, and early apoptosis increases respectively from 4.57%
Being added to 9.34%, 19.3%, 14.3%, late apoptic increases respectively to 10.7%, 19.1%, 34.3% from 5.67%.
Medicine effect A498 cell 72 hours, early apoptosis increases respectively to 9.75%, 19.1%, 25.2% from 2.86%,
Late apoptic increases respectively to 17.1%, 45.8%, 53.1% from 6%.(Fig. 2)
3.3CONPs can significantly inhibit kidney cancer cell and migrate and invasion and attack
In cell migration assay, medicine effect 48 hours, in A498 cell, the every high power field of control group (525.9
± 9.1), 1.25 μ g/ml group (293.1 ± 11.3), 2.5 μ g/ml group (181.6 ± 15.9), 5 μ
G/ml group (13.5 ± 3.7), 10 μ g/ml group (1.6 ± 1.1), often group (P < 0.001) compared with control group.
In 786-O cell, the every high power field of control group (346.9 ± 7.8), 1.25 μ g/ml group (231.7 ± 12.6),
2.5 μ g/ml group (166 ± 9.1), 5 μ g/ml group (29.1 ± 10.2), 10 μ g/ml group (5.5 ± 2.2),
And often (P < 0.001) between group and control group.
In cell invasion test, medicine effect 48 hours, in A498 cell, the every high power field of control group (242.7
± 8.4), 1.25 μ g/ml group (130.3 ± 7.0), 2.5 μ g/ml group (55.7 ± 9.2), 5 μ g/ml
Group (17.2 ± 4.7), 10 μ g/ml group (3.5 ± 2.4), often (P < 0.001) between group and control group.
In 786-O cell, the every high power field of control group (273.9 ± 9.4), 1.25 μ g/ml group (126.5 ± 12.5),
2.5 μ g/ml group (82.3 ± 6.9), 5 μ g/ml group (26 ± 6.2), 10 μ g/ml group (3 ± 2.4),
Often group (P < 0.001) compared with control group.(Fig. 4)
3.4CONPs can cause kidney cancer cell Cycle Arrest
In cell cycle experiment, to 786-O cell, set 1.25 μ g/ml, 2.5 μ g/ml, 5 μ g/ml respectively
Concentration gradient, medicine effect 48 and 72 hours are all set, the S phase draws compared with control group,
1.25 μ g/ml48h (P=0.032), 2.5 μ g/ml48h and 1.25 μ g/ml72h (P=0.001), its
Remaining each group compared with control group equal (P < 0.001)；The G2 phase draws 1.25 μ g/ml48h (P=0.022),
1.25 μ g/ml72h (P=0.001), remaining each group compared with control group equal (P < 0.001).Thin at A498
In born of the same parents, the S phase, 1.25 μ g/ml72h (P=0.003), remaining each group compared with control group equal (P < 0.001).
The G2 phase, 1.25 μ g/ml48h (P=0.02), 1.25 μ g/ml72h (P=0.001), remaining is respectively organized with right
(P < 0.001) is compared all according to group.Statistical analysis understands, along with the raising of drug concentration, and action time
Extending, the G1 phase is gradually reduced, and the ratio of S phase and G2 phase gradually steps up, it can thus be appreciated that kidney cancer cell is all
Phase is blocked with S phase and G2 phase.
3.5CONPs processes the express spectra sequencing analysis result of kidney 786-O clone
In KEGG database, CONPs acts on human renal carcinoma cell, and the signal path of rise mainly has: endoplasm
Plectin is processed, cell endocytic, and virus infects, spliceosome, the circulation of Vibrio cholerae infection, synaptic vesicle,
The signal paths such as rheumatoid arthritis, proteasome, mineral absorption, concetrated pipe acidic materials secretion (figure
5)。
In gene-ratio figure, the signal path that prompting is raised mainly has: endoplasmic reticulum Protein processing, intracellular
Gulping down, virus infects, spliceosome, the circulation of rheumatoid arthritis, synaptic vesicle, proteasome, cholera arc
The signal paths (Fig. 6) such as bacterium infection, mineral absorption, concetrated pipe acidic materials secretion.
Same in KEGG database, the signal path that display is lowered mainly has: cancer, virus causes
Cancer occurs, systemic loupus erythematosus, alcoholism, and cancer of pancreas, non-small cell lung cancer, mucopolysaccharide are biological
Synthesis, chondroitin sulfate biosynthesis, dermatan sulfate biosynthesis, chronic granulocytic leukemia, thin
The signal paths (Fig. 7) such as born of the same parents' cycle, base excision repair, alcoholism.
In ratio chart, the signal path of downward is main still: tumour, alcoholism, virus resulted tumour
Generation, systemic loupus erythematosus, cell cycle, cancer of pancreas, chronic granulocytic leukemia, non-small cell
Lung cancer, base excision repair, mucopolysaccharide biosynthesis, chondroitin sulfate biosynthesis, dermatan sulfate life
The signal paths (Fig. 8) such as thing synthesis.
In GO database, the signal path of rise mainly has: organic metabolism process, metabolism mistake
Journey, the metabolic process of heterocycle, gene expression, the metabolic process of cell nitrogen-containing compound, cellular process,
Cellular macromolecule metabolic process, protein folding, to the reaction of topoisomerase albumen and to non-collapsible egg
The metabolic pathways such as white reaction (Fig. 9).
In the enrichment times figure of GO database, the signal path of rise mainly has: protein refolding, bag
Contain body assembles, inclusion body assembles regulation, glucose starvation is reacted, to cadmium metal and zinc ion by cell
The metabolic pathways (Figure 10) such as reaction.
In the ratio chart of GO database, the signal path of rise mainly has: metabolism, cell metabolism, organic matter
The generation such as metabolism, cellular macromolecule metabolism, cell nitrogen-containing compound metabolism, heterocycle metabolism and gene expression
Thank to path (Figure 11).
In GO database, the signal path of downward mainly has: single creature body metabolic process, primary generation
Apologize for having done sth. wrong journey, organic metabolism process, nitrogen-containing compound metabolic process, cellular macromolecule metabolic process, metabolism
And the signal path (Figure 12) such as cellular process.
Multiple enrichment figure in, the signal path of downward relates generally to: ubiquinone metabolism, ubiquinone biosynthesis,
The proteosome of SCF dependence and the protein metabolism of ubiquitin dependence, the reaction of folic acid, respiratory chain complex four
Assembling, the regulation that connects of spindle, quinones biosynthesis, albumen be transported into peroxisome and
The metabolic pathways (Figure 13) such as phosphatidyl glycerol biosynthesis.
In ratio chart, the signal path of downward mainly has: metabolism, organic metabolism, primary metabolite, cell
Metabolism, cellular macromolecule metabolism, nitrogen-containing compound metabolism, cell nitrogen-containing compound metabolism, heterocycle metabolism,
The signal paths (Figure 14) such as single organism metabolism.
The above is only the preferred embodiment of the present invention, it is noted that common for the art
Technical staff, on the premise of without departing from the inventive method, it is also possible to makes some improvement and supplements, these
Improve and supplement and also should be regarded as protection scope of the present invention.
Claims (8)
1. cuprous nano grain application in preparation treatment kidney medicine.
The cuprous nano grain the most according to claim 1 application in preparation treatment kidney medicine, its
Being characterised by, the particle diameter of described cuprous nano grain is between 50-100nm.
The cuprous nano grain the most according to claim 1 application in preparation treatment kidney medicine, its
Being characterised by, described kidney is clear cell type kidney, granular cell type kidney, mixed cell type kidney
Cancer, neoblast type kidney.
The cuprous nano grain the most according to claim 3 application in preparation treatment kidney medicine, its
Being characterised by, described kidney is clear cell type kidney.
5. the medicine treating kidney, it is characterised in that described medicine is by cuprous nano grain and pharmaceutically
Acceptable carrier or excipient composition.
A kind of medicine treating kidney the most according to claim 5, it is characterised in that described cuprous oxide
The particle diameter of nanoparticle is between 50-100nm.
A kind of medicine treating kidney the most according to claim 5, it is characterised in that described kidney is
Clear-cells type kidney, granular cell type kidney, mixed cell type kidney, neoblast type kidney.
A kind of medicine treating kidney the most according to claim 7, it is characterised in that described kidney is
Clear-cells type kidney.
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|---|---|---|---|---|
| CN108785326A (en) * | 2018-07-18 | 2018-11-13 | 许传亮 | Cuprous nano grain is preparing the application in treating bladder cancer drug |
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Application publication date: 20160810 |