CN101297966B - Tumor vaccine for intestinal cancer containing rich chaperone-antigenic peptide complexes and preparation thereof - Google Patents
Tumor vaccine for intestinal cancer containing rich chaperone-antigenic peptide complexes and preparation thereof Download PDFInfo
- Publication number
- CN101297966B CN101297966B CN2008100635095A CN200810063509A CN101297966B CN 101297966 B CN101297966 B CN 101297966B CN 2008100635095 A CN2008100635095 A CN 2008100635095A CN 200810063509 A CN200810063509 A CN 200810063509A CN 101297966 B CN101297966 B CN 101297966B
- Authority
- CN
- China
- Prior art keywords
- chaperone
- intestinal cancer
- cell
- antigenic peptide
- tumor vaccine
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a tumour vaccine of intestinal cancer, which is a compound rich in chaperone molecules-antigenic peptide, and a preparation method thereof. The tumour vaccine is a compound which is rich in protein molecules combined with antigenic peptide, and the compound is the tumour vaccine of the intestinal cancer. The preparation method is that: after being added with elemene and routinely cultured, intestinal cancer cells are treated with heat treatment at the temperature of 39 DEG C to 42 DEG C for 30 minutes and routinely cultured; and then the intestinal cancer cells are irradiated by high-energy X-rays and routinely cultured for one day. Cell lysis and dialysis are carried out to remove the molecules, the molecular weight of which is less than 60kD or more than 180kD. Gel filtration combined with SDS-PAGE is carried out to collect liquid egg whites at the crest segments of 70kD, 90kD, 110kD and 170kD to be combined and condensed to obtain the compound rich in the chaperone molecules-antigenic peptide. The tumour vaccine of the invention has simple method, low cost and high productivity, and can better extract and prepare various chaperone molecules-antigenic peptides; the obtained tumour vaccine has comparatively strong immunological effect and lymphocyte proliferation effect, comparatively high NK and CTL activity and comparatively good application value in the treatment and the relapse prevention of the intestinal cancer; and good economic benefits and social benefits are expected to be realized.
Description
(1) technical field
The present invention relates to a kind of intestinal cancer containing rich chaperone-antigenic peptide complexes tumor vaccine and preparation method thereof.
(2) background technology
Known cytotoxic T lymphocyte (CTL) acts in the antineoplastic immune and plays a major role.But different tumors has the CTL epi-positions of different sudden changes, and so CTL effect does not possess versatility, and its separation and evaluation are also difficult.Some chaperone molecule of discovered in recent years such as the effect of multiple heat shock protein (HSP) in the tumor antigen submission.Down a large amount of synthetic this type chaperones of heat stress have the characteristic that combines the various tumor antigen polypeptide (CTL epi-position) that produce by suddenling change in the tumor cell.Form the HSP-antigenic peptide complexes thus.Get into APC by combining with the surperficial hsp receptor of antigen presenting cell (APC).Different to exogenous antigenic processing with common APC, the tumor antigen peptide of this moment is mainly offered to supply CD8 on the APC surface with " polypeptide-HSP-MHCI " complex by the MHCI molecular pathways
+T cell recognition and activation.Thereby induce special antitumor CTL reaction.This process is found by multiple chaperone albumen such as HSP70 at present, HSP90, and HSP110, gp96, grp170 in too many levels, approach, coordinates to accomplish each other.The preparation of chaperone albumen tumor vaccine needn't separate and identify tumor antigen.In addition, HSP also has the ripe activation of the BMDC of inducing (DC), and short secrete cytokines transmits effects such as signal and enhancing tumor cell immunogenicity.Experiment and clinical cancer therapy have also proved its many superiority.
But present research is conceived to a certain chaperone albumen more, can not fully take into account multiple HSP molecule coordinative role, and also not taking into account different tumor antigens needs different chaperones in different links; And the chaperone-antigenic peptide amount is few and weak immunogenicity, affects curative effect.These have all limited the clinical effectiveness of chaperone-antigenic peptide tumor vaccine.So chaperone is inducing the body antitumor immune particularly to play an important role in the anticancer immunity of specificity.The tumor vaccine that extracts the chaperone preparation carries the antigenic peptides storehouse of individual tumors, becomes the multivalence tumor vaccine, because of no MHC-I class antigen restriction, also need not separate antigenic peptides (antigenic peptides is combined on the chaperone), and have unique superior property.
For this reason, improving the immunogenicity of tumor cell, consider the harmony between the chaperone and obtain enough tumor antigens, is very important for chaperone tumor vaccine antineoplastic immune.In recent years it is swollen to find that the Chinese medicine extract elemene can improve the immunogenicity of tumor cell, thereby improves the synthetic of tumor antigen peptide; The heat stress of radiotherapy and simulation thermotherapy can not only improve the immunogenicity of tumor cell, but more inducing tumor cell synthesizes chaperones such as heat shock protein in a large number; In the experiment formerly, we find that the raising of chaperone local density helps the antineoplastic specificity immunity and forms; Formation in view of the tumour-specific immunity needs many chaperones to coordinate to work at too many levels again; The present invention handles colon cancer cell with the heat stress of elemene, non-lethal dose roentgenization and different temperatures; Extract the known main chaperone that in anticancer specific immunity forms, has important function; Remove the protein molecular that part possibly suppress antitumor immune, concentrate the tumor vaccine of preparation rich chaperone-antigenic peptide complexes.Be expected to a certain degree to solve amount, the density of tumor antigen peptide and present in to the requirement of many chaperones.
Further, Comprehensive Treatment is the developing direction of intestinal cancer treatment, how to carry out Comprehensive Treatment and obtains focus and the difficult point that the optimum synergistic effect is research always.In the present invention, elemene has been applied to clinical because of it directly suppresses tumor growth as chemotherapeutics; Radiotherapy, thermotherapy become clinical antitumor means commonly used because of killing tumor cell in a large number; So; Expect that this experiment helps to form a kind of new method of effective Comprehensive Treatment intestinal cancer: elemene whole body administration anticancer (new adjuvant chemotherapy); Local thermotherapy, surgical operation therapy then before the while art; Utilize the Colorectal Carcinoma of excision to prepare the rich chaperone antigenic peptide complexes at last, feed back patient, further treatment and prophylaxis of cancer recurrence with its activation DC cell.
Intestinal cancer is common malignancy, occupies first three, and its morbidity in recent years is lasting rising situation again.Intestinal cancer treatment difficult point is cancerous cell chemotherapy resistance and transfer and relapse; Immunization therapy has its special advantages, and is more effective reliable to transfer or drug-fast cancerous cell after the chemotherapy.The therefrom condensed just application and development Journal of Sex Research achievement of coming out of the present invention.Being expected in its recurrence of treatment colorectal cancer and prevention has using value preferably, produces good economic and social benefit.
(3) summary of the invention
The object of the invention provides a kind of intestinal cancer tumor vaccine that contains multiple, a large amount of chaperone-antigenic peptides and preparation method thereof.
The technical scheme that the present invention adopts is:
A kind of intestinal cancer containing rich chaperone-antigenic peptide complexes tumor vaccine is obtained by following method: it is 3 μ g/ml that intestinal cancer CT26 cell suspension adds elemene to elemene concentration, 5%CO
2, 37 ℃ cultivated for 3 weeks, through 39 ℃～42 ℃ water bath processing after 40 minutes, 5%CO
2, 37 ℃ cultivated 8 hours, handled 30 seconds 5%CO again with sigmatron (30Mv) vertical irradiation
2, 37 ℃ cultivated 24 hours; Ultrasonic degradation cell in the ice bath; 4 ℃ of low temperature dialysis bag dialysis are removed molecular weight and are lower than 60kD and the protein molecular that is higher than 180kD; Gel filtration combines SDS-PAGE to analyze to collect 70,90,110 and 170kD crest segment place protein liquid and merging, and concentrates (concentrating is in order to remove a large amount of moisture in the protein liquid removal, and generally being concentrated into volume is that 1/15～1/10 of protein liquid volume get final product) and gets rich chaperone-antigenic peptide complexes; As anti-tumor vaccine (tumor vaccine), be said intestinal cancer containing rich chaperone-antigenic peptide complexes tumor vaccine.
The chemotherapeutic elemene is the effective ingredient that from the Chinese medicine Rhizoma Curcumae, extracts, and can improve tumor-cell antigen property, thereby makes the stronger antitumor immune of tumor cell induction body; But chaperones such as the synthetic in a large number heat shock protein of heat stress and radiation inducing tumor cell, also can improve tumor antigen property (show as tumor antigen peptide is synthetic to be increased).Therefore; The present invention is with elemene, heat stress and the radiation of different condition; External sequential processing colon-cancer cell, improve the antigenicity of cancerous cell and promote various chaperones---the formation of tumor antigen peptide complexes is beneficial to that antineoplastic specificity is immunoreactive induces.This imagination has novelty, is first technical problem that the present invention solves.Further, the present invention is with different condition elemene, heat stress and radiation, and the antigenic substance (chaperone-antigenic peptide) of tumor cell is synthetic under the external sequential processing, gropes best synthetic condition.This is another technical problem that the present invention solves, and this has solved the antigenicity of tumor antigen and the problem of amount to a certain extent.
Key problem in technology of the present invention is that extraction from the colon-cancer cell lysate after above-mentioned processing, preparation contain multiple, a large amount of chaperone-antigenic peptides.This can further solve the tumor antigen amount of activation body antitumor immune and the problem of density, and the problem that requires many chaperones to coordinate in the antigen presentation.
The invention still further relates to the method for preparing of described intestinal cancer containing rich chaperone-antigenic peptide complexes tumor vaccine, said method is following: intestinal cancer CT26 cell suspension, adding elemene to elemene concentration is 3 μ g/ml, 5%CO
2, 37 ℃ cultivated for 3 weeks, behind 39 ℃～42 ℃ water bath processing 30～40min, 5%CO
2, 37 ℃ cultivated 8 hours, handled 30 seconds 5%CO again with the sigmatron vertical irradiation
2, 37 ℃ cultivated 24 hours; Cell lysis; Remove molecular weight and be lower than 60kD and the protein molecular that is higher than 180kD, gel filtration combine SDS-PAGE to analyze to collect 70,90,110 and 170kD crest segment place protein liquid merge, concentrate rich chaperone-antigenic peptide complexes (being tumor vaccine).
Preferably, said method is following: get cell concentration 1 * 10
3The intestinal cancer CT26 cell suspension of individual/mL, adding elemene to elemene concentration is 0.003mg/mL, 5%CO
2, 37 ℃ cultivated for 3 weeks, through 42 ℃ of water bath processing after 40 minutes, 5%CO
2, 37 ℃ cultivated 8 hours, handled 30 seconds 5%CO again with 30Mv sigmatron vertical irradiation
2, 37 ℃ cultivated 24 hours; Ultrasonic degradation cell in the ice bath; 4 ℃ of low temperature dialysis bag dialysis are removed molecular weight and are lower than 60kD and the protein molecular that is higher than 180kD; Gel filtration combine SDS-PAGE to analyze to collect 70,90,110 and 170kD crest segment place protein liquid merge, concentrate rich chaperone-antigenic peptide complexes, be said tumor vaccine.
The present invention handles colon-cancer cell with roentgenization, elemene and heat stress; Improve its antigenicity and induce a large amount of synthetic chaperones; Adopt low temperature dialysis, gel filtration to combine SDS-PAGE slightly to carry these chaperones, with the loss of the amount that reduces chaperone-antigenic peptide; Remove the protein molecular (being mainly the albumen of small-molecular weight) that part possibly suppress antitumor immune; Mix the tumor vaccine that concentrates back preparation rich chaperone-antigenic peptide complexes, in the hope of overcoming antigen amount and density issue and many chaperones coordination problem to a certain extent.The result shows, compares with ion-exchange process with affinity chromatograph commonly used at present, and method of the present invention is easier, cost is low, and output is high.The various chaperone-antigenic peptides of better extract and prepare.
Laboratory observation of the present invention the influence that chaperone is expressed of different heat stress, it is the strongest to find that 42 ℃ of inductive tumor cells synthesize the chaperone effect.Further, observe roentgenization, elemene and treatment of different temperature and made up the immunological effect of inducing chaperone-antigenic peptide.The experiment proof; Roentgenization, different temperatures heat stress and elemene are handled and all can effectively be improved the inductive immunology effect of tumor vaccine; Wherein the inductive immunological effect of tumor vaccine with roentgenization, 42 ℃+elemene group is the strongest, demonstrates the strongest lymphocytic hyperplasia effect, and the highest NK and CTL are active.
Intestinal cancer is common malignancy, occupies first three, and its morbidity in recent years is lasting rising situation again.
Tumor vaccine shift after to chemotherapy or drug-fast cancerous cell more effective reliably.The present invention successfully finds out the method for preparing of extracting the rich chaperone-antigenic peptide complexes of high immunogenicity from the tumor cell lysate.Formed novel intestinal cancer tumor vaccine, the animal model test result indicates that tumor vaccine has using value preferably in its recurrence of treatment intestinal cancer and prevention at present, and expection produces good economic and social benefit.
Beneficial effect of the present invention is mainly reflected in: method is easy, cost is low, and output is high, but the various chaperone-antigenic peptides of better extract and prepare; The gained tumor vaccine has stronger immunological effect and lymphocytic hyperplasia effect, and higher NK and CTL are active, in its recurrence of treatment intestinal cancer and prevention, using value is preferably arranged, and expection produces good economic and social benefit.
(4) description of drawings
Fig. 1 is the cell pyrolysis liquid protein electrophoresis figure of different heat treatment; 1 swimming lane: 39.5 ℃; 2 swimming lanes: 37 ℃; 3 swimming lanes: 42 ℃, 4 swimming lanes: 43 ℃;
Fig. 2 is the rich chaperone-antigenic peptide Western Blotting figure of different sequential Processing of Preparation; 1 swimming lane: albumen Marker; 2 swimming lanes: 42 ℃; 3 swimming lanes: 37 ℃;
Fig. 3 is the tumor bulk-growth of each group tumor vaccine immunized mice;
Fig. 4 is the existence prolongation effect of each group tumor vaccine to mice with tumor;
Fig. 5 is that the NK in each tumor vaccine immanoprotection action is active;
Fig. 6 is that the CTL in each tumor vaccine immanoprotection action is active.
(5) specific embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Reagent and material:
Sterilizing room, super-clean bench, high speed low temperature centrifugal machine, molecular cut off are respectively bag filter, sephadex G-200, low temperature dialysis device and the consumptive material (East China medicine) of 50KD and 300KD; DYCP-31D type electrophoresis tank, DYCZ-40D type electrophoretic blotting groove, CO
2Incubator, 10% hyclone RPMI-1640, and pancreatin purchase company in GIBO; Chaperone: HSP70, gp96, the fluorescent monoclonal antibody of HSP110 and HSP170 purchase the company in Sigma; Examine Ma Shi light blue protein determination reagent, albumen marker (worker company is given birth in Shanghai); Lactic dehydrogenase enzyme reagent kit and BCA protein quantification test kit (Sigma company); MTT (AMRESCO company); ELIASA EXL-800 (BIO-TEK, the U.S.); Elemene injection is available from Kingsoft, Shanghai pharmaceutical factory; The CT-26 colon cancer cell line is purchased the Shanghai cell research institute in the Chinese Academy of Sciences; BALB/c mouse is purchased in branch center, Shanghai, national laboratory rodent center; Centrifuge (HITACHI), high speed centrifuge (BECKMAN), electrophresis apparatus (BIO-RAD), cell culture incubator (SHELL), inverted microscope (NIK0N) and ultrasonic cell disintegration appearance (the new sesame bio tech ltd in Ningbo);
The cell pyrolysis liquid of pH7.5 is formed: 20mmol/L Tris-Cl; 20mmol/L NaCl; 0.1mmol/L ethylenediaminetetraacetic acid (ED-TA); 0.1mmol/L dithiothreitol, DTT (DTT); 0.5mmolHexadecyltrimethylammonium bromide.
Embodiment 1: different sequential processing cancerous cell:
Method and process:
The CT26 cell routine is cultivated go down to posterity (RPMI-1640 culture medium, 5%CO
2, 37 ℃), the compound Digestive system of EDTA-trypsin (available from GIBICO) digestion attached cell.Harvesting is with PBS centrifuge washing (2000g, 5 minutes); Process cell suspension with the RPMI-1640 culture medium, the adjustment cell concentration is 1 * 10
3/ ml, in culture medium, adding elemene injection to its final concentration is 3mg/L, is sub-packed in the 50ml culture bottle, the 20ml/ bottle divides 4 groups to place 37 ℃, 39.5 ℃, 42 ℃ and 43 ℃ of water bath processing respectively 30 minutes, the conventional (5%CO that cultivates
2, 37 ℃) spend the night, next day with sigmatron (30MV) from culture bottle top vertical irradiation 0.5 minute (reaching 200cGy dosage).Conventional (the 5%CO that cultivates
2, 37 ℃) and after 24 hours, the compound Digestive system harvesting of EDTA-trypsin; The CT26 cell that does not add elemene also divides 4 groups of same heat stresss and ray to handle.Form 8 groups of cells altogether, every group of culture bottle number is identical.
Embodiment 2: the preparation of rich chaperone tumor antigen peptide complexes
Method and process:
With each group cell routine results and low temperature (ice bath) Ultrasonic Pulverization cracking: 300W, 10 seconds/inferior, 10 seconds at interval, 200 times/pipe, the lysate low-temperature and high-speed was centrifugal, got supernatant SDS-PAGE and analyzed, and the chaperone protein expression of 42 ℃ of groups of (see figure 1) prompting as a result is the highest.By combination and supernatant; Saturated ammonium sulfate salting out method protein isolate; Institute's precipitate that obtains (being protein) is with 0.0002mol/LpH7.4 PBS dissolving, and moving into molecular cut off is 4 ℃ of low temperature dialysis of 50KD bag filter 48 hours (dialysis solution is the PBS of 0.00002mol/L pH7.4), removes to be lower than the 50KD macromole; It is the same low temperature dialysis of 300KD bag filter that the bag filter content is transferred to molecular cut off, collects and has removed the dialysis solution that is higher than the 300KD molecule.Move into 4 ℃ of 50KD bag filters again, add the Polyethylene Glycol powder of 60,000 molecular weight, with the liquid volume that concentrates bag filter to about 250ml.With the conventional dress of sephadex G-200 post (column diameter 2cm; Long 1 meter); With this 250ml dialysis solution gel filtration, SDS-PAGE is made in each collecting pipe liquid sampling analyze, according to electrophoretic band; Collect and merge nearly 70,90,110 and the filtrate of 170KD crest segment place pipe, do with 70,90,110 and the labelling of 170KD.Each is organized each crest segment collection liquid and is 30ml.
Embodiment 3: rich chaperone tumor antigen peptide complexes is identified
Method and process:
Above-mentioned 8 groups of cells are through the cracking dialysis, and every group is filtered and be divided into nearly 70,90,110 and the 4 bassoon filtrate (each bassoon is 30ml) at 170KD crest segment place, every combination and 4 bassoon filtrate, the 120ml mixed liquor, Polyethylene Glycol method concentrated liquid volume is to 10ml.Western blotting is to obtaining mixed liquor protein urine, and the result is as shown in Figure 2, and prepared product is rich in chaperone albumen HSP70, HSP90, HSP110 and HSP170, and luminous the most outstanding with 42 ℃ of heat stress+irradiation group, content is the highest.Conventional BCA method is measured protein concentration, and 42 ℃ of heat stress+irradiation group are 3g/L, and 37 ℃ of heat stress+irradiation group are 0.1g/L.
The separation of embodiment 4:DC, induce and activation
6 of execution BALBL/c mices in 7 age in week, get femur and extract medullary cell liquid the sterile working, and medullary cell liquid is collected in together, with 0.83%tris-NH
4Cl is broken red; The PBS washing.Every group of conventional Ficoll density direct separation and purification of individual nucleus:
Method:
1. in the brachymedial pipe, add an amount of lymphocyte separation medium 3ml;
2. extracting marrow cell liquid and the abundant mixing of equivalent RPMI1640 slowly are superimposed on the laminated fluid level along tube wall with dropper, note keeping clearly interface.The centrifugal 2000rpm of level * 20 minutes.The increase of centrifuge speed and minimizing evenly, steadily make to keep interface clearly;
3. be divided into three layers in the pipe of centrifugal back, the upper strata is not for containing cell bone marrow fluid and RPMI1640 culture fluid liquid, and it is each phase cell with grain that lower floor is mainly red system.The middle level is a lymphocyte separation medium, has one to be the main narrow band of white cloud and mist layer with the mononuclearcell at the interface in last, middle level, and mononuclearcell comprises lymphocyte and mononuclear cell.In addition, also contain platelet;
4. be inserted into the cloud and mist layer with blood capillary, draw mononuclearcell.Insert in another brachymedial pipe, add 5 times of RPMI1640,1500rpm * 10 minute, washed cell 2 times with upper volume;
Last centrifugal after, abandon supernatant, add the RPMI1640 contain 10% calf serum, re-suspended cell.Get a cell suspension and expect that with one 0.2% blue dye liquor mixes, on blood counting chamber, count the TCS in four big grids;
Mononuclearcell concentration (cell number/1 ml cells suspension)=(4 big grid inner cell sum/4) * 10
4* 2 (extension rates);
6. cell viability detects: dead cell can be dyed blue color, and living cells is not painted.Count 200 lymphocytes.Calculate the living cells percentage rate;
Living cells percentage rate=viable count/total cell number * 100%
The result: the living cells percentage rate is 96%.
Reagent and material:
10% calf serum RPMI1640 is available from Hangzhou Ji Nuo company, 0.2% blue dyeing liquor of phenol, and lymphocyte separation medium (Ficoll-Hypaque of proportion 1.077 ± 0.001) is given birth to worker company available from Shanghai.Phosphate buffer (PB) preparation
Stock solution:
A liquid: 0.2M NaH
2PO
4Solution
NaH
2PO
427.6 gram
Or NaH
2PO
42H
2O 31.2 grams
Dissolved in distilled water to 1000 milliliter
B liquid: 0.2M Ha
2HPO
4Solution
Ha
2HPO
412H
2O 71.6 grams
Dissolved in distilled water to 1000 milliliter
Actual preparation (0.1M, PH7.2 PBS):
28 milliliters of A liquid
72 milliliters of B liquid
Adding distil water 100ml
Through 10 pounds, 10 minutes autoclavings.4 ℃ of preservations are subsequent use.
RPMI-1640 with 10%FCS adjusts to 3 * 10
6/ ml adds in 6 well culture plates (2ml/ hole), 10 hole .5%CO totally
2, to cultivate after two hours for 37 ℃, sucking-off is attached cell not, uses the culture medium fine laundering, and sucking-off suspension cell-80 is ℃ frozen.Attached cell is adopted and contains GM-CSF (100 μ g/L), IL-4 (50 μ g/L), the RPMI-1640 of 10%FCS, 5%CO
2, 37 ℃ of cultivations were changed liquid once in 3 days.The rich chaperone peptide complexes solution 0.2ml of purification is respectively organized in the back adding of one week, continues to cultivate 5 to 7 days harvestings, is activatory DC.
Embodiment 5: the animal vivo antitumor experiment of tumor vaccine
In 8 age in week BALB/c mouse the right hind back injected the DC cell suspension (cell concentration 1 * 10 of 100ul embodiment 4 preparation in subcutaneous the 3rd, 6,9,12,15 day respectively
4), and respective packets (totally 5 groups, 8/group).3 Mus are put to death in the last injection after 3 days, get spleen and separating Morr. cell (seeing embodiment 6 for details).All the other 5 mices are with the attack (1 * 10 of the CT26 colon-cancer cell of living
5Individual living cells/inferior, subcutaneous vaccination).Observe mouse tumor growing state, mice life span etc.Result's (seeing Fig. 3, Fig. 4) prompting: the tumor vaccine of the heat stress+irradiation group of different temperatures all has tangible anti-tumor attack function after the elemene pretreatment.And the strongest with the anti-tumor attack function of the tumor vaccine of 42 ℃ of heat stress+irradiation group, show as minimum tumor growth and the longest mice life span.
Embodiment 6: the separation of immune mouse spleen cell
Every group is taken off 3 of cervical vertebra execution BALB/C mices, totally 15, the sterilization of 75% soak with ethanol; The taking-up spleen of cutting open the belly under the aseptic condition is given a baby a bath on the third day after its birth time with aseptic PBS, is cut into diameter 5～10mm fritter; Place 120 order stainless (steel) wires, stainless steel mesh is placed in the plate and adds the RPMI-1640 culture medium, with sterile test tube end extrusion tissue gently; Harvesting, 0.83%Tris-NH
4Cl is broken red, moulds plate and sticks and remove macrophage, with RPMI-1640 complete medium re-suspended cell, is made into 2 * 10
6The lymphocyte suspension of/ml concentration is for use.
Embodiment 7: the immunological effect of tumor vaccine (rich chaperone-antigenic peptide complexes)
1) the cell proliferation stimulating effect of tumor vaccine:
Stimulating was the chaperone-antigenic peptide complexes 100ul that handles through different condition originally, and proliferative cell is normal BALB/c Mus splenocyte, and separation method is made into 2 * 10 with embodiment 6
6The lymphocyte suspension of ml.Both respectively get 50 μ l and mix to add 96 orifice plate Tissue Culture Plates, 3 every group multiple holes, with culture plate place 37,5%CO
2, 100% damp condition cultivates after 3 days, with MTT colorimetric method for determining light absorption value (A490nm) down.With proliferation index (PI) expression, PI=(experimental port A value-tumor cell hole), lymphocyte hole A value.Add the conventional cultivation of 96 orifice plates 3 days, with MTT colorimetric method for determining light absorption value (A), PI representes cell propagation effect with proliferation index; PI=experimental group A value/control group A value.Result such as following table:
Table 1: the body outer cell proliferation stimulating effect of tumor vaccine
Table 1 result shows:
The experiment of vitro lymphocyte proliferation stimulating effect shows the tumor vaccine of being developed: the tumor vaccine of the heat stress+irradiation group of different temperatures all has tangible lymphopoiesis stimulating effect after the elemene pretreatment.And it is the most powerful with this cell proliferation stimulating effect of 42 ℃+ray+elemene group.
2) detection of NK cytoactive
The YAC-I cell is as target cell; With isolating immune mouse spleen cell among the embodiment 6 is the effector lymphocyte, and lactic acid dehydrogenase method for releasing (LDH method) detects the NK cytoactive: by test kit (available from promega company) explain requirement with target cell, effector lymphocyte, 1640 and NP-40 add 100 μ l respectively and (make target cell and effector lymphocyte be 2 * 10 in respective aperture
5Individual), the conventional cultivation.1000 rev/mins centrifugal 5 minutes, get supernatant 50 μ l to new hole, add zymolyte.Every hole adds stop buffer 50 μ l after placing 37 ℃ of 10min, and ELIASA is measured its OD value (A).Killing activity=(A kills and wounds hole-A target cell release aperture)/(the maximum release aperture of A target cell-A target cell release aperture) * 100%.The result sees Fig. 5:
The result shows:
The tumor vaccine of being developed: the tumor vaccine of the heat stress+irradiation group of different temperatures all has the active effect of NK of tangible immune stimulatory Mus after the elemene pretreatment.And it is the most powerful with this activating effect of 42 ℃+ray+elemene group.
3) mensuration of CTL killing activity
Splenocyte (2 * 10 with the 1ml immunized mice
6/ ml) mix with the chaperone-tumor antigen peptide complexes 1ml of 6 kinds of different condition preparations respectively, add 6 orifice plates in 37 ℃, 5%CO
2, 100% damp condition cultivated 7 days down, set up contrast, collected in the culture hole suspension cell as CTL effector lymphocyte.The CT26 cell carries out 4-h as target cell to live
51Cr discharges killing experiments: the CT26 cell is used 200uCiNa
51CrO
4Labelling 2h, cell is washed 3 times with serum-free medium then, and target cell is by different effects: target is than (E: T) educate altogether with the effector lymphocyte.Get supernatant and on the γ calculating instrument, survey the cpm value.BALB/c mouse B lymphoma A20 cell is as the contrast target cell.Maximum release group adds 1%SDS for target cell; Spontaneous release group adds culture medium for target cell.Killing activity=(the spontaneous release group of experimental group cpm-cpm)/(the spontaneous release group of maximum release group cpm-cpm) * 100%.The result sees Fig. 6.
The result shows:
Each tumor vaccine of being developed: the tumor vaccine of the heat stress+irradiation group of different temperatures all has the CTL active effect of tangible raising immunized mice after the elemene pretreatment.And it is the most powerful with this effect of 42 ℃+ray+elemene group.
Claims (2)
1. intestinal cancer containing rich chaperone-antigenic peptide complexes tumor vaccine is obtained by following method: get intestinal cancer CT26 cell suspension, adding elemene to concentration is 3 μ g/ml, 5%CO
2, 37 ℃ cultivated for 3 weeks, behind 39 ℃～42 ℃ water bath processing 30～40min, 5%CO
2, 37 ℃ cultivated 8 hours, handled 5%CO 30 seconds with the sigmatron vertical irradiation of 30Mv again
2, 37 ℃ cultivated 24 hours; The ultrasonic degradation cell; 4 ℃ of low temperature dialysis are removed molecular weight and are lower than 60kD and the protein molecular that is higher than 180kD; Gel filtration combines SDS-PAGE to analyze to collect 70,90,110 and 170kD crest segment place protein liquid, merge protein liquid concentrate said intestinal cancer containing rich chaperone-antigenic peptide complexes tumor vaccine.
2. the method for preparing of intestinal cancer containing rich chaperone-antigenic peptide complexes tumor vaccine as claimed in claim 1, said method is following: get intestinal cancer CT26 cell suspension, adding elemene to concentration is 3 μ g/ml, 5%CO
2, 37 ℃ cultivated for 3 weeks, through 39 ℃～42 ℃ water bath processing after 30～40 minutes, 5%CO
2, 37 ℃ cultivated 8 hours, handled 5%CO 30 seconds with the sigmatron vertical irradiation of 30Mv again
2, 37 ℃ cultivated 24 hours; Ultrasonic degradation cell in the ice bath; 4 ℃ of low temperature dialysis bag low temperature dialysis are removed molecular weight and are lower than 60kD and the protein molecular that is higher than 180kD; Gel filtration combine SDS-PAGE to analyze to collect 70,90,110 and 170kD crest segment place protein liquid merge, concentrate the rich chaperone-antigenic peptide complexes tumor vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100635095A CN101297966B (en) | 2008-06-16 | 2008-06-16 | Tumor vaccine for intestinal cancer containing rich chaperone-antigenic peptide complexes and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100635095A CN101297966B (en) | 2008-06-16 | 2008-06-16 | Tumor vaccine for intestinal cancer containing rich chaperone-antigenic peptide complexes and preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101297966A CN101297966A (en) | 2008-11-05 |
| CN101297966B true CN101297966B (en) | 2012-03-21 |
Family
ID=40078001
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008100635095A Expired - Fee Related CN101297966B (en) | 2008-06-16 | 2008-06-16 | Tumor vaccine for intestinal cancer containing rich chaperone-antigenic peptide complexes and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101297966B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2413953B1 (en) * | 2009-04-03 | 2017-11-08 | Agenus Inc. | Methods for preparing and using multichaperone-antigen complexes |
| CN104211772A (en) * | 2013-12-18 | 2014-12-17 | 深圳市康尔诺生物技术有限公司 | Compound of colorectal cancer antigen peptides and heat shock proteins and use thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1167440A (en) * | 1994-09-30 | 1997-12-10 | 纽约城市大学辛乃山医科学校 | Immunotherapeutic stress protein-peptide complexes against cancer |
-
2008
- 2008-06-16 CN CN2008100635095A patent/CN101297966B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1167440A (en) * | 1994-09-30 | 1997-12-10 | 纽约城市大学辛乃山医科学校 | Immunotherapeutic stress protein-peptide complexes against cancer |
Non-Patent Citations (2)
| Title |
|---|
| 黄常新等.膜表达热休克蛋白70的肠癌瘤苗抗肿瘤治疗作用研究.中华胃肠外科杂志8 3.2005,8(3),255-258. |
| 黄常新等.膜表达热休克蛋白70的肠癌瘤苗抗肿瘤治疗作用研究.中华胃肠外科杂志8 3.2005,8(3),255-258. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101297966A (en) | 2008-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Apuzzo et al. | Immunological aspects of intrinsic glial tumors | |
| US10869922B2 (en) | Alhydrogel-sodium chloride compound immunologic adjuvant, preparation method and use thereof | |
| Cheever et al. | Tumor neutralization, immunotherapy, and chemoimmunotherapy of a Friend leukemia with cells secondarily sensitized in vitro | |
| CN104262459B (en) | Acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide, and vaccine and pharmaceutical application of acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide | |
| ES2246566T3 (en) | APPLICATION OF HSP70 PROTEINS. | |
| CN101830968A (en) | Bursa of Fabricius heptapeptide with immune regulation effect | |
| CN107488235A (en) | A kind of preparation and application of new enhanced antigen combined polypeptid induction liver cancer-specific CTL cells | |
| CN113244383B (en) | Preparation method of DC tumor vaccine and application of DC tumor vaccine in tumor treatment | |
| CN106892974A (en) | A kind of peptide ERE1 long based on tumour antigen ECM1 and its application in immunotherapy of tumors | |
| CN101297966B (en) | Tumor vaccine for intestinal cancer containing rich chaperone-antigenic peptide complexes and preparation thereof | |
| CN109153974A (en) | Enhance the composition to abnormal cell lethality and its application | |
| CN101626781A (en) | Preparation has the method for the cell mass of anti-tumor immune response | |
| CN107417809A (en) | Chondroitin sulfate is used to expand CIK cell, prepare CIK cell amplifing reagent and be coated with the purposes of culture vessel | |
| CN104211772A (en) | Compound of colorectal cancer antigen peptides and heat shock proteins and use thereof | |
| CN111777677A (en) | EGFR T790M new antigen epitope peptide and application thereof in treating tumors | |
| US20190038731A1 (en) | Therapeutic cancer vaccine based on stress proteins rendered immunogenic | |
| CN113368226B (en) | Pharmaceutical composition containing DC tumor vaccine and application thereof in treating cancer | |
| CN105175498A (en) | Heat shock protein complex associated with cervical cancer and application of heat shock protein complex | |
| CN104306976A (en) | Efficient individual tumor immunotherapy and application thereof | |
| CN102775495A (en) | Polypeptide mixture, T cell vaccine and application thereof | |
| CN105176957A (en) | Complexes formed by RCC (renal cell carcinoma) related peptide and HSPs (heat shock proteins) as well as applications of complexes | |
| CN102838679B (en) | The preparation and application of Her2 neu antigen positive tumor therapeutic vaccines | |
| CN103952443A (en) | Tumor-treatment adenovirus vaccine taking hTERT (human telomerase reverse transcriptase) as target point | |
| AU2003277641B2 (en) | Remedy for cancer | |
| CN107080837A (en) | Tumor vaccine, dendritic cell tumor vaccine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Huang Changxin Inventor after: Wu Zhenghu Inventor before: Huang Changxin |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: HUANG CHANGXIN TO: HUANG CHANGXIN WU ZHENGHU |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120321 Termination date: 20130616 |