CN107961381A - Application of the DNA tetrahedrons in terms of cell anti-aging is promoted - Google Patents
Application of the DNA tetrahedrons in terms of cell anti-aging is promoted Download PDFInfo
- Publication number
- CN107961381A CN107961381A CN201711213058.4A CN201711213058A CN107961381A CN 107961381 A CN107961381 A CN 107961381A CN 201711213058 A CN201711213058 A CN 201711213058A CN 107961381 A CN107961381 A CN 107961381A
- Authority
- CN
- China
- Prior art keywords
- dna
- cell
- aging
- inflammatory
- tetrahedrons
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of application of DNA tetrahedrons in terms of cell anti-aging is promoted, and the DNA tetrahedrons are by four single-stranded self assemblies of DNA, four single stranded sequences such as SEQ ID NO:Shown in 14.Cell anti-aging process includes anti-inflammatory and antioxidation process.DNA tetrahedrons, which promote the anti-inflammatory effect of cell, to be played a role by the iNOS expressing quantities for reducing the release of inflammatory cytokine, suppressing iNOS gene sums；The antioxidation for promoting cell is played a role by suppressing the expression quantity of the phosphorylation of the albumen ERK1/2, P38, JNK in MAPK/ERK signal paths downstream and promotion induction Heme oxygenases-1.DNA tetrahedrons have good bioavilability and biocompatibility, while have anti-inflammatory and anti-oxidation function, so as to delay the aging of cell.
Description
Technical field
The invention belongs to cell anti-inflammatory anti-oxidation tech field, and in particular to a kind of DNA tetrahedrons are promoting cell anti-ageing
The application of old aspect.
Background technology
With developing rapidly for DNA nanometer technologies, various DNA nanostructures are manufactured.DNA tetrahedrons
(tetrahedral DNAnanostructures, TDNs) is considered recently as one of most promising DNA nanostructure
It is the biomaterial with extensive potential application.TDNs is by four high degree of specificity and programmable DNA single-stranded (ssDNA) from group
Dress forms.The design of single stranded DNA is matched with accurate complementary base, sequence design and complementary function sequence it is specific miscellaneous
Hand over.Different from ssDNA, TDNs can be into mammalian cell and without liposome transfection, and has been kept in cytoplasm
It is whole at least 48 it is small when, this for medicine delivery and its in cell activity control be important properties.As with stabilization
Property the nanostructured for being easy to modification, TDNs be used to by immunopotentiator CpG transporte to cells to induce immunostimulation to make
With.Delivered in vivo in addition, TDNs and siRNAs can be self-assembled into targeting DNA/siRNA nano-particles, cause stable base
Because of silence.In our previous studies, we have demonstrated that TDNs is promoting cell migration, chondrocyte phenotype is maintained, is promoted
Play an important role into stem cell skeletonization.But and have no DNA tetrahedrons in the report applied to the anti-oxidant aspect of cell anti-inflammatory.
The content of the invention
For the above-mentioned problems in the prior art, the present invention provides a kind of DNA tetrahedrons and is promoting cell anti-aging
The application of aspect, can effectively facilitate cell anti-inflammatory antioxidation, and then prevent cell from producing disease, delay its aging.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is：
Application of the DNA tetrahedrons in terms of cell anti-aging is promoted, the DNA tetrahedrons are closed by the single-stranded self assemblies of four DNA
Into four single stranded sequences such as SEQ ID NO:Shown in 1-4.
Further, cell anti-aging process includes anti-inflammatory and antioxidation process.
Further, it is by reducing inflammatory cytokine (such as NO, IL-1 that DNA tetrahedrons, which promote the anti-inflammatory effect of cell,
β, IL-6 and TNF-α) release play a role.
Further, DNA tetrahedrons, which promote the anti-inflammatory effect of cell, is played by suppressing the expression quantity of iNOS genes
Effect.
Further, DNA tetrahedrons, which promote the anti-inflammatory effect of cell, is played by suppressing the expression quantity of iNOS albumen
Effect.
Further, it is to suppress MAPK/ERK signal paths by inducing that DNA tetrahedrons, which promote the antioxidation of cell,
The phosphorylation of the albumen ERK1/2, P38, JNK in downstream plays a role.
Further, it is the expression by inducing DELTA rHO-1 that DNA tetrahedrons, which promote the antioxidation of cell,
Measure to play a role.
Further, DNA four sides used bulk concentration is 100-300nM in cell anti-inflammatory antioxidation process.
Application of the DNA tetrahedrons provided by the invention in terms of cell anti-aging is promoted, has the advantages that：This hair
It is bright to stimulate RAW264.7 cells using LPS, inflammatory response model is established, the DNA tetrahedrons of specified quantitative are acted on into inflammatory conditions
Cell after, the expression of various inflammatory factors such as NO, IL-1 β, IL-6 and TNF-α etc. substantially reduces, and may also suppress at the same time
The expression quantity of iNOS genes and albumen, and then promote the anti-inflammatory effect of cell.
It is to suppress the albumen in MAPK/ERK signal paths downstream by inducing that DNA tetrahedrons, which promote the antioxidation of cell,
The phosphorylation of ERK1/2, P38, JNK plays a role；It can additionally be sent out by improving the expression quantity of DELTA rHO-1
The effect of waving.
DNA tetrahedrons used in the present invention have good bioavilability and biocompatibility, at the same have anti-inflammatory and
Anti-oxidation function, so as to delay the aging of cell.
Brief description of the drawings
Fig. 1 is the tetrahedral polyacrylamide gel electrophoresis figures of DNA；Marker, single-stranded S1 are wherein from left to right followed successively by,
Single-stranded S2, single-stranded S3, single-stranded S4, DNA tetrahedrons, Marker, DNA tetrahedrons are represented in red circle.
Fig. 2 characterizes the tetrahedral success composite result figures of DNA for transmission electron microscope；Wherein, Yellow triangles represent DNA four sides
Body, Polymer represent the polymer produced in building-up process.
Fig. 3 is the tetrahedral grain-size graphs of DNA.
Fig. 4 is the tetrahedral potential energy diagrams of DNA.
Fig. 5 is the NO contents after the TDNs of LPS and various concentrations handles RAW264.7 cells 12h.
Fig. 6 is the NO contents after LPS and TDNs handles RAW264.7 cells 24h and 48h.
Fig. 7 is the influence knot after Western blotting detection DNA tetrahedron effects 24h to iNOS expressing quantities
Fruit.
Fig. 8 is the influence knot after observation DNA tetrahedrons effect 24h to iNOS expressing quantities under laser confocal microscope
Fruit.
Fig. 9 is the influence result after fluorescence quantitative PCR detection DNA tetrahedrons effect 24h to iNOS expressing quantities.
Figure 10 is the inflammatory cytokine secretory volume after LPS and TDNs handles cell with Elisa detections.
Figure 11 is to handle CCK-8 results of the RAW264.7 in 12 and 24h by LPS (1ug/ml) and TDNs (250nM).Number
Average value ± SD (n=4) is represented according to this.
Figure 12 is Flow cytometry RAW264.7 intracellular ROS levels.
Figure 13 is the RAW264.7 intracellular ROS levels that fluorescence microscopy Microscopic observation handles 24h by LPS and TDNs.
Figure 14 is the expression of results of fluorescence quantitative PCR detection HO-1mRNA.
Figure 15 TDNS suppress p38 in the RAW264.7 cells that LPS is induced, the phosphorylation of ERK1/2, JNK1/2/3.
Embodiment
The tetrahedral synthesis of embodiment 1DNA and characterization
1st, the tetrahedral synthesis of DNA
By single-stranded being added to according to equimolar ratio containing 100ul TM buffer of tetrahedral four special designings of DNA
In the 200 μ lEP pipes of (10mMTris-HCl, 50mM MgCl2, pH 8.0), mixed system is then maintained into 10min at 95 DEG C,
Then fast cooling has synthesized DNA tetrahedrons, can have been preserved in 4 DEG C long-term to 4 DEG C of maintenance 20min.
Four the single-stranded particular sequences of DNA are as follows：
S1：
5’-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3’
(SEQ ID NO:1)；
S2：
5’-ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG-3’
(SEQ ID NO:2)；
S3：
5’-ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC-3’
(SEQ ID NO:3)；
S4：
5’-ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG-3’
(SEQ ID NO:4)。
2nd, polyacrylate hydrogel electrophoresis, dynamic light scattering, transmission electron microscope characterize DNA tetrahedrons
The DNA tetrahedrons that success synthesizes carry out 8% polyacrylamide gel electrophoresis characterization first, and loading runs glue, constant pressure
100V runs glue 80min, then using 1:50 Goldview carries out dyeing exposure observation.
The DNA tetrahedrons that success synthesizes are diluted to 250nM with redistilled water and then divide in Zetasizer Nano ZS90
The essential characteristic of TDNs is detected in analyzer, such as zeta potential and is averagely hydrated particle size.
Directly observed after the DNA tetrahedron negative staining that success synthesizes with transmission electron microscope (TEM).
Experimental result：8% non denatured polyacrylamide gel electrophoresis the results show successfully synthesizes DNA tetrahedrons, and
Size is about 150bp (Fig. 1).The form (Fig. 2) of TDNs is characterized by TEM, based on TEM image, it is observed that triangle is received
Rice corpuscles and some polymer, Yellow triangles represent DNA tetrahedrons, and Polymer represents the polymer produced in building-up process.
In addition, understand the average hydration particle diameter about 21.33nm of TDNs by dynamic light scattering result, and TDNs with 0.313 it is scattered
Coefficient (PDI) (Fig. 3).DNA tetrahedrons have the negative charged surface (Fig. 4) of -3.89 ± 0.118mV.From the above results, it can be seen that, I
Successfully synthesized TDNs.
Embodiment 2Griess methods detect influence of the DNA tetrahedrons to inflammatory model cell secretion inflammatory factor NO
The biological effect of NO depends on the slight change of its concentration and intraor extracellular.Under normal physiological conditions, it is few
The NO of amount is beneficial to body.However, the NO of high concentration can cause tissue damage, malignant tumour, rheumatoid arthritis and infection
Property shock.In order to study whether TDNs works in inflammatory reaction, we with lipopolysaccharides (LPS) processing RAW264.7 cells with
Establish inflammatory response model.The generation for checking inflammatory mediator NO is measured by Griess.
Experimental method：Experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, with the medium culture 8h containing 10%FBS, so
The culture medium containing 1%FBS is changed to afterwards, and after balancing 1h, the DNA tetrahedrons added to experimental group one, in experimental group two after filtering are (dense
Spend for 250nM), isometric culture medium is added in negative control group and positive controls.Act on 1 it is small when after, in positive control
LPS solution is added in group and experimental group two, it is 1 μ g/ml to make LPS concentration in culture medium, and negative control group and experimental group one add
Isometric culture medium.Effect after a certain period of time, is produced using using Griess methods detection NO.Take the supernatant of 25 μ l nutrient solutions
Liquid, is separately added into Griess I liquid and GriessII liquid, and nitrite is detected by measuring the absorbance at 540nm after incubation
Generation.Using sodium nitrite as standard test as a result, the NO for then calculating processing cell is horizontal.
Experimental result：LPS stimulates the significant increase for causing NO contents in supernatant, and TDNs reduces NO and produces, particularly
Under the dosage of 250nM, best results.When only being handled with TDNs, it is horizontal non-with being observed in control group that NO produces level
Normal similar (Fig. 5).Therefore, we select to carry out next step research using the DNA tetrahedrons of 250nM concentration.We pass through detection
The NO releases of different time points, it is found that (Fig. 6, left side 24h, right side is interior persistently performance anti-inflammatory effect TDNs when 48 is small
48h).Result above shows that TDNs can resist NO excessive in the inflammation RAW264.7 cells that LPS is induced and produce.Thus can
To draw a conclusion, TDNs has vital effect to adjusting the balance that NO is produced.
Anti-inflammatory mechanisms of the 3 researching DNA tetrahedron of embodiment to RAW264.7 cells
L-arginine can be converted into L-citrulline and NO by endogenous NO essentially from L-arginine, three kinds of isoenzymes, i.e.,
Neuron NO synzyme, Endothelial cell NO synthase and inducible NO-synthase (iNOS) 31,32.INOS is distributed widely in human body, special
It is not macrophage, liver cell, astroglia and smooth muscle cell.Therefore, we further by Laser Scanning Confocal Microscope,
Western Blotting, fluorescence quantitative PCR detection iNOS genes inquire into influences of the TDNs to iNOS levels with protein expression.
Test method：
(1) experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, 37 DEG C are based on the culture containing 10%FBS,
5%CO28h is cultivated in incubator, is then changed to the culture medium containing 1%FBS, after balancing 1h, to experimental group one, in experimental group two
Add DNA (250nM) tetrahedron (isometric culture medium is added in negative control group and positive controls) after filtering.Effect
After 1h, LPS solution is added in positive controls and experimental group two, it is 1 μ g/ml (negative control groups to make LPS concentration in culture medium
Isometric culture medium is added with experimental group one).Continue to collect total protein of cell, Western Blotting methods after cultivating 36h
Detect the change of albumen iNOS expression quantity.
(2) experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, 37 DEG C are based on the culture containing 10%FBS,
5%CO28h is cultivated in incubator, is then changed to the culture medium containing 1%FBS, after balancing 1h, to experimental group one, in experimental group two
Add DNA (250nM) tetrahedron (isometric culture medium is added in negative control group and positive controls) after filtering.Effect
After 1h, LPS solution is added in positive controls and experimental group two, it is 1 μ g/ml (negative control groups to make LPS concentration in culture medium
Isometric culture medium is added with experimental group one).Continue to carry out immunofluorescence dyeing immediately after cultivating 36h, then using copolymerization
Focusing microscope observes the change of albumen iNOS expression quantity.
(3) experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, 37 DEG C are based on the culture containing 10%FBS,
5%CO28h is cultivated in incubator, is then changed to the culture medium containing 1%FBS, after balancing 1h, to experimental group one, in experimental group two
Add DNA (250nM) tetrahedron (isometric culture medium is added in negative control group and positive controls) after filtering.Effect
After 1h, LPS solution is added in positive controls and experimental group two, it is 1 μ g/ml (negative control groups to make LPS concentration in culture medium
Add isometric culture medium with experimental group one), continue culture 24 it is small when after, supernatant is abandoned in suction, collects total serum IgE, is purified, and is reversed
Record as cDNA, then carry out fluorescence quantitative PCR detection.
Experimental result：
(1) WesternBlotting is the results show that the expression quantity of iNOS type albumen has significantly after LPS inductions are added
Rise, illustrates that inflammatory response model is successfully set up.And after DNA tetrahedrons are added, the expression quantity of iNOS significantly reduces, bright
It is aobvious to be higher than control group (not adding DNA tetrahedrons group) (Fig. 7), illustrate that DNA four sides physical efficiency effectively suppresses the expression of iNOS albumen.
(2) laser co-focusing is the results show that nucleus is blueness in Fig. 8, and F- actins are green, and iNOS albumen is green
Color.In positive test group, the green fluorescence particle of state of aggregation is more and obvious in cell, on the contrary, in test group one and test group
There was only seldom green fluorescence particle in two groups, further illustrate that DNA four sides physical efficiency effectively suppresses the expression of iNOS albumen.
(3) fluorescent quantitative PCR result is shown, consistent with the above results, the up-regulation of LPS induction iNOSmRNA expression, however,
After adding TDNs, decayed this process (Fig. 9, data are represented with average value ± SD (n=4)).This result shows that, TDNs exists
Suppress NO by iNOS transcriptional regulatories to produce, and further lower the generation of NO.
The ELISA detections of 4 inflammatory factor of embodiment secretion
Test method：Experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, 37 DEG C are based on the culture containing 10%FBS,
5%CO28h is cultivated in incubator, is then changed to the culture medium containing 1%FBS, after balancing 1h, to experimental group one, in experimental group two
Add DNA (250nM) tetrahedron (isometric culture medium is added in negative control group and positive controls) after filtering.Effect
After 1h, LPS solution is added in positive controls and experimental group two, it is 1 μ g/ml (negative control groups to make LPS concentration in culture medium
Isometric culture medium is added with experimental group one), continue to extract supernatant after cultivating 24h and 48h, carry out ELISA detections, as a result
See Figure 10 (Control, LPS, TDNs, LPS+TDNs are from left to right followed successively by column diagram).
We have detected DNA tetrahedrons by ELISA and some inflammatory cytokines (IL-1 β, IL-6 and TNF-α) secreted
Influence.In order to exclude the possibility for the suppression that the tetrahedral cytotoxicities of DNA cause these cell factors to produce, we pass through
CCK-8, which is measured, standardizes the result of ELISA, the result is shown in Figure 11 (be from left to right followed successively by column diagram Control, LPS,
TDNs、LPS+TDNs)。
Figure 11 is the CCK-8 for handling RAW264.7 cells 12h and 24h respectively by LPS (1 μ g/ml) and TDNs (250nM)
As a result, data are represented with average value ± SD (n=4).
Experimental result：As shown in Figure 10, the time point when 24 is small and when 48 is small, can be reduced with TDNs pretreatments
The release for the inflammatory cytokine (IL-1 β, IL-6 and TNF-α) that LPS is stimulated, therefore deduces that, DNA tetrahedrons have certain
Anti-inflammatory effect.
5 oxidative stress of embodiment
Oxidative stress refers to that intracellular ROS is produced and anti-oxidant unbalance.It causes protein to be repaiied by the generation of free radical
Decorations, lipid peroxidation and subsequent cell dysfunction, and an important factor for be aging and disease.In order to whether study TDNs
Work in anti-oxidant, DCFH-DA fluorescence probes are attached to cell by us, and reactive oxygen species can be aoxidized without glimmering
The DCFH probes of light have the DCF of fluorescence.Therefore, the abundance of intracellular ROS can be green based on being sent from intercellular spaces
Color fluorescence determines that fluorescence intensity can be detected by flow cytometry or directly be observed by fluorescence microscope.
Experimental method：
(1) experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, 37 DEG C are based on the culture containing 10%FBS,
5%CO28h is cultivated in incubator, is then changed to the culture medium containing 1%FBS, after balancing 1h, to experimental group one, in experimental group two
Add DNA (250nM) tetrahedron (isometric culture medium is added in negative control group and positive controls) after filtering.Effect
After 1h, LPS solution is added in positive controls and experimental group two, it is 1 μ g/ml (negative control groups to make LPS concentration in culture medium
Isometric culture medium is added with experimental group one).Continue culture 12 it is small when after, cell and DCFH-DA are incubated 25 minutes, then
Digest and wash 3 times.Finally, by cell be suspended in 0.5ml PBS and by flow cytometer (FC500Beckman, IL,
USA) tested, analyzed by Flowjo softwares, data are represented (n=4) with average value ± SD, the result is shown in Figure 12.
(2) experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, 37 DEG C are based on the culture containing 10%FBS,
5%CO28h is cultivated in incubator, is then changed to the culture medium containing 1%FBS, after balancing 1h, to experimental group one, in experimental group two
Add DNA (250nM) tetrahedron (isometric culture medium is added in negative control group and positive controls) after filtering.Effect
After 1h, LPS solution is added in positive controls and experimental group two, it is 1 μ g/ml (negative control groups to make LPS concentration in culture medium
Add isometric culture medium with experimental group one), continue culture 12 it is small when after, cell and DCFH-DA are incubated 25 minutes, used
Fluorescence microscope adaptive immune fluoroscopic image, the result is shown in Figure 13.
Experimental result：Flow cytometry (Figure 12) and fluorescence microscope (Figure 13) directly observation result are shown, with feminine gender
Control group is compared, and the intracellular ROS that LPS is stimulated produces significant increase, and due to the application of TDNs, the ROS yields of LPS inductions
Significantly reduce.In the case where lacking LPS, TDNs is produced without significantly affecting on ROS's.
Embodiment 6 probes into the mechanism of anti-oxidation stress
Being presently used for the potential treatment agent of anti-oxidation stress includes antioxidant supplement, enzyme, nano particle and NADPH oxygen
Change enzyme.They are by directly removing or neutralizing ROS or play antioxidation by suppressing the downstream product of ROS42.At present,
HO-1's is anti-oxidant, and anti-apoptotic and anti-inflammatory effect have been widely studied, its mechanism is probably due to the drop of antioxidant bilirubin
The common induction of solution or ferritin.In order to further inquire into the mechanism of TDNs anti-oxidation stress, we are commented by quantitative fluorescent PCR
The mRNA level in-site of enzyme DELTA rHO-1 (HO-1) is estimated to probe into the mechanism of anti-oxidation stress.
Experimental method：Experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, 37 DEG C are based on the culture containing 10%FBS,
5%CO28h is cultivated in incubator, is then changed to the culture medium containing 1%FBS, after balancing 1h, to experimental group one, in experimental group two
Add DNA (250nM) tetrahedron (isometric culture medium is added in negative control group and positive controls) after filtering.Effect
After 1h, LPS solution is added in positive controls and experimental group two, it is 1 μ g/ml (negative control groups to make LPS concentration in culture medium
Add isometric culture medium with experimental group one), continue culture 24 it is small when after, supernatant is abandoned in suction, collects total serum IgE, is purified, and is reversed
Record as cDNA, then carry out fluorescence quantitative PCR detection, data are represented (n=4) with average value ± SD.Statistical analysis：**p<
0.01, * p<0.05, the result is shown in Figure 14.
Experimental result：It can be drawn from fluorescent quantitative PCR result, in the expression of addition DNA tetrahedron groups HO-1mRNA
Significantly rise (Figure 14).It follows that the tetrahedral antioxidations of DNA are mainly by improving DELTA rHO-1 (HO-
1) expression plays a role.
Embodiment 7TDNs suppresses the MAPK/ERK passage downstream protein phosphorylations that LPS is induced in RAW264.7 cells
In inflammatory reaction, mitogen-activated protein kinase (MAPK) signal transduction is to proinflammatory cytokine and medium
Adjusting has a major impact.ERK1/2, JNK1/2/3 and p38 are referred to as MAPK subtribes.Adjusted to further elucidate DNA tetrahedrons
For macrophage to prevent the molecular pathways of oxidative stress and inflammation, activation/2/3 of p38, ERK1/2 and JNK1 pass through western
Trace inspection.
Experimental method：Experiment is divided into four groups, is respectively：Negative control group, positive controls, experimental group one, experimental group two.
First by RAW264.7 cell inoculations in 96 porocyte culture plates, 37 DEG C are based on the culture containing 10%FBS,
5%CO28h is cultivated in incubator, is then changed to the culture medium containing 1%FBS, after balancing 1h, to experimental group one, in experimental group two
Add DNA (250nM) tetrahedron (isometric culture medium is added in negative control group and positive controls) after filtering.Effect
After 1h, LPS solution is added in positive controls and experimental group two, it is 1 μ g/ml (negative control groups to make LPS concentration in culture medium
Isometric culture medium is added with experimental group one).Continue culture 24 it is small when after, rinse sample, then surveyed by full cell cracking
Fixed (KeyGen, Nanjing, China) harvest total protein.By protein example and sample-loading buffer with 4：1 ratio mixing, is then boiled
Boiling.Then, target protein is separated by electrophoresis using SDS-PAGE (10%).Target protein passes through the transport of pvdf membrane, at BSA (5%)
It is middle incubate 1 it is small when after, pvdf membrane and iNOS (ab178945,1:1000；Abcam, Cambridge, UK), ERK (ab184699,
1:1000；Abcam), p38 (ab32142,1:1000；Abcam), JNK (ab179461,1:1000；Abcam), p-ERK
(ab201015,1:1000；Abcam), p-p38 (ab195049,1:1000；Abcam) and p-JNK (ab124956,1:1000；
) etc. Abcam antibody is in 4 DEG C of overnight incubations, then add secondary antibody (Beyotime, Shanghai, China) (3000 times of dilution, each
Band adds 1-2ml) continue be incubated 1 it is small when.Then, with TBST detergent bar bands.Finally, using Bio-Rad detecting systems, the result is shown in
Figure 15.In Figure 15 (a) be the RAW264.7 cells through different disposal total ERK1/2, phosphorylated CREB 1/2, total p38, phosphorylation
The western blot analysis of p38, total JNK1/2/3 and phosphorylation JNK1/2/3.(b-d) it is p38, ERK1/2, JNK phosphorylation water
Flat semi-quantitative analysis.Data are represented (n=4) with average value ± SD.Statistical analysis：*p<0.05, * * p<0.01.We are special
It marked the significant difference between LPS groups and LPS TDNs groups.
Experimental result：LPS substantially have activated MAPK subfamily phosphorylations.However, by the application of TDNs, MAPK has been lowered
The phosphorylation of subfamily (ERK, P38 and JNK), i.e. TDNs are mainly protected by lowering the MAPK subfamilies phosphorylation of LPS inductions
Macrophage is protected from oxidative stress and inflammatory reaction.
Sequence table
<110>Sichuan University
<120>Application of the DNA tetrahedrons in terms of cell anti-aging is promoted
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atttatcacc cgccatagta gacgtatcac caggcagttg agacgaacat tcctaagtct 60
gaa 63
<210> 2
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
acatgcgagg gtccaatacc gacgattaca gcttgctaca cgattcagac ttaggaatgt 60
tcg 63
<210> 3
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
actactatgg cgggtgataa aacgtgtagc aagctgtaat cgacgggaag agcatgccca 60
tcc 63
<210> 4
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60
ccg 63
Claims (8)
- Application of the 1.DNA tetrahedrons in terms of cell anti-aging is promoted, the DNA tetrahedrons by four single-stranded self assemblies of DNA, Four single stranded sequences such as SEQ ID NO:Shown in 1-4.
- Application of the 2.DNA tetrahedrons in terms of cell anti-aging is promoted, it is characterised in that cell anti-aging process include anti-inflammatory and Antioxidation process.
- 3. application of the DNA tetrahedrons according to claim 2 in terms of cell anti-aging is promoted, it is characterised in that DNA tetra- Face body, which promotes the anti-inflammatory effect of cell, to be played a role by reducing the release of inflammatory cytokine.
- 4. application of the DNA tetrahedrons according to claim 2 in terms of cell anti-aging is promoted, it is characterised in that DNA tetra- Face body, which promotes the anti-inflammatory effect of cell, to be played a role by suppressing the expression quantity of iNOS genes.
- 5. application of the DNA tetrahedrons according to claim 2 in terms of cell anti-aging is promoted, it is characterised in that DNA tetra- Face body, which promotes the anti-inflammatory effect of cell, to be played a role by suppressing the expression quantity of iNOS albumen.
- 6. application of the DNA tetrahedrons according to claim 2 in terms of cell anti-aging is promoted, it is characterised in that DNA tetra- It is albumen ERK1/2, the P38 by suppressing MAPK/ERK signal paths downstream that face body, which promotes the antioxidation of cell, JNK's Phosphorylation plays a role.
- 7. application of the DNA tetrahedrons according to claim 2 in terms of cell anti-aging is promoted, it is characterised in that DNA tetra- Face body, which promotes the antioxidation of cell, to be played a role by inducing the expression quantity of DELTA rHO-1.
- 8. application of the DNA tetrahedrons according to claim 2 in terms of cell anti-aging is promoted, it is characterised in that cell DNA four sides used bulk concentration is 100-300nM in anti-inflammatory antioxidation process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711213058.4A CN107961381B (en) | 2017-11-28 | 2017-11-28 | Application of the DNA tetrahedron in terms of promoting cell anti-aging |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711213058.4A CN107961381B (en) | 2017-11-28 | 2017-11-28 | Application of the DNA tetrahedron in terms of promoting cell anti-aging |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107961381A true CN107961381A (en) | 2018-04-27 |
| CN107961381B CN107961381B (en) | 2019-11-12 |
Family
ID=61998880
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711213058.4A Active CN107961381B (en) | 2017-11-28 | 2017-11-28 | Application of the DNA tetrahedron in terms of promoting cell anti-aging |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107961381B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110292644A (en) * | 2019-07-23 | 2019-10-01 | 四川大学 | A kind of drug prevented and treated myocardial ischemia-reperfusion injury or treat heart ischemia disease |
| CN114288316A (en) * | 2022-01-27 | 2022-04-08 | 四川大学 | Use of tetrahedral framework nucleic acid in medicine for treating pancreatitis |
| CN116139285A (en) * | 2022-07-07 | 2023-05-23 | 四川大学 | Biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid and preparation method and application thereof |
| CN116327627A (en) * | 2023-03-10 | 2023-06-27 | 四川大学 | Tetrahedral framework nucleic acid-glabridin complex and application thereof in skin whitening |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107335065A (en) * | 2017-06-23 | 2017-11-10 | 郑州大学 | A kind of targeting DNA nanoassembles body carries NF kB decoy ODNs preparation method and applications |
-
2017
- 2017-11-28 CN CN201711213058.4A patent/CN107961381B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107335065A (en) * | 2017-06-23 | 2017-11-10 | 郑州大学 | A kind of targeting DNA nanoassembles body carries NF kB decoy ODNs preparation method and applications |
Non-Patent Citations (1)
| Title |
|---|
| DAN ZHU等: "PolyA-Mediated DNA Assembly on Gold Nanoparticles for Thermodynamically Favorable and Rapid Hybridization Analysis", 《ANALYTICAL CHEMISTRY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110292644A (en) * | 2019-07-23 | 2019-10-01 | 四川大学 | A kind of drug prevented and treated myocardial ischemia-reperfusion injury or treat heart ischemia disease |
| CN114288316A (en) * | 2022-01-27 | 2022-04-08 | 四川大学 | Use of tetrahedral framework nucleic acid in medicine for treating pancreatitis |
| CN114288316B (en) * | 2022-01-27 | 2022-12-30 | 四川大学 | Use of tetrahedral framework nucleic acid in preparation of medicine for treating pancreatitis |
| CN116139285A (en) * | 2022-07-07 | 2023-05-23 | 四川大学 | Biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid and preparation method and application thereof |
| CN116139285B (en) * | 2022-07-07 | 2024-05-31 | 四川大学 | Biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid and preparation method and application thereof |
| CN116327627A (en) * | 2023-03-10 | 2023-06-27 | 四川大学 | Tetrahedral framework nucleic acid-glabridin complex and application thereof in skin whitening |
| CN116327627B (en) * | 2023-03-10 | 2024-06-07 | 四川大学 | Tetrahedral framework nucleic acid-glabridin complex and application thereof in skin whitening |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107961381B (en) | 2019-11-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107961381B (en) | Application of the DNA tetrahedron in terms of promoting cell anti-aging | |
| Mendes et al. | Quantification of left ventricular myocardial collagen system in children, young adults, and the elderly | |
| Zhou et al. | Omentin-1 protects against bleomycin-induced acute lung injury | |
| Zhang et al. | Hypoxia preconditioned renal tubular epithelial cell-derived extracellular vesicles alleviate renal ischaemia-reperfusion injury mediated by the HIF-1α/Rab22 pathway and potentially affected by microRNAs | |
| Pleskova et al. | The interaction between human blood neutrophil granulocytes and quantum dots | |
| CN107988150A (en) | A kind of identification method and system of Myocytes Anoxia-reoxygenation | |
| Ge et al. | DNA damage in thyroid gland cells of rats exposed to long-term intake of high fluoride and low iodine | |
| CN107961243A (en) | Application of the DNA tetrahedrons in terms of Induces Autophagy | |
| Ferrero et al. | Cellfood™ improves respiratory metabolism of endothelial cells and inhibits hypoxia-induced reactive oxygen species (ROS) generation | |
| Grossini et al. | Exposure to plasma from non-alcoholic fatty liver disease patients affects hepatocyte viability, generates mitochondrial dysfunction, and modulates pathways involved in fat accumulation and inflammation | |
| Wei et al. | Metabolic reprogramming of immune cells at the maternal-fetal interface and the development of techniques for immunometabolism | |
| Huang et al. | Dual-targeting nanotherapeutics antagonize hyperinsulinemia-promoted tumor growth via activating cell autophagy | |
| Ding et al. | Exosomal miR-125a-5p regulates T lymphocyte subsets to promote silica-induced pulmonary fibrosis by targeting TRAF6 | |
| WO2021179952A1 (en) | Application of gw8510 in preparation of drugs for prolonging lives, improving cognitive ability, and the like of mammals in natural aging | |
| TWI819235B (en) | Fermentation broth of natural raw materials and uses thereof for inhibiting allergic reaction or for enhancing immunity | |
| Zhang et al. | Anti-aging potency correlates with metabolites from in vitro fermentation of edible fungal polysaccharides using human fecal intestinal microflora | |
| Yang et al. | S100A1 is involved in myocardial injury induced by exhaustive exercise | |
| CN109207556A (en) | Application of the Nrf2-ARE access in quickly detection ambient particle object toxicity | |
| Wang et al. | A Composite Nanomaterial with the Ability to Regulate Oxidative Stress and Anti‐inflammatory for the Treatment of Osteoarthritis | |
| CN103550197A (en) | Application of guluronic acid oligosaccharide | |
| Zhang et al. | Riboflavin ameliorates intestinal inflammation via immune modulation and alterations of gut microbiota homeostasis in DSS-colitis C57BL/6 mice | |
| CN115501205B (en) | Radiotherapy sensitization nanoparticle and preparation method thereof | |
| CN102604946B (en) | Small interfering ribonucleic acid (siRNA) for inhibiting tumor metastasis and application thereof | |
| He et al. | Metabolic alteration of Tetrahymena thermophila exposed to CdSe/ZnS quantum dots to respond to oxidative stress and lipid damage | |
| CN108113995A (en) | The application of microRNA hsa-mir-1-3p and the like and the application for expressing the microRNA carrier |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230406 Address after: 213000 No.28 Shuanglong Road, Jintan District, Changzhou City, Jiangsu Province Patentee after: Changzhou Zhisheng Biotechnology Co.,Ltd. Address before: 610064, No. 24, south section of first ring road, Chengdu, Sichuan, Wuhou District Patentee before: SICHUAN University |
|
| TR01 | Transfer of patent right |