CN108836980A - Graphene oxide is preparing the application in dendritic cell function promotor - Google Patents
Graphene oxide is preparing the application in dendritic cell function promotor Download PDFInfo
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Abstract
It is maturing dendritic cell degree and/or transfer ability that the invention discloses graphene oxides preparing the application in adoptive dendritic cell function promotor, the dendritic cell function.Present invention discover that graphene oxide is activated by the common incubation and stimulation in vitro with derived from bone marrow immature dendritic cell, promote that its costimulatory molecules, chemokine receptor expression be horizontal and inflammatory response ability, so that it be promoted to migrate in body and to lymphocyte homing ability.Compared with Cocktail of cytokines, graphene oxide has low in cost, stable structure, is easily obtained and forms relatively single advantage, is more advantageous to the clinical conversion in dendritic cell vaccine preparation.
Description
Technical field
The present invention relates to field of nano biotechnology, more particularly to graphene oxide in preparation Dendritic Cells, especially
It is adoptive Dendritic Cells, the application in adoptive dendritic cell vaccine function promoter.
Background technique
Dendritic Cells (Dendritic Cells, DCs) is the bridge for connecting the innate immunity and acquired immunity, and
It is a kind of antigen presenting cell (the Antigen Presenting that can uniquely activate Naive T cells (Naive T Cells)
Cells,APCs).Dendritic Cells can transfer panimmunity resistance mechanisms, such as cytotoxic T cell, CD4+T helper cell,
Natural kill (NK) cell and natural killer T (NKT) cell etc..These Dendritic Cells all have identification and kill illness thin
The effect of born of the same parents and release guard cell factor (such as IFN-γ, TNF-α).
DCs plays antigen presentation and needs to undergo two vital processes in the physiological state：1) to invasion disease
Poison or bacterium identified, swallow and antigen processing and submission and maturity state transformation, i.e., chemokine expression spectrum conversion and
The up-regulated expression of surface co-stimulatory molecules；2) DCs leaves peripheral tissues and goes back to the nest to lymphoid tissue, and provides for the activation of T cell
Second signal.Immature DC receive pathogen associated molecular pattern (PAMPs) or danger signal stimulation after up-regulation chemotactic factor (CF) by
Body 7 (CCR7) expression, obtains lymphocyte homing ability, once reaching lymph node T cell area, mature DC is being expressed in CD4+T
It is activated under the stimulation of the CD40L of cell surface by " approval ".CD40 signal stimulus raises DC surface co-stimulatory molecules, obtains DC
It obtains and most preferably activates CD8+The ability of T cell.
Immunization therapy (DCs-based immunotherapy) based on DCs, also referred to as DCs vaccine refer to for swollen
The characteristics of tumor and the immunocompromised and disorder of chronic infective patient, carries out induction training to DCs in vitro using biological means
Feeding, antigen load and stimulation are mature, then feed back into patient's body, align immune order simultaneously activation antigen specificity T to reach
The purpose of cell effect.The auxiliary that DCs vaccine is often used for after patient's Radiotherapy chemotherapy or hematopoietic stem cell transplantation, organ transplant is controlled
In treatment；By complicated in vitro culture and cell engineering method, culture proliferation carried out to primary cell in vitro, and by functionalization
Immunocyte feed back to the process of patient's body and be known as " adoptive ", the functionalization DCs for feeding back to patient's body was referred to as
After property Dendritic Cells (referred to as " adoptive DC ").Therefore, adoptive Dendritic Cells infusion is as the immune treatment to reach its maturity
Method will play a greater and greater role in the prevention and treatment of tumour and chronic infectious disease.
The activation of adoptive DC and to T cell enrichment region go back to the nest be influence DCs functionalization key factor.DCs activation
Afterwards often a variety of costimulatory molecules in its surface of up-regulated expression (such as CD40, CD80, CD86 etc.) and secretion Th1 type cytokines (IL-
1 β, TNF-α, IL-6 etc.) carry out inducing T cell immune activation, and immature adoptive DC is not only unable to activating T cell, instead
It can make T cell functionally inactive, inducing T cell tolerance.Currently, Cocktail of cytokines (the Cytokine authenticated through FDA
Cocktail, IL-1 β, TNF-α, IL-6, PGE2) and improvement Cocktail of cytokines (IL-1 β, TNF-α, IL-6, polyI：C,
CpG ODN) it is the mature most common promotor of the adoptive DC of stimulation.The promotor can improve to a certain degree adoptive DC at
Ripe degree, the secretion of up-regulated expression and certain proinflammatory factors including costimulatory molecules.But the maximum defect of the promotor is not
Adoptive DC can be effectively facilitated to go back to the nest and migrate to lymphoid tissue, a large amount of document (such as O ' Neill D.W.et.al.Blood
2004,104,2235-2246) prove only less than 5%Cytokine-Cocktail load DCs can go back to the nest through lymphatic drainage to
Neighbouring lymph node, this is also the one of the major reasons that adoptive DC treats clinically response rate low (10-15%) in recent years.
In addition to this, the problems such as Cocktail of cytokines is complicated there is also preparing and producing purifying process, with high costs.
Therefore, exploitation effectively promotes agent, farthest improves the maturity of adoptive DC activation, promotes it in body
Transfer ability to more present antigen to T cell to induce immune response, be the key that improve its clinical efficacy.
Summary of the invention
The purpose of the present invention is being directed to technological deficiency existing in the prior art, the new use of one kind of graphene oxide is provided
On the way, i.e., graphene oxide is preparing the application in dendritic cell function promotor, and the dendritic cell function is dendron shape
Cellular maturity and/or transfer ability.
The graphene oxide has Micro-sheet Structure, and purity is not less than 99%.
The piece diameter of the graphene oxide is having a size of 50-1500nm, preferably 50-500nm, more preferable 50-200nm.
The lamellar spacing of the graphene oxide is about 0.8-1.2nm.
The dendritic cell function promotor is that graphene oxide is dispersed in solvent or culture medium by ultrasound to obtain
It arrives.
The graphene oxide be by graphene through strong oxidizer (such as potassium permanganate) oxidation processes obtain have it is microcosmic
The two-dimension nano materials of lamellar structure.
The preparation process of the graphene oxide is as follows：Flaky graphite, NaNO3With dense H2SO4It is stirred in ice bath after mixing
It mixes, KMnO is added435 DEG C of stirring 2h afterwards, then 60 DEG C of stirring 2h are to slowly warm up to, it is then added in 200mL water, is stirred at 80-90 DEG C
5h is mixed, the H of 30wt% is added2O2Aqueous solution to bubble-free generates, and after filtering while hot, first cleans filter cake with the dilute hydrochloric acid of 5wt%,
Then it is washed with deionized, until filtrate is in neutrality and without SO4 2-, filter cake is freeze-dried, single-layer graphene oxide is obtained.
Further by with Micro-sheet Structure graphene oxide or single-layer graphene oxide ground and aoxidized
Graphene powder；Graphene oxide powder is dispersed again sonicated in deionized water.
The Dendritic Cells is immature dendritic cell；When the immature dendritic cell is derived from bone marrow,
With the CD11c positive rate evaluation of its surface specific expression, purity should reach 65% or more.
The graphene oxide and immature dendritic cell are incubated for altogether with promote the maturing dendritic cell degree and/
Or migrate, graphene oxide final concentration of 3.9-62.5 μ g/mL, preferably 7.8-31.2 μ g/mL in total incubation system, most preferably
7.8-15.6μg/mL。
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention provides a kind of promotors for the adoptive treatment of Dendritic Cells --- graphene oxide
(graphene oxide, GO), the maturity and infusion of adoptive Dendritic Cells can be promoted as promotor before infusion
Transfer ability afterwards, and without obvious toxic-side effects.The present invention utilizes the good biocompatibility of stannic oxide/graphene nano material and piece
The characteristics such as diameter size adjustable are used as part or the lymph under venoclysis that promotor can greatly improve adoptive DC vaccine
Organ, which is gone back to the nest, rate and goes back to the nest efficiency, provides new selection and path for clinical convert of dendritic cell vaccine.The present invention
The new application of the graphene oxide composite material of proposition demonstrates it to DCs stimulated in vitro and function by the way of in-vitro screening and evaluation
Therefore the effect of energyization can get preferably expected in clinical use.
Detailed description of the invention
Fig. 1 show the characterization result of the graphene oxide of different diameter sizes：Wherein A width is transmission electron microscope photo, B width
For dynamic light scattering curve graph, C width is atomic force microscopy；
Fig. 2 show imDCs metamorphosis photo of the Dendritic Cells through Fiber differentiation different time；
Fig. 3 show imDCs cell survival rate figure (horizontal seat in streaming figure after the graphene oxide processing of different diameter sizes
Mark indicates the Annexin V of Fitc label)；
Transmission electron microscope photo after A width is swallowed for the graphene oxide of different diameter sizes by DCs in Fig. 4, B width are difference
The graphene oxide of piece diameter size swallowed by DCs after Switzerland-Giemsa staining photo；
The graphene oxide and DCs chemokine receptor expression level view after DCs effect that Fig. 5 show different diameter sizes
(note：The abscissa of streaming figure indicates the surface of dendritic cells molecule of fluorescent molecule and its label；* with Ctrl-DCs group ratio
Compared with # is compared with S-DCs group, and & is compared with M-DCs group, P < 0.05)；
Fig. 6 show the graphene oxide of different diameter sizes and inflammatory cytokine 1L-6 in system after DCs effect divides
The flat histogram of bleeding (for * compared with Ctrl-DCs group, # is compared with S-DCs group, P < 0.05)；
The graphene oxide and DCs surface chemokine receptor CCR7 table after DCs effect that Fig. 7 show different diameter sizes
Up to level；
Fig. 8 show the comparison of DCs transfer ability after foot pad is transfused after the graphene oxide processing of different diameter sizes：
Wherein A width is annotated map of the foot pad infusion with luminous site after the imDCs of firefly luciferase reporter gene；B width is DCs
Dynamic migration cross photo；C width be DCs migrate to the histogram of PLN percentage (* with Ctrl-DCs group compared with, # and S-DCs group
Compare, P < 0.05).
Specific embodiment
Graphene is a kind of two-dimentional carbon nanomaterial with unique superior electrical, optics and mechanical property, in electronics device
Part, biochemistry sensing and materials science field are with a wide range of applications.Graphene oxide (graphene oxide, GO)
It is that graphene through strong oxidizer handles resulting derivative, after oxidation processes, graphene oxide still keeps the layer of graphene
Shape structure, but many oxygen-containing functional groups, in addition to synthesis technology is relatively easy, cost are introduced on each layer of graphene monolithic
It is cheap outer, also have both the characteristics such as stronger hydrophily, apparent height Raman scattering ability, high surfaces function plasticity.Therefore,
Graphene oxide becomes the hot spot biomaterial of the application studies such as oncotherapy and biosensor.However, graphene oxide pair
The phenotype and function of Dendritic Cells can have an impact not someone report, more without graphene oxide can promote imDCs at
Ripe and activation report.
The invention proposes a kind of new applications of graphene oxide --- the promotion as the adoptive treatment of Dendritic Cells
Agent, and by external and animal body it is demonstrated experimentally that graphene oxide treated immature dendritic cell (immature
Dendritic cells, imDCs), surface co-stimulatory molecules CD40, CD80 and CD86 expression are raised, inflammatory cytokine
The obvious up-regulation of IL-6 secretion, the significant up-regulation of CCR7 expression；It is observed after graphene oxide is handled under Laser Scanning Confocal Microscope, DCs
Microfilament, micro-pipe are reset and sticking phenomenon is obvious；The mechanism that above-mentioned phenomenon generates should active oxygen (reactive in incubation system together
Oxygen species, ROS) generation it is related；DCs is promoted about from the speed that foot pad is migrated Zhi popliteal nest lymph node after GO processing
1.5-3 times, adoptive DC vaccine goes back to the nest efficiency in animal body being effectively promoted to less than 5% by traditional adoptive DC vaccine
24%.In addition, being deduced by related experiment：It is straight as promotor or immune function controlling agents using nano material compared to other
Immune nano vaccine is connect, due to there was only the small amounts graphene nano particle for swallowing or sticking in Dendritic Cells (usually
Less than the 10% of incubation amount) it can enter in animal pattern or human body, therefore compared with other vaccine adjuvants, promotion of the invention
A possibility that agent has better biological safety, increases the product to clinical expansion and application.
Below in conjunction with specific embodiment, the content of the present invention will be explained in more detail, and the present invention is further elaborated, but
These embodiments limit the invention absolutely not.
The source of used biology and nano material is extensive in embodiment, any to keep on the right side of the law and moral ethics
The biology and nano material that can be obtained can be replaced according to the prompt in embodiment.It is any related fields have through
The people tested, can principle according to the invention, using other graphene oxides, such as multilayer graphene oxide, other piece diameter rulers
Very little single-layer graphene oxide or above-mentioned mixture, and the graphite oxide olefinic base material after other reagents or molecular modification
Deng the application realized in the adoptive maturing dendritic cell degree of " graphene oxide " raising and transfer ability described in the invention.
In addition, Dendritic Cells described in the present invention, does not limit to the Dendritic Cells with bone marrow derived, other sources (people yet
Or other animals) Dendritic Cells be also included, these are without departing from the basic conception that describes of the present invention.Therefore, these
Modification or different applications are all within protection scope of the present invention.
Method therefor is conventional method unless otherwise instructed.Unless otherwise instructed, in each embodiment same names material
Material or reagent content are identical.
The preparation of embodiment one, different piece diameter size single-layer graphene oxides (graphene oxide, GO)
It can refer to prior art for the graphene oxide in the present invention to be prepared.It can be by graphene through strong oxidizer
The two-dimension nano materials for the lamellar structure that (such as potassium permanganate) oxidation processes obtain, or using the potassium permanganate and stone in the concentrated sulfuric acid
Ink powder end after oxidation reaction, obtain brown edge have derivative carboxyl and in the plane be mainly phenolic hydroxyl group and epoxy group
The graphite flake of group, it is graphene oxide that this graphene layers can be vigorously stirred removing through ultrasound or high shear, and in water
Form stable, sundown single-layer graphene oxide suspension.
Illustrate how to obtain the graphene oxide of different diameter sizes with specific example below：
1g flaky graphite, 1g NaNO3, the dense H of 46mL2SO4It is stirred 30 minutes in ice bath after mixing, is slowly added to 4.0-
6.0g KMnO4, reaction system is gone into 35 DEG C of stirring 2h in oil bath after 1h, then be to slowly warm up to 60 DEG C of stirring 2h.By reactant
System adds in 200mL water, and 5h is stirred at 80-90 DEG C, and 30wt%H is added2O2Aqueous solution bubble-free into reaction system generates.This
When solution brown become by yellow, filter while hot, first clean filter cake with the dilute hydrochloric acid of 5wt%, be then washed with deionized, directly
It is in neutrality to filtrate and has no SO4 2-(BaCl can be used2Detection).After filter cake is freeze-dried to get arrive single-layer graphene oxide.
By single-layer graphene oxide grind into powder obtained and disperse in deionized water, to be ultrasonically treated through 300W, root
The graphene oxide of different diameter sizes can be obtained according to processing length of time, piece diameter size can be obtained in 2 hours in such as ultrasonic treatment
The oxidation stone that piece diameter size is about 200nm-500nm can be obtained in 10 hours in the about graphene oxide of 50-200nm, ultrasonic treatment
The graphene oxide that piece diameter size is about 500-1500nm can be obtained in 18 hours in black alkene, ultrasonic treatment.
It is measured with transmission electron microscope (TEM), dynamic light scattering (DLS), atomic force Electronic Speculum (AFM) and Zeta potential
Instrument characterizes the GO for the third-grade film diameter size that above method obtains, the result is shown in Figure 1.
Transmission electron microscope results are as shown in figure 1 shown in A width, it can be seen that the GO size difference of third-grade film diameter is obvious, in water
It is uniformly dispersed in solution, is irregular polygon shape.
DLS result is as shown in figure 1 shown in B width, it can be seen that the average piece diameter of tertiary oxidation graphene is respectively
103.1nm (S-GO), 362.5nm (M-GO), 1192.1nm (L-GO).(note：In embodiment, using S-GO, M-GO, L-GO
The DCs group of processing is respectively labeled as S-DCs, M-DCs, L-DCs；Control DCs group echo is Ctrl-DCs.)
AFM characterization result is as shown in figure 1 shown in C width, it can be seen that the GO average thickness of third-grade film diameter size is 1-
2nm meets single layer GO structure feature.
The Zeta potential characterization result of the GO of the different piece diameter sizes of table 1
| Name | S-GO | M-GO | L-GO |
| Hydrodynamic radius (nm) | 103.1 | 362.5 | 1192.1 |
| Zeta potential (mV) | -32.76±1.19 | -31.58±1.34 | -30.74±0.82 |
Zeta potential characterization result is shown in Table 1, it is seen that the Zeta potential absolute value of the GO of three kinds of piece diameter sizes in aqueous solution
It is all larger than 30mV, illustrates that GO colloidal stability obtained by the above method is preferable.
The culture and identification of embodiment two, bone marrow derived immature dendritic cell
1, the culture of bone marrow derived immature dendritic cell and induction are broken up
Cervical dislocation put to death L2G85.C57BL/6J mouse (6-8 weeks), be immersed in 75% (v/v) alcohol be placed on it is ultra-clean
Its femur and shin bone are separated in workbench, are soaked in PBS, are removed muscle and epiphysis with sterile scissors and tweezers, injected with 2mL
Device syringe needle blows out the marrow in ossis, 1640 culture medium repeated flushing ossis is drawn, until ossis bleaches.With note
Emitter gently dispels bone marrow cell, and is filtered with 40 μm of filter into centrifuge tube.
By the bone marrow cell of dispersion with 2 × 106Cells/mL is incubated in 6 orifice plates, and 1640 complete mediums are added in every hole
(fetal calf serum containing 10wt%, 5ng/mL IL-4,10ng/mL GM-CSF).Half amount changes liquid when cultivating third day, and half is old
Culture medium is replaced with 1640 new complete mediums of equivalent；At the 5th day half amount changes liquid, and the old culture medium of half is new with equivalent
1640 complete mediums replace.Then in 37 DEG C, 5%CO2It is cultivated in cell incubator.Cultivating the 6th day collection cell is bone
The immature Dendritic Cells in marrow source.
2, the identification of bone marrow derived immature dendritic cell
The form that immature dendritic cell (imDCs) is observed under inverted phase contrast microscope, is as a result shown in Fig. 2：In culture the
One day, the bone marrow precursor of imDCs was small and circle cell, and cell membrane is smooth, without protrusion.3rd day half amount is changed visible when liquid
Majority is attached cell, there is a small amount of half small suspension cell colony.Visible cell colony increases, increases when half amount changes liquid within 5th day,
Most suspension growths, cell volume increase, surface hairiness thorn-like protrusion.
Due to the specific expressed CD11c of the imDC of bone marrow derived, with the purity of CD11c positive rate evaluation DC.?
The 6th day of imDC culture, cultivating imDC purity in cell can reach 65% or more, illustrates to induce successfully, carries out next step test.
The effect experiment of embodiment three, graphene oxide
The acquisition of graphene oxide GO and immature dendritic cell imDCs are referring to embodiment one used in the present embodiment
With embodiment two.
1, influence of the graphene oxide to Dendritic Cells survival rate in incubation system altogether
It is respectively S-GO (50-200nm), the M- of 7.8 μ g/ml, 15.6 μ g/ml, 31.2 μ g/ml, 62.5 μ g/ml by concentration
Embodiment two is added containing the successful imDCs of induction in GO (200-500nm), L-GO (500-1500nm) third-grade film diameter GO aqueous solution
Culture medium in altogether be incubated for (altogether be incubated for be will additional substance (such as graphene oxide) be added the culture medium containing imDCs in
The process being incubated for together, incubation system is the culture medium system containing additional substance and imDCs altogether) (respectively S- after 48h
DCs group, M-DCs group and L-DCs group), it is total to the cell survival rate in incubation system with Flow cytometry, as a result such as Fig. 3 institute
Show.
As can be seen from Figure, compared with control group imDCs (Ctrl-DCs group), 7.8 μ g/ml concentration GO processing
S-DCs group, M-DCs group and L-DCs group cell survival rate be respectively (93.29 ± 0.99) %, (93.29 ± 0.99) %,
(91.89 ± 2.97) %, 15.6 μ g/ml concentration treated cell survival rate be respectively (91.65 ± 1.65) %, (91.56 ±
1.60) %, (91.89 ± 1.32) %, 31.2 μ g/ml concentration treated cell survival rate be respectively (83.71 ± 1.66) %,
(83.62 ± 1.65) %, (83.48 ± 1.98) %, 62.5 μ g/ml concentration treated cell survival rate be respectively (78.93 ±
0.33) %, (78.72 ± 0.29) %, (76.88 ± 3.22) %.
Because the cell survival rate of S-DCs group, M-DCs group and L-DCs group is high under 7.8 μ g/ml, 15.6 μ g/ml concentration
In 90%, to obtain better facilitation effect, preferably 15.6 μ g/ml are the working concentration of GO in embodiment.
This experiment survival rate also shows using graphene oxide GO to Dendritic Cells without obvious toxic-side effects.
2, phagocytosis of the Dendritic Cells to graphene oxide
Binding experiment 1 is observed different diameter GO using transmission electron microscope and Switzerland-Giemsa staining and is absorbed by DCs
The case where, to compare graphene oxide (S-GO (50-200nm), M-GO (200-500nm), L-GO (500- of three kinds of piece diameters
The 1500nm)) difference of endocytosis mode, as a result as shown in Figure 4.
A width (enlarged drawing that second row is Blocked portion in first row picture) in Fig. 4 as can be seen that observe under a lens
It is wrapped in DCs intracellular vesicle in Filamentous S-GO and M-GO, S-GO vacillates to the region of nearly core, and L-GO is in a pencil trip
From in outside DCs cell membrane.Lens inspection to Dendritic Cells in graphene oxide size and using preceding independent GO material ruler
It is very little to be consistent, illustrate that GO does not degrade in culture medium or cell matter, is swallowed with original state by Dendritic Cells.
B width in Fig. 4 also observes the DCs after taking in different diameter GO by Switzerland-Giemsa staining, intuitively
Phagocytosis to DCs to different diameter GO, and after this phagocytosis occurs, GO in the form of cluster (in figure arrow signified) in cytoplasm
Middle enrichment analysis is exactly that this phagocytosis behavior promotes the maturation of DCs, and then promotes its transfer ability in vivo.
This experiment proves that Dendritic Cells to the phagocytic activity of graphene oxide, based on being incubated for after 1 couple of incubation 48h of experiment
The calculating of graphene oxide concentration in system knows that roughly the amount of the stannic oxide/graphene nano particle swallowed in Dendritic Cells is small
In the 10% of graphene oxide dosage (incubation amount).
3, dendritic cell co-stimulatory molecules expression and inflammatory cytokine IL-6 after being incubated for altogether with graphene oxide
Expression
Binding experiment 1, this experiment are evaluated by the expression rate of CD40, CD80 and CD86 in monitoring CD11c positive cell
The maturity of DCs, as a result as shown in figure 5, the expression of inflammatory cytokine IL-6 is as shown in Figure 6.
As seen from Figure 5, the gene expression abundance of control group imDCs (Ctrl-DCs group) surface C D40, CD80 and CD86 point
It Wei not (44.27 ± 3.46) %, (32.67 ± 1.70) % and (32.73 ± 1.52) %；S-DCs group surface C D40, CD80 and
The gene expression abundance of CD86 is respectively (53.43 ± 0.55) %, (34.93 ± 1.01) % and (43.83 ± 4.01) %；M-DCs group
The gene expression abundance of costimulatory molecules CD40, CD80 and CD86 of imDCs respectively (54.43 ± 2.48) %, (45.10 ±
4.19) % and (47.97 ± 0.85) %；L-DCs group then for (57.13 ± 2.70) %, (46.93 ± 0.86) % and (49.97 ±
0.76) %.After illustrating GO stimulation imDCs, surface co-stimulatory molecules CD40, CD80 and CD86 expression rate are compared with Ctrl-DCs
Group is significant to be increased, the statistically significant (P of difference<0.05), show graphene oxide can be promoted immature dendritic cell at
Ripe degree.
As shown in fig. 6, secreted in S-DCs, M-DCs, L-DCs group supernatant IL-6 (respectively 42617.02 ±
341.24pg/mL, 51709.04 ± 10.78pg/mL, 48080.82 ± 4856.53pg/mL) it is above Ctrl-DCs (imDCs)
Group (37361.91 ± 5357.26pg/mL) illustrates that GO can promote the maturation of immature dendritic cell when inflammation occurs
Degree shows that graphene oxide can promote immature dendritic cell and play antigen presentation, and present antigen is to T cell, activation
T cells with antigenic specificity is reacted to induce immune response, to adjust the inflammatory response mode of DCs.
4, Dendritic Cells chemokine receptor expression is horizontal after being incubated for altogether with graphene oxide
ImDCs surface chemokine receptor expresses, Binding experiment 1 related to its transfer ability, this experiment passes through monitoring DCs
Surface chemokine receptor CCR7 expression rate evaluates influence of the GO to DCs shift function, as a result sees Fig. 7 and table 2.
As shown in table 2 and Fig. 7, S-DCs group, M-DCs group and L-DCs group CCR7 gene expression abundance be respectively 20.00 ±
2.53%, 15.54 ± 4.75,18.71 ± 4.75, hence it is evident that be higher than control group imDCs (Ctrl-DCs group) 8.66 ± 2.06, difference
Statistically significant (P<0.05).Experimental result illustrates the surface C CR7 expression rate of the imDCs of GO stimulation compared in control group expression
It adjusts, shows that GO can enhance DCs transfer ability.
2 DCs surface chemokine receptor CCR7 expression rate of table
Dendritic Cells is in body migration and distribution pattern after example IV and graphene oxide are incubated for altogether
It is adopted by experimental verification GO processing in animal body to going back to the nest in DCs body and migration model bring facilitation
With the FVB transgenic mice of constitutive expression firefly luciferase (firefly luciferase, Fluc)
(Fluc.L2G85) immature dendritic cell of derived from bone marrow carries out the total incubation process with graphene oxide.
To carry the untreated Dendritic Cells of firefly luciferase reporter gene as negative control group, positive drug
The mature DCs that lipopolysaccharides (lipopolysaccharide, LPS) stimulation obtains are implemented as positive controls (LPS-DCs group)
S-DCs group, M-DCs group and the L-DCs group of example three are GO processing group, the Fluc+DCs that control group and each GO processing group are obtained
(negative control imDCs, similarly hereinafter) is infused in the wild type C57BL/6J mouse to have lost hair or feathers through foot pad, small animal living body at
As Technique dynamic monitoring DCs moves to from infusion site the cell quantity of regional nodes, Fig. 8 is as a result seen.Experiment monitors respectively
Luminous situation after DCs infusion after 4,24,48 and 72h.Each lymphoid tissue strong light degree is analyzed using living imaging software
According to further calculating out the migration quantity of DCs.
The area Tu8Zhong a (popliteal nest lymph node position) luminous signal intensity can indicate that DCs moves shifting to popliteal nest lymph node from foot pad
(PLN) quantity, the area b (foot pad location) luminous signal intensity can indicate that DCs remains in quantity (the A width and B in Fig. 8 of foot pad
Width).The results show that the cell migration of S-DCs group Zhi popliteal nest lymph node region signal strength for 24 hours, 48h and 72h be above L-
DCs group, M-DCs group and negative control group, signal strength are more than positive control (B width and C width in Fig. 8) in 72h.Luminous signal
Intensity system analyzes to obtain by living imaging software, is computed, S-DCs group, L-DCs group, M-DCs group and negative control group, sun
Property control group DCs is respectively 0.17 ± 0.02,0.12 ± 0.06,0.11 ± 0.05,0.07 in the percentage that 48h moves to PLN
±0.01,0.17±0.01；72h move to the percentage of PLN be respectively 0.24 ± 0.05,0.16 ± 0.09,0.15 ± 0.05,
0.08±0.02,0.23±0.08；Illustrate that migration rate sequence is LPS-DCs ≈ S-DCs group > L-DCs group > M-DCs group >
Control group, group difference are statistically significant (P < 0.05).The embodiment proves that graphene oxide can enhance h inf
It the local migration of DCs and goes back to the nest ability to lymphoid tissue.
Embodiment five：Toxicity test
It has been confirmed that cell survival rate is greater than 90% under 7.8 μ g/ml, 15.6 μ g/ml concentration in 3 part 1 of embodiment,
Reach generally acknowledged nano material no cytotoxicity standard.It is further demonstrated that in 3 part 2 of embodiment and actually enters organism
The 10% of interior graphene oxide deficiency incubation amount calculates, only the oxygen less than 0.2mg/kg according to the DCs amount of adoptive infusion
Graphite alkene enters in Mice Body, well below the maximum tolerated dose of glucan-modified GO, i.e., between 50-125mg/kg
(Biomaterials 35(2014)7022-7031)).In addition, animal state does not occur in the zoopery of example IV
It is affected or the case where life cycle is affected, illustrates that graphene oxide of the invention has hypotoxicity and high bio-safety
Property.
The above is only a preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
Also the contents of the present invention be should be regarded as.
Claims (10)
1. graphene oxide is preparing the application in dendritic cell function promotor, which is characterized in that the Dendritic Cells
Function is maturing dendritic cell degree and/or transfer ability.
2. applying according to claim 1, which is characterized in that the graphene oxide has Micro-sheet Structure, and purity is not
Less than 99%.
3. applying according to claim 2, which is characterized in that the piece diameter of the graphene oxide is excellent having a size of 50-1500nm
Select 50-500nm, more preferable 50-200nm.
4. being applied according to Claims 2 or 3, which is characterized in that the lamellar spacing of the graphene oxide is about 0.8-
1.2nm。
5. -4 any application according to claim 1, which is characterized in that the dendritic cell function promotor is by super
Graphene oxide is dispersed in solvent or culture medium and obtains by sound.
6. -5 any application according to claim 1, which is characterized in that the graphene oxide is by graphene through Strong oxdiative
The two-dimension nano materials with Micro-sheet Structure that agent (such as potassium permanganate) oxidation processes obtain.
7. -5 any application according to claim 1, which is characterized in that the preparation process of the graphene oxide is as follows：Squama
Shape graphite, NaNO3With dense H2SO4It is stirred in ice bath after mixing, KMnO is added435 DEG C of stirring 2h afterwards, then it is to slowly warm up to 60 DEG C
2h is stirred, is then added in 200mL water, is stirred 5h at 80-90 DEG C, the H of 30wt% is added2O2Aqueous solution to bubble-free generates, and takes advantage of
After heat filtering, filter cake first is cleaned with the dilute hydrochloric acid of 5wt%, is then washed with deionized, until filtrate is in neutrality and without SO4 2-,
Filter cake is freeze-dried, single-layer graphene oxide is obtained.
8. being applied described according to claim 6 or 7, which is characterized in that further by the graphite oxide with Micro-sheet Structure
Alkene or single-layer graphene oxide are ground to obtain graphene oxide powder；Graphene oxide powder is dispersed in deionized water again
In it is sonicated.
9. -8 any application according to claim 1, which is characterized in that the Dendritic Cells is that prematurity dendron shape is thin
Born of the same parents；When the immature dendritic cell is derived from bone marrow, evaluated with the CD11c positive rate of its surface specific expression,
Purity should reach 65% or more.
10. -9 any application according to claim 1, which is characterized in that by the graphene oxide and prematurity dendron shape
Cell is incubated for altogether to promote the maturing dendritic cell degree and/or migration, and graphene oxide is final concentration of in incubation system altogether
3.9-62.5 μ g/mL, preferably 7.8-31.2 μ g/mL, most preferably 7.8-15.6 μ g/mL.
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Cited By (3)
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| CN111330004A (en) * | 2020-03-04 | 2020-06-26 | 中国人民解放军军事科学院军事医学研究院 | Application of molybdenum disulfide nanosheet layer in preparation of dendritic cell function promoter |
| CN113069541A (en) * | 2021-04-09 | 2021-07-06 | 中国人民解放军军事科学院军事医学研究院 | Application of graphene oxide sheet in preparation of DC vaccine immunoadjuvant, DC vaccine and preparation method thereof |
| CN116333989A (en) * | 2023-05-23 | 2023-06-27 | 中国人民解放军军事科学院军事医学研究院 | Application of sodium magnesium lithium silicate in preparation of dendritic cell function promoter |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111330004A (en) * | 2020-03-04 | 2020-06-26 | 中国人民解放军军事科学院军事医学研究院 | Application of molybdenum disulfide nanosheet layer in preparation of dendritic cell function promoter |
| CN111330004B (en) * | 2020-03-04 | 2023-02-10 | 中国人民解放军军事科学院军事医学研究院 | Application of molybdenum disulfide nanosheet layer in preparation of dendritic cell function promoter |
| CN113069541A (en) * | 2021-04-09 | 2021-07-06 | 中国人民解放军军事科学院军事医学研究院 | Application of graphene oxide sheet in preparation of DC vaccine immunoadjuvant, DC vaccine and preparation method thereof |
| CN116333989A (en) * | 2023-05-23 | 2023-06-27 | 中国人民解放军军事科学院军事医学研究院 | Application of sodium magnesium lithium silicate in preparation of dendritic cell function promoter |
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