CN101406701A - Adjuvant capable of improving dendritic cell vaccine efficiency - Google Patents
Adjuvant capable of improving dendritic cell vaccine efficiency Download PDFInfo
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Abstract
The invention relates to an adjuvant for improving the effectiveness of a dendritic cell vaccine, which is a traditional Chinese medicine monomer, namely evodiamine.
Description
Technical field
The present invention relates to a kind of new vaccine adjuvant that improves dendritic cell vaccine efficiency and cellular immunization effect, this adjuvant is Chinese medicine monomer a---rutaecarpin.
Background technology
Dendritic cell (dendritic cell, DC) the strongest as the angtigen presentation ability, also be unique antigen presenting cell that can activate primary tape T cell, become one of focus that biomedical sector receives much concern.
With other antigen presenting cell such as B cell, macrophage (macrophage, M φ), (langerhans cell LC) compares, and DC has more the innate advantage as vaccine: (1) DC is the strongest cell of angtigen presentation ability to langhans' cells.Except by engulf, the pinocytosis mode antigen uptaking, DC also has picked-up and transports antigenic special film receptor, so DC can capture the very low corresponding antigens of concentration efficiently.Have the scholar to prove, mice DC promotes that the T ability of cell proliferation is respectively 100 times of M φ, 1000 times of activating B cell.Have to experiment showed, the stimulation T cytological effect that will reach same, the B cell needs antigenic amount will exceed more than 20 times than DC, and needs longer stimulation time.(2) DC can significant stimulation primary tape T cell (native T cell), and M φ, B cell only can stimulate activatory or memory t cell.(3) M φ can catch exogenous antigen, but can not carry out I classpath processed to antigen, and DC then can carry out the processing of I, II classpath and presents in maturation process exogenous antigen.(4) to take in the back at antigen be 24-48h to time of effective angtigen presentation to DC, and M φ then can only encumbrance hour, and it is by the transporter chamber realization of adjusting MHC-II quasi-molecule.Antigenic delay is offered, and helps the performance of immune surveillance function.
The adjuvant of DC vaccine can help antigen stimulation fast, effectively, the immunoreation of longer duration, thereby improve the efficient and the cellular immunization effect of vaccine.Found more DC vaccine adjuvant at present, as the endotoxin of low-molecular-weight immunoreation ornamental equivalent R-848, CpG oligodeoxynucleotide (CpGODN), IL-4/GM-CSF, I type IFN (α or β), antibacterial, monophogphoryl lipid A (monophosphoryl lipid A) etc.Its mechanism of action that strengthens DC angtigen presentation ability can reduce following two aspects: (1) induces the DC maturation, and cell surface CD80, CD86, CD40 and HLA-DR expression are improved; (2) induce DC secrete cytokines (IL-6, IL-12, TNF-α, IFN-α) and chemotactic factor (IL-8, MIP-1 α, MIP-1); Therefore these effective DC vaccine adjuvants can be used for the cellular immunization treatment.
The Chinese medicine Fructus Evodiae has the analgesia of dispeling cold, preventing or arresting vomiting, antidiarrheal and vasodilatory effect.(evodiamine is one of most important alkaloid component of Fructus Evodiae Evo) to rutaecarpin, has the various biological activity, and its immunoregulatory activity is the new discovery of Recent study.Studies show that, rutaecarpin can suppress to activate that the NADP oxidase relies on active chalcogen and iNOS dependency NO product in the inflammatory cell, and synthesizing of inflammatory factor bring into play anti-inflammatory effect by suppressing proinflammatory cytokine secretion prostate E2, leukotriene etc., in addition, rutaecarpin also can suppress the inductive allergic disease of IgE such as allergic dermatitis, rhinitis.Rutaecarpin have similar biologic activity to Fructus Capsici, can competitively demonstrate the effect of anti-nociceptive pain in conjunction with capsaicin receptor when high dose, and this effect is to mediate by anti-inflammatory response.But whether rutaecarpin can be to immune important member---the function of DC exerts an influence, and does not appear in the newspapers as yet.
We successfully develop and a kind ofly can effectively promote DC vaccine of spinal cord injury function recovery and preparation method thereof, see patent 200810110498.1, on this basis, found that further rutaecarpin is not influencing under the sophisticated prerequisite of DC, can pass through to change the secretion pattern of DC, and then promote the multiplication capacity of T cell and improve DC vaccine stress efficacy in vivo.
Summary of the invention
The purpose of this invention is to provide a kind of new vaccine adjuvant---rutaecarpin.
Rutaecarpin can pass through to change the secretion pattern of dendritic cell under the prerequisite that does not influence maturing dendritic cell, and then promotes the multiplication capacity of T cell and improve dendritic cell vaccine stress efficacy in vivo.
The invention provides the application of rutaecarpin in the preparation vaccine adjuvant for this reason.
Vaccine of the present invention, preferably dendritic cell vaccine.
Dendritic cell vaccine of the present invention is meant that the gene with single antigen or hybrid antigen or coding for antigens imports in the dendritic cell, is fed back in the body then.
Dendritic cell of the present invention be in body immune organ or/and direct isolating dendritic cell and by bone marrow, peripheral blood lymphocytes and the inductive dendritic cell of stem cell the immunocyte.
The preparation method of dendritic cell vaccine of the present invention, the process following steps:
1) medullary cell separates, 2) induce medullary cell to be divided into immature dendritic cell with granulocyte-macrophage colony stimutaing factor, 3) preparation spinal cord homogenate albumen, 4) the spinal cord homogenate protein load is in immature dendritic cell.
Wherein medullary cell described in the step 1 is that extraction separation comes out from human or animal's the skeleton that contains bone marrow.
Wherein immature dendritic cell differentiates from medullary cell described in the step 2.
Wherein the albumen of spinal cord homogenate described in the step 3 is that spinal cord extraction separation from syngeneic animal comes out.
Wherein load described in the step 4 is to use culture medium culturing a period of time jointly after spinal cord homogenate albumen and immature dendritic cell mix, and the former is absorbed by the latter.
Detailed step is as follows:
1), obtaining of medullary cell: get Mus femur and tibia, after the PBS flushing, ethanol is fixed, and cleans again, puts in the RPMI1640 culture fluid standby; Extract RPMI RPMI-1640 flushing medullary cavity with aseptic empty needle, medullary cell is flushed in the culture dish, filter through sterilized cotton ball; Contain medullary cell RPMI RPMI-1640 after collect filtering, centrifugal, abandon supernatant, add erythrocyte cracked liquid, add isopyknic PBS then, stop lytic response; Centrifugal, abandon supernatant, add RPMI RPMI-1640 mixing, centrifugal, abandon supernatant.
2), inducing of medullary cell: the medullary cell after will washing is mixed with cell suspension with containing calf serum RPMI RPMI-1640, transfers concentration; Get 6 orifice plates, add cell suspension, add simultaneously and contain calf serum RPMI RPMI-1640, add the Mus granulocyte-macrophage colony stimutaing factor at last, putting the CO2 incubator cultivated 10 days, during this time, change liquid, replenish equivalent simultaneously and contain calf serum RPMI RPMI-1640 and M-CSF.
3), the evaluation of dendritic cell: the 10th day, collect non-adherent cell and centrifugal, identify through flow cytometer, in this experiment the DCs that uses CD11c after testing
+Cell＞94%.With CD11c
+Cell circle door is done I-A/I-E, CD40, CD80, the detection of CD86 surface marker, judges Maturity.Adopt immature DC s in this experiment.
4), the load of dendritic cell: the spinal cord that obtains syngeneic animal, conventional preparation spinal cord homogenate albumen is also quantitative, immature DC s put into contain the proteic DC culture medium culturing of spinal cord homogenate and hatch 2h jointly, fresh DC culture medium flushing, preserve standby on ice, before the injection, cell carries out centrifugal, and is suspended in PBS.
Concrete steps are as follows:
(1) obtaining of medullary cell:
Get two of mices, dislocation of cervical vertebra is put to death, and behind the immersion 3min, mice is fixed on super-clean bench in 75% ethanol, sterilization strip off skin removes muscle, takes out femur and tibia, after the PBS flushing, at the fixing 3min of 75% ethanol, clean with PBS again, put in the RPMI RPMI-1640 standby.Use the tweezers fixed backbone, shears cuts off the bone two ends, and the aseptic empty needle of reuse extracts RPMI1640 culture fluid flushing medullary cavity, 2-3 time repeatedly, medullary cell is flushed in the culture dish, at last the RPMI RPMI-1640 that contains medullary cell in the culture dish is filtered through sterilized cotton ball, remove its component of organization, contain medullary cell RPMI1640 culture fluid after collect filtering, the centrifugal 15min of 1500rpm, abandon supernatant, add erythrocyte cracked liquid 4ml, cracking 3min, add isopyknic PBS then, fully mixing stops lytic response, the centrifugal 10min of 900rpm, abandon supernatant, add RPMI RPMI-1640 mixing, the centrifugal 10min of 900rpm abandons supernatant;
(2) inducing of medullary cell:
With containing the cell suspension that calf serum RPMI RPMI-1640 is mixed with 1ml, cell counting is transferred concentration to 1 * 10 with the medullary cell after the washing
6Individual/ml, get 6 orifice plates, add cell suspension 0.5ml/ hole, add simultaneously and contain calf serum RPMI RPMI-1640 2ml/ hole, the Mus granulocyte-macrophage colony stimutaing factor 10 μ l/ hole final concentrations that add concentration at last and be 5mg/ml are 20 μ g/ml, put CO
2Incubator was cultivated 10 days for 37 ℃, and changed liquid at the 3rd, 6,8 day, replenished equivalent simultaneously and contained calf serum RPMI RPMI-1640 and mGM-CSF;
(3) evaluation of dendritic cell:
The 10th day, collect non-adherent cell and centrifugal, identify through flow cytometer, in this experiment the DCs that uses CD11c after testing
+Cell＞94%, DCs are divided into two groups, and one group is immature DC s, and another is organized for the mature DCs behind the lipopolysaccharide stimulation 24h (1 μ g/ml), with CD11c
+Cell circle door is done I-A/I-E, CD40, CD80, the detection of CD86 surface marker, judges Maturity, adopts immature DC s in this experiment;
(4) load of dendritic cell:
Obtain the spinal cord of syngeneic animal, conventional preparation spinal cord homogenate albumen is also quantitative, and immature DC s is put into the DC culture medium culturing that contains spinal cord homogenate albumen 1 μ g/ μ l, and (do not add cytokine mGM-CSF, cell concentration is 2 * 10
6/ ml) hatch 2h jointly, fresh DC culture medium flushing is preserved standbyly on ice, and before the injection, cell carries out centrifugal, and is suspended in PBS (5 * 10
5/ 5 μ l PBS are for local injection, 1 * 10
6/ 0.3ml PBS is for lumbar injection).
The using method of dendritic cell vaccine is to be expelled in health, and before the injection, cell carries out centrifugal, and is suspended in PBS for injection.
The molecular formula of rutaecarpin of the present invention is as follows: C
19H
17N
3O
Rutaecarpin of the present invention is fields such as the regeneration of infection, antitumor and the spinal cord/nerve that promotes damage and reparation as the range of application of the vaccine of adjuvant.
Wherein said infection is antibacterial, fungus, viral infection etc.; Wherein said tumor is hepatocarcinoma, gastric cancer, esophageal carcinoma, breast tumor, bladder cancer, tumor of prostate etc.; Spinal cord/the nerve of wherein said damage is death of axonal degeneration disintegrate, neuron, astrocyte and the oligodendrocyte of spinal cord/nerve due to the various mechanicalness violences (comprising compressing power and traction force) etc.
The present invention also comprises the pharmaceutical composition that contains adjuvant of the present invention.
Pharmaceutical composition of the present invention can contain the medicine acceptable carrier in case of necessity.
Pharmaceutical composition of the present invention is that the rutaecarpin that adds proper proportion in the preparation process of dendritic cell vaccine obtains.
Add in the described preparation process be the load of dendritic cell finish preceding, load simultaneously, load finishes back adding rutaecarpin.
Described proper proportion is that the part by weight of dendritic cell and rutaecarpin is 1-1000: 1-1000
Preferred ratio is 1-100: 1-100, and preferred ratio is 1-1000: 1, particularly preferably be 50: 1
Rutaecarpin is seen embodiment to the potentiation of the dendritic cell vaccine of reparation Spinal Cord.
Description of drawings
Fig. 1 is the structural formula of rutaecarpin.
The DC that Fig. 2 handles for rutaecarpin promotes the experimental result of T cell proliferation.
Fig. 3 is the influence experimental result of rutaecarpin to the MHC II expression of DC surface
Fig. 4 is the influence experimental result of rutaecarpin to DC surface C D40 expression
Fig. 5 is the influence experimental result of rutaecarpin to DC surface C D80 expression
Fig. 6 is the influence experimental result of rutaecarpin to DC surface C D86 expression
Fig. 7 is the influence experimental result of rutaecarpin to DC IL-12 (P40) secretory function
Fig. 8 is the influence experimental result of rutaecarpin to DC IL-12 (P70) secretory function
Fig. 9 is the influence experimental result of rutaecarpin to DC IL-10 secretory function
Figure 10 is the antibody chip testing result of rutaecarpin to the DC secrete cytokines
The specific embodiment
Further specify the present invention by the following examples, but not as limitation of the present invention.
The physicochemical property of embodiment 1, rutaecarpin
Molecular weight 303.4Da, water insoluble, dissolve in organic solvents such as dimethyl sulfoxide, ethanol, ether, chloroform, fusing point is 278-279 ℃.
Structural formula is seen Fig. 1.
The DC that embodiment 2, rutaecarpin are handled promotes the experiment of T cell proliferation
Separate the Balb/c bone marrow cells in mice, handle and be divided into through different factors through 10 days immature DC cell (iDC) of the external evoked cultivation of GM-CSF: matched group (I), rutaecarpin group (Evo) (II), endotoxin (LPS) group (III), Evo+LPS (IV).Wherein the Evo final concentration is 10 μ M, and the LPS final concentration is 1 μ g/ml, acts on 24 hours.Collect above-mentioned four groups of cells, add ametycin (final concentration is 25 μ g/ml) and put CO
2After 37 ℃ of incubators were hatched 30min, centrifugal, counting was adjusted cell concentration to 1 * 10 with the RPMI-1640 that contains calf serum
6Individual/ml is as irritation cell, and 100 μ l/ holes add in 96 well culture plates.The C57BL/6 mouse spleen non-adherent cell for preparing is centrifugal, counting are adjusted concentration to 4 * 10
6Individual/ml is as reacting cells, and 100 μ l/ holes add in 96 well culture plates.Put CO
2Incubator is cultivated 72h for 37 ℃.Take out culture plate, add MTT (5mg/ml) 20 μ l/ holes, continue to cultivate 4～6h.The taking-up culture plate adds dissolving back, 20%SDS 50 μ l/ holes and goes up microplate reader in 570nM place survey absorbance (OD) value.
The result shows that LPS group and Evo group mixed lymphocyte reaction value are all apparently higher than matched group; Evo+LPS group mixed lymphocyte reaction value shows that also apparently higher than the LPS group Evo has not only promoted the breeder reaction of immature DC to the T cell, has also promoted the breeder reaction of ripe DC (LPS stimulates ripe) to the T cell simultaneously.See Fig. 2.
Embodiment 3, rutaecarpin are to the potentiation of the DC vaccine of repairing Spinal Cord
1, each treatment group motor function evaluation of spinal cord injury mice
Adult BALB/c mouse is used the microsurgery bulldog clamp to make T10 sections severe clamp and is hindered the SCI model; Routinely the homology bone marrow cells in mice is induced into immature DC (simple DC group), part immature DC load spinal cord homogenate albumen (100 μ g/mL) 2h (DC vaccine group), the part immature DC albumen of load spinal cord homogenate again 2h (Evo+DC vaccine group) after rutaecarpin (10 μ M) acts on 24 hours in addition, fresh DC culture medium flushing is preserved standby on ice.Before the injection, cell carries out centrifugal, and is suspended in PBS (5 * 10
5/ 5 μ l PBS are for local injection, 1 * 10
6/ 0.3ml PBS is for lumbar injection).Laboratory animal divides 3 groups to accept above-mentioned 3 kinds of DC treatment respectively.Hindered BBB＜2 fens persons that mark, part or lumbar injection DC (part: 1 * 10 respectively back 1 day
6Cells/5 μ L; Abdominal cavity: 1 * 10
6Cells/0.3mL); Each animal carries out the BBB scoring weekly, observes the neurological functional recovery situation.
The result shows, model success rate 86.5%, and each is organized mice and complete paraplegia all occurs after wound, and 1 all left and right sides BBB scorings recover gradually.Wherein, it is the fastest that Evo+DC vaccine group mice recovers, and the BBB scoring is significantly higher than simple DC group and DC vaccine group.Lumbar injection and local injection BBB appraisal result no difference of science of statistics.The results are shown in Table 1.
Table 1 mice BBB scoring record result (n=90)
A: compare p＜0.01 with simple DC group; B: compare p＜0.05 with the DC vaccine group.
2, the pathological change of each treatment group 84dpi spinal cord stringer sagittal slices is observed
Mice is respectively at 28,56,84 days (the days post injection in injection back, dpi) excessive anesthesia is put to death, each time point is put to death 4 animals at random, washes down blood with normal saline 100ml through the left ventricle perfusion earlier, and the perfusion of reuse 100ml 4% paraformaldehyde is fixing.With the spinal cord injury district is that spinal cord 1.5cm is got at the center, fixing behind the 4% paraformaldehyde liquid, paraffin embedding, and stringer section (5 μ m) continuously, damage back damage zone pathological change and pathological structure are observed in HE dyeing.Immunohistochemical staining (IHC) is observed damage back glial fibrillary acidic protein (glial fibrillary acidic protein with the two mark methods of immunofluorescence histochemistry, GFAP), Nestin, neurofilament (neurofilament, NF), measure glial scar thickness at the damage zone expression.
HE dyeing shows that each group is basic identical in the pathologic structure of corresponding time point.At 28dpi, damage zone cellularity confusion, the stringer arrangement architecture of forfeiture normal spinal cord conductive beam, damage zone is unclear with the scope of demarcating on every side; At 56dpi, damage zone forms blister cavities, and the border is clearer, has the strip glial scar to stretch in the blister cavities, and more amorphous composition and cell component are arranged in the blister cavities, and the blister cavities wall has more complete ependymocyte to cover; At 84dpi, injury region blister cavities clear border, cyst wall glial scar hypertrophy, cyst wall is smooth, and interior is liquid component, and the blister cavities wall that injury region forms has more new vessels.
84dpi cuts into slices after observing mice SCI, the two marks of NF and GFAP fluorescence, astrocyte with hypertrophy is a labelling, thickness to blister cavities wall glial scar is measured, the result shows, DC vaccine group, Evo+DC vaccine group all obviously alleviate in damage zone scope, empty area, the simple DC group of glial scar thickness, and the degree that the Evo+DC vaccine group alleviates is more obvious.The results are shown in Table 3.
Table 284dpi respectively organizes damage zone scope, cavity, glial scar thickness measure
A: compare p＜0.05 with simple DC group; B: compare p＜0.05 with the DC vaccine group.
Embodiment 4, rutaecarpin are tested the influence of DC maturation and costimulatory molecules
DC induce and packet transaction with embodiment 2, each organizes cell centrifugation, abandons supernatant, with PBS washing 3 times, adjusting cell concentration is 1 * 10
6Individual/ml, add Fc receptor blocking agent (1 μ g/ml) earlier, hatch 15min at 4 ℃, add corresponding antibody cell surface binding molecule (MHC II, CD40, CD80 and CD86) then respectively, and do the contrast of corresponding homotype, with PBS washing 2 times, flow cytometer detects DC surface molecular MHC II, CD40, CD80 and CD86 after 4 ℃ of lucifuges are hatched 30min.
The result shows that LPS group MHC II, CD40, CD80 express all apparently higher than matched group; Evo group compare with matched group CD80, CD86 down-regulated expression; The Evo+LPS group compares each index no significant difference with the LPS group.Show that rutaecarpin has suppressed the expression of immature DC part costimulatory molecules, but MHC II molecule and the costimulatory molecules of ripe DC do not had obvious influence.See Fig. 3,4,5,6.
DC induce and packet transaction with embodiment 2, each is organized cell and cultivates 24h, 48h respectively.Collect all culture fluid, the centrifugal 10min of 400rpm collects supernatant ,-20 ℃ of preservations, the content of ELISA kit measurement cytokine IL-12, IL-10.
The result shows that the Evo group is extremely low with cellular control unit factor secretion level, exceeds detection sensitivity, changes with LPS group cytokine secretion level so only compare the Evo+LPS group.No matter two groups of cells are to cultivate 24h, still cultivate 48h, and its IL-12 (P40), IL-12 (P70) and IL-10 level be equal no change difference between two groups.Show that Evo does not have obviously influence to cytokine IL-12, the IL-10 secretion of ripe DC.See Fig. 7,8,9.
Embodiment 6, rutaecarpin detect the antibody chip of DC secrete cytokines
DC induce and packet transaction with embodiment 2, each organizes all culture fluid of cell harvesting, the centrifugal 10min of 400rpm collects supernatant ,-20 ℃ of preservations, use mouse cell factor antibody chip (
Mouse Cytokine Antibody ArrayIII,
Mouse Cytokine Antibody Array IV) relative amount of detection cytokine (being respectively 62 kinds, 34 kinds).
The result shows, Evo group and matched group relatively, cytokine secretion increases has Eotaxin-2 (5.7 times), a VEGF (3.4 times) more than 2 times; Evo+LPS group and LPS group relatively, cytokine secretion increases has Eotaxin-2 (2.6 times), an IL-13 (2.1 times) more than 2 times.Show that Evo all has significantly short secretory action to the Eotaxin-2 of immature DC, ripe DC, simultaneously to the VEGF of immature DC, the IL-13 of ripe DC is also had significantly short secretory action.See Figure 10.
The preparation of embodiment 7, vaccine
The weight that adds rutaecarpin wherein in the preparation process of dendritic cell vaccine is that 50: 1 ratio is calculated according to the part by weight of dendritic cell and rutaecarpin.
Add in the described preparation process is to add rutaecarpin before the load of dendritic cell is finished.
The preparation of embodiment 8, vaccine
The weight that adds rutaecarpin wherein in the preparation process of dendritic cell vaccine is that 100: 1 ratio is calculated according to the part by weight of dendritic cell and rutaecarpin.
Add in the described preparation process is that load in dendritic cell adds rutaecarpin simultaneously.
The preparation of embodiment 9, vaccine
The weight that adds rutaecarpin wherein in the preparation process of dendritic cell vaccine is that 1000: 1 ratio is calculated according to the part by weight of dendritic cell and rutaecarpin.
Add in the described preparation process is to add rutaecarpin after the load of dendritic cell is finished.
The preparation of embodiment 10, vaccine
The weight that adds rutaecarpin wherein in the preparation process of dendritic cell vaccine is that 10000: 1 ratio is calculated according to the part by weight of dendritic cell and rutaecarpin.
Add in the described preparation process be the load of dendritic cell finish preceding, load simultaneously, load finishes back adding rutaecarpin.
Claims (10)
1, the application of rutaecarpin in the preparation vaccine adjuvant.
2, the application of claim 1 is characterized in that, described vaccine is a dendritic cell vaccine.
3, the application of claim 2 is characterized in that, described dendritic cell vaccine prepares through following steps:
1) medullary cell separates, 2) induce medullary cell to be divided into immature dendritic cell with granulocyte-macrophage colony stimutaing factor, 3) preparation spinal cord homogenate albumen, 4) the spinal cord homogenate protein load is in immature dendritic cell.
Wherein medullary cell described in the step 1 is that extraction separation comes out from human or animal's the skeleton that contains bone marrow.
Wherein immature dendritic cell differentiates from medullary cell described in the step 2.
Wherein the albumen of spinal cord homogenate described in the step 3 is that spinal cord extraction separation from syngeneic animal comes out.
Wherein load described in the step 4 is to use culture medium culturing a period of time jointly after spinal cord homogenate albumen and immature dendritic cell mix, and the former is absorbed by the latter.
4, the application of claim 2 is characterized in that, described preparation: the process following steps:
1), obtaining of medullary cell: get Mus femur and tibia, after the PBS flushing, ethanol is fixed, and cleans again, puts in the RPMI RPMI-1640 standby; Extract RPMI RPMI-1640 flushing medullary cavity with aseptic empty needle, medullary cell is flushed in the culture dish, filter through sterilized cotton ball; Contain medullary cell RPMI RPMI-1640 after collect filtering, centrifugal, abandon supernatant, add erythrocyte cracked liquid, add isopyknic PBS then, stop lytic response; Centrifugal, abandon supernatant, add RPMI RPMI-1640 mixing, centrifugal, abandon supernatant.
2), inducing of medullary cell: the medullary cell after will washing is mixed with cell suspension with containing calf serum RPMI RPMI-1640, transfers concentration; Get 6 orifice plates, add cell suspension, add simultaneously and contain calf serum RPMI RPMI-1640, add the Mus granulocyte-macrophage colony stimutaing factor at last, putting the CO2 incubator cultivated 10 days, during this time, change liquid, replenish equivalent simultaneously and contain calf serum RPMI RPMI-1640 and M-CSF.
3), the evaluation of dendritic cell: the 10th day, collect non-adherent cell and centrifugal, identify through flow cytometer, in this experiment the DCs that uses CD11c after testing
+Cell＞94%.With CD11c
+Cell circle door is done I-A/I-E, CD40, CD80, the detection of CD86 surface marker, judges Maturity.Adopt immature DC s in this experiment.
4), the load of dendritic cell: the spinal cord that obtains syngeneic animal, conventional preparation spinal cord homogenate albumen is also quantitative, immature DC s put into contain the proteic DC culture medium culturing of spinal cord homogenate and hatch 2h jointly, fresh DC culture medium flushing, preserve standby on ice, before the injection, cell carries out centrifugal, and is suspended in PBS.
5, the application of claim 2 is characterized in that, described preparation: the process following steps:
(1) obtaining of medullary cell:
Get two of mices, dislocation of cervical vertebra is put to death, and behind the immersion 3min, mice is fixed on super-clean bench in 75% ethanol, sterilization strip off skin removes muscle, takes out femur and tibia, after the PBS flushing, at the fixing 3min of 75% ethanol, clean with PBS again, put in the RPMI RPMI-1640 standby.Use the tweezers fixed backbone, shears cuts off the bone two ends, and the aseptic empty needle of reuse extracts RPMI RPMI-1640 flushing medullary cavity, 2-3 time repeatedly, medullary cell is flushed in the culture dish, at last the RPMI RPMI-1640 that contains medullary cell in the culture dish is filtered through sterilized cotton ball, remove its component of organization, contain medullary cell RPMI RPMI-1640 after collect filtering, the centrifugal 15min of 1500rpm, abandon supernatant, add erythrocyte cracked liquid 4ml, cracking 3min, add isopyknic PBS then, fully mixing stops lytic response, the centrifugal 10min of 900rpm, abandon supernatant, add RPMI RPMI-1640 mixing, the centrifugal 10min of 900rpm abandons supernatant;
(2) inducing of medullary cell:
With containing the cell suspension that calf serum RPMI RPMI-1640 is mixed with 1ml, cell counting is transferred concentration to 1 * 10 with the medullary cell after the washing
6Individual/ml, get 6 orifice plates, add cell suspension 0.5ml/ hole, add simultaneously and contain calf serum RPMI RPMI-1640 2ml/ hole, the Mus granulocyte-macrophage colony stimutaing factor 10 μ l/ hole final concentrations that add concentration at last and be 5mg/ml are 20 μ g/ml, put CO
2Incubator was cultivated 10 days for 37 ℃, and changed liquid at the 3rd, 6,8 day, replenished equivalent simultaneously and contained calf serum RPMI RPMI-1640 and mGM-CSF;
(3) evaluation of dendritic cell:
The 10th day, collect non-adherent cell and centrifugal, identify through flow cytometer, in this experiment the DCs that uses CD11c after testing
+Cell＞94%, DCs are divided into two groups, and one group is immature DC s, and another is organized for the mature DCs behind the lipopolysaccharide stimulation 24h (1 μ g/ml), with CD11c
+Cell circle door is done I-A/I-E, CD40, CD80, the detection of CD86 surface marker, judges Maturity, adopts immature DC s in this experiment;
(4) load of dendritic cell:
Obtain the spinal cord of syngeneic animal, conventional preparation spinal cord homogenate albumen is also quantitative, and immature DC s is put into the DC culture medium culturing that contains spinal cord homogenate albumen 1 μ g/ μ l, and (do not add cytokine mGM-CSF, cell concentration is 2 * 10
6/ ml) hatch 2h jointly, fresh DC culture medium flushing is preserved standbyly on ice, and before the injection, cell carries out centrifugal, and is suspended in PBS (5 * 10
5/ 5 μ l PBS are for local injection, 1 * 10
6/ 0.3ml PBS is for lumbar injection).The using method of dendritic cell vaccine is to be expelled in health, and before the injection, cell carries out centrifugal, and is suspended in PBS for injection.
6, a kind of vaccine combination is characterized in that, contains dendritic cell and rutaecarpin.
7, the vaccine combination of claim 6 is characterized in that, the part by weight of dendritic cell and rutaecarpin is 1-1000: 1-1000.
8, the vaccine combination of claim 7 is characterized in that, the part by weight of dendritic cell and rutaecarpin is 1-100: 1-100.
9, the vaccine combination of claim 8 is characterized in that, the part by weight of dendritic cell and rutaecarpin is 1-1000: 1.
10, be fields such as the regeneration of infection, antitumor and the spinal cord/nerve that promotes damage and reparation with rutaecarpin as the range of application of the vaccine of adjuvant, wherein said infection is antibacterial, fungus, viral infection etc.; Wherein said tumor is hepatocarcinoma, gastric cancer, esophageal carcinoma, breast tumor, bladder cancer, tumor of prostate etc.; Spinal cord/the nerve of wherein said damage is death of axonal degeneration disintegrate, neuron, astrocyte and the oligodendrocyte of spinal cord/nerve due to the various mechanicalness violences (comprising compressing power and traction force) etc.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101991575A (en) * | 2010-11-05 | 2011-03-30 | 中国人民解放军第三军医大学第三附属医院 | Application of evodiamine in preparing medicine for inhibiting aryl hydrocarbon receptor |
| CN105622608A (en) * | 2016-01-12 | 2016-06-01 | 沈阳药科大学 | Nitrate NO donor type evodiamine derivatives with anti-tumor activity |
| WO2017164407A1 (en) * | 2016-03-25 | 2017-09-28 | 国立大学法人東北大学 | Indole alkaloid compound having immune checkpoint inhibitory action |
| CN107510838A (en) * | 2017-08-31 | 2017-12-26 | 广东颜值科技有限公司 | A kind of cell preparation and its preparation method and application |
| CN113136366A (en) * | 2021-04-15 | 2021-07-20 | 厦门大学 | Method for enhancing dendritic cell cross presentation by 27-bit lysine mutant ubiquitin molecule |
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2008
- 2008-11-28 CN CNA2008101805197A patent/CN101406701A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101991575A (en) * | 2010-11-05 | 2011-03-30 | 中国人民解放军第三军医大学第三附属医院 | Application of evodiamine in preparing medicine for inhibiting aryl hydrocarbon receptor |
| CN105622608A (en) * | 2016-01-12 | 2016-06-01 | 沈阳药科大学 | Nitrate NO donor type evodiamine derivatives with anti-tumor activity |
| CN105622608B (en) * | 2016-01-12 | 2017-04-26 | 沈阳药科大学 | Nitrate NO donor type evodiamine derivatives with anti-tumor activity |
| WO2017164407A1 (en) * | 2016-03-25 | 2017-09-28 | 国立大学法人東北大学 | Indole alkaloid compound having immune checkpoint inhibitory action |
| US10550120B2 (en) | 2016-03-25 | 2020-02-04 | Tohoku University | Indole alkaloid compound having immune checkpoint inhibitory action |
| CN107510838A (en) * | 2017-08-31 | 2017-12-26 | 广东颜值科技有限公司 | A kind of cell preparation and its preparation method and application |
| CN113136366A (en) * | 2021-04-15 | 2021-07-20 | 厦门大学 | Method for enhancing dendritic cell cross presentation by 27-bit lysine mutant ubiquitin molecule |
| CN113136366B (en) * | 2021-04-15 | 2022-05-31 | 厦门大学 | Method for enhancing dendritic cell cross presentation by 27-bit lysine mutant ubiquitin molecule |
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