CN105363027A - Dendritic cell tumor vaccine and preparing method thereof - Google Patents
Dendritic cell tumor vaccine and preparing method thereof Download PDFInfo
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- CN105363027A CN105363027A CN201410437169.3A CN201410437169A CN105363027A CN 105363027 A CN105363027 A CN 105363027A CN 201410437169 A CN201410437169 A CN 201410437169A CN 105363027 A CN105363027 A CN 105363027A
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Abstract
The invention provides a dendritic cell tumor vaccine obtained by co-culturing dendritic cells and cancer stem cells. The invention further provides a preparing method of the dendritic cell tumor vaccine. The preparing method includes the steps that the cancer stem cells are separated from a tumor tissue and are screened through a flow cytometer, and after being irradiated by radioactive rays, the target cancer stem cells are cultured together with the dendritic cells to obtain the dendritic cell tumor vaccine.
Description
Technical field
The present invention relates to a kind of tumor vaccine, particularly a kind of dendritic cell tumor vaccine for cancer stem cell.
Background technology
Conventional tumor therapeuticing method comprises operative treatment, radiation cure and chemotherapy etc.Tumor vaccine is the method for the another kind treatment tumor beyond above-mentioned Therapeutic Method, be through and activate patient's self immune system, utilize specific cellular immunity and the humoral immune reaction of tumor cell or tumor antigen material induction body, the anti-cancer ability of enhancing body, stop the growth of tumor, diffusion and recurrence, to reach the object removed or control tumor.
Dendritic cell (dendriticcell) is that generally acknowledge can the cell of antigen expressed, can antigen decompose after present to the T cell that can play immunological effect and killing ability, make immune system produce reaction.Dendritic cell tumor vaccine is the functional dendritic cell carrying tumor antigen information, can activate specificity T cell and produce immunoreation, play Graft Versus Tumor and immunological memory function.
Dendritic cell tumor vaccine of the prior art, all take tumor cell as target, namely through the antigen that cracking tumor cell obtains, and by this antigen load in the dendritic cell of patient, but the dendritic cell tumor vaccine so obtained is used for the treatment of cancer, often can not solve the problem of tumor recurrence, main cause is not all killed by the cancer stem cell in tumor, and the growth causing cancerous tissue revivable, even via the formation of the cancer stem cell driving tumor of only a few.
Summary of the invention
An aspect of of the present present invention relates to a kind of preparation method of dendritic cell tumor vaccine, comprises the following step: (a) carries out primary separation to a tumor tissues and cultivation obtains tumor cell; B () has the cancer stem cell of specific cells surface antigen from tumor cell screening with flow cytometer; C cancer stem cell that step (b) obtains by () irradiates lonizing radiation; (d) isolating dendritic cells; E (), by the cancer stem cell co-cultivation of dendritic cell with (c) step gained, obtains the dendritic cell tumor vaccine mixing dendritic cell and cancer stem cell.
According to one embodiment of the invention, wherein tumor tissues is glioma.
According to another embodiment of the present invention, wherein cancer stem cell is glioma stem cell.
According to one more embodiment of the present invention, wherein specific cells surface antigen is CD133 and CD15.
According to still another embodiment of the invention, wherein dose radiation is 100Gy.
Another aspect of the present invention relates to a kind of dendritic cell tumor vaccine, is that a dendritic cell and a cancer stem cell are obtained by mixing.
According to one embodiment of the invention, wherein dendritic cell is autologous dendritic cell.
According to another embodiment of the present invention, wherein cancer stem cell is autologous cancer stem cell.
According to another embodiment of the present invention, wherein cancer stem cell is CD133 positive cell, particularly glioma stem cell.
By this, the preparation method of dendritic cell tumor vaccine of the present invention can be used for the preparation that various tumor tissue in vitro carries out dendritic cell tumor vaccine, particularly glioma, and obtained dendritic cell tumor vaccine not only has malicious tumoricidal ability, more for cancer stem cell, there is specific toxic action, be that after the tumor vaccine of target still exists chemotherapy and radiation treatment, tumor resistance improves with tumor cell to solve, and the problem that relapse rate is high and the rate of transform is high.And dendritic cell tumor vaccine of the present invention is because autologous cancer stem cell and autologous dendritic cell, low for the immunologic rejection for the treatment of in the future, the comprehensive and specificity of immunity is also better.
Foregoing invention content aims to provide the simplification summary of this disclosure, possesses basic understanding to make reader to this disclosure.This summary of the invention is not the complete overview of this disclosure, and its purpose is not being pointed out the key/critical assembly of the embodiment of the present invention or defining scope of the present invention.
Accompanying drawing explanation
Know that for making the present invention above-mentioned and other object, feature, advantage and embodiment can become apparent, and are described as follows accompanying drawing:
Fig. 1 is the monolayer cell culture of glioma stem cell and the displaing micro photo figure of neural ball cultivation.
Fig. 2 A is the flow cytometer result figure of dendritic cell tumor vaccine for CD133 negative glial oncocyte toxic action of comparative example.
Fig. 2 B is the flow cytometer result figure of dendritic cell tumor vaccine for CD133 positive neurons glioma cell toxic action of comparative example.
Fig. 3 is comparative example and the dendritic cell tumor vaccine of the present invention displaing micro photo figure for glioma stem cell toxic action.
Fig. 4 is the displaing micro photo figure of dendritic cell tumor vaccine of the present invention to glioma stem cell toxic action.
Fig. 5 is the quantification figure of dendritic cell tumor vaccine of the present invention to glioma stem cell toxic action.
Detailed description of the invention
This description disclosure proposes a kind of dendritic cell tumor vaccine and preparation method thereof, it is from tumor tissues separating tumor cell, and screen cancer stem cell further, after again cancer stem cell being irradiated lonizing radiation, with the dendritic cell co-cultivation of separator well, obtain the dendritic cell tumor vaccine mixing dendritic cell and cancer stem cell.
Aforementioned alleged " cancer stem cell (the cancerstemcells of the present invention, CSCs) " be the cell that there is minute quantity in tumor tissues and there is self-renewal capacity (self-renewal) and differentiation potential (differentiationpotency), cancer stem cell also possesses radio-resistant (radioresistance) and chemoresistance (chemoresistance), for tumor is after accepting radiation cure or chemotherapy, present the clinical picture of resistance, cause cancer sufferer to produce resistance for treatment, tumor easily recurs, and the important key of transfer.
The present invention aforementioned alleged " glioblastoma multiforme (glioblastomamultiforme, GBM) " is the pernicious astrocytoma in glioma (glioma), is one the most serious in malignant brain tumor.The method of traditional treatment glioblastoma multiforme is operation, X-ray therapy and chemotherapy, but because the wellability of glioblastoma multiforme is very high, so-called " wellability " is because the glial cell in brain is one of composition unit in nervous system, provide support, supply nutrition, maintenance environment is constant and provide the functions such as insulation, therefore neurogliocyte tightly can envelope neural axon, because aixs cylinder is very long, if neurogliocyte generation canceration, cancerous cell can spread afield along aixs cylinder.Because operation cannot excise the remote wetted part of cancerous cell, easily cause residual tumor so postoperative, postoperatively need be aided with chemistry and radiotherapy again, but because there is the cancer stem cell of tool radio-resistant and chemoresistance, after treatment, relapse rate is quite high.
The present invention aforementioned alleged " CD133 " is a glycoprotein with 5 trans-membrane region, first in adult blood, bone marrow and embryonic stem cell in isolated CD34 positive precursor cells by identification, and be regarded as the mark of hematopoietic stem cell, then CD133 is more considered to the cell surface marker of leukemia, the brain cancer, retinoblastoma, renal cancer, pancreatic cancer, carcinoma of prostate, hepatocarcinoma, medulloblastoma and gliomatous cancer stem cell.The hypertrophy of the CD133 positive and self-renewal capacity are more better than general tumor cell, and have the characteristic that tumorigenicity (tumorigenicity) and spheroid form cancer stem cells such as (sphereformation).
Multiple test example is hereafter proposed so that some aspect of the present invention to be described, in order to be conducive to persons of ordinary skill in the technical field of the present invention, can the complete utilization put into practice the present invention when not needing over-read, and these test examples should be considered as limitation of the scope of the invention.
Test example 1: the separation and ientification of glioma stem cell
Collection glioblastoma multiforme tissue, first cleans with normal saline solution and is shredded, then being broken up by cell with pepsin digestion system (WorthingtonBiochemical) test kit.The glioblastoma multiforme cell broken up [is added B27 replenisher, 10ng/mL epidermal growth factor (epidermalgrowthfactor with stem cell medium, and 10ng/mL basic fibroblast growth factor (basicfibroblastgrowthfactor EGF), bFGF) neuronal cell cultures base (Neurobasal-Amedium)] settling flux at least 6 hours, make glioblastoma multiforme cell express cell surface antigen again.Add be connected to PE or APC CD133 and CD15 antibody (MiltenylBiotec.Inc.) in the above-mentioned glioblastoma multiforme cell broken up, to demarcate CD133 and CD15 cell surface antigen, and with flow cytometer (fluorescence-activatedcellsorting, FACS) or magnetic bead screen the cell simultaneously with CD133 and CD15 cell surface antigen, this is glioma stem cell.The glioma stem cell filtered out is incubated in stem cell medium, and is placed in 37 DEG C containing 5%CO
2in incubator.
Test example 2: the qualification of glioma stem cell
So that in vitro and in vivo test is further, this test example must confirm that screened cell is for glioma stem cell.In in vitro tests, to limit dilution method (limitingdilutionassay), gene expression analysis and western blot method analysis of cells characteristic.In in vivo test, by screened cell via subcutaneous injection (subcutaneousinjection) with cell to be injected the tumorigenicity that former position (orthotopicinjection) carrys out the cell that evaluation and screening arrives.
Test example 2-1: restriction dilution method
Glioma stem cell is carried out neural ball cultivation, first by 3 × 10
6seeded with living celis in 6 centimeters of Tissue Culture Dishs, again sphaerocyst be separated and be inoculated in 96 porose discs after diluting, 0.2mL stem cell medium is contained in each hole, and the dilution range of cell is 200 to 1 cells/well, the stem cell medium that can add 0.025mL afterwards for every 2 days again until the 7th day, and calculates every 1 hole and forms cell quantity needed at least 1 neural ball in each hole.Test, to confirm whether screened cell has cancer stem cell characteristic during this analytic process be separated in glioma stem cell the 0th day.
With reference to Fig. 1, for the displaing micro photo figure that monolayer cell culture and the neural ball of glioma stem cell are cultivated, visible neuroglial cytoma is incubated in the culture fluid containing hyclone, cell attachment is on Tissue Culture Dish, and cell kenel is outwards stretch, when irradiating lonizing radiation, glioma stem cell starts to assemble, and the kenel of neuroglial cytoma does not change, between cell and cell, still possess distance.And when cultivating neuroglial cytoma with stem cell medium, neuroglial cytoma is still attached on Tissue Culture Dish, but glioma stem cell is then formed spherical, when irradiating lonizing radiation, spherical cell kenel is more obvious, neuroglial cytoma then cause death and have no the cell of attaching.
Test example 2-2: gene expression analysis
For determining the labelling of the glioma stem cell screened, first extract with RNA the RNA that test kit (Qiagen) extracts glioma stem cell, and detect the gene expression of OLIG2, Sox2, CD133 and Nestin with RT-PCR, and be internal reference group with GAPDH.
Test example 2-3:Western blotting
Confirm that the protein labeling of the glioma stem cell screened is expressed with western blot method further again.By cell with the RIPA buffer solution broken cell containing protease inhibitor and inhibitors of phosphatases (Roche), and analyze glioma stem cell Connexin45, GFAP, S100b, SOX2, Nestin and OLIG2 protein expression with western blot method.
Test example 2-4: in vivo test
By 3 × 10
6the glioma stem cell screened in the PBS of 100 μ L, with subcutaneous injection to the right side of Scid/bg mice, and by 1 × 10
8the glioma stem cell screened in the PBS of 2 μ L, with stereotaxical injection to the right side striatum of Scid/bg mice.Mice is sacrificed respectively at 1 to 10 week after injection, carry out brain tissue's frozen section, and with Hematoxylin-eosin dyeing (Hematoxylinandeosinstain) and immunohistochemical staining (immunohistochemistry), determine whether screened glioma stem cell can form tumor in Mice Body.
Comparative example: just for the separation and ientification of neuroglial cytoma
Collection glioblastoma multiforme tissue, first clean with normal saline solution and shredded, in room temperature with enzyme [the Hanks Balanced Salt buffer (Hankbalancedsaltsoultion) containing 40mg the 4th collagen type enzyme and 100U the 5th type hyaluronic acid enzyme] digestion 3 hours, and with the sieved filter of cell, to break up as the cell suspending liquid of individual cells or small fragment is with the minimal medium (Dulbecco ' sModifiedEagleMedium containing 10% patients serum, DMEM), 37 DEG C are incubated at containing 5%CO
2in incubator.
Test example 3: the preparation of dendritic cell
Cells Derived from Dendritic is peripheral blood mononuclear glomus cell (peripheralbloodmononucleatedcells, PBMC), collect the peripheral blood of patient, and separated in peripheral blood by monocyte cell by exclusion, and the patients serum collecting 50-100mL is using the use as subsequent in vitro cell culture.By isolated monocyte cell, with 2 × 10
6the density of cell/mL is incubated in the AIM-V culture medium (Invitrogen) containing 2% patients serum, in 37 DEG C after 2 hours, monocyte cell can be attached on plastic cell culture dish, wash the monocyte cell do not attached with PBS, and collect remaining monocyte cell and be first frozen in-196 DEG C of liquid nitrogens and save backup.
By the monocyte cell of above-mentioned separator well, with graininess M-CSF (granulocytemacrophagecolonystimulatingfactor, GMCSF; And interleukin 4 (interleukin4, IL-4 BectonDickinson); BectonDickinson) be induced to differentiate into dendritic cell, by monocyte cell in the AIM-V culture medium containing 50ng/mLGMCSF, 1000U/mLIL-4 and 2% patients serum, be placed in 37 DEG C containing 5%CO
2cultivate after 7 days in incubator, with flow cytometry analysis cell, whether there is MHCI and MHCII cell surface antigen, to confirm whether cell is divided into dendritic cell.
Test example 4: the preparation of dendritic cell tumor vaccine
Be 100Gy's respectively with dosage
137caesium irradiates glioma stem cell with just for neuroglial cytoma, the glioma stem cell after lonizing radiation and just centrifugal for neuroglial cytoma will be irradiated respectively, and break up with cell culture fluid, mix co-cultivation in 37 DEG C containing 5%CO with the ratio of 1:1 with ripe dendritic cell
2in incubator after 18 to 24 hours, the dendritic cell tumor vaccine that load of the present invention has glioma stem cell can be obtained, and comparative example is just for the dendritic cell tumor vaccine of neuroglial cytoma antigen.Diluted with human serum albumin by above-mentioned dendritic cell tumor vaccine and be divided into 10 pipes, often pipe is containing 2 to 5 × 10
7dendritic cell, and be frozen in-196 DEG C of liquid nitrogens and save backup.
Test example 5: dendron shape tumor vaccine is to the toxic action of glioma stem cell
With reference to figure 2A and Fig. 2 B, the flow cytometer result figure of dendritic cell tumor vaccine for CD133 positive neurons glioma cell toxic action of Fig. 2 A to be the flow cytometer result figure of dendritic cell tumor vaccine for CD133 negative glial oncocyte toxic action of comparative example, Fig. 2 B be comparative example.Wherein the cell mass of white background represents neuroglial cytoma, and the cell of black matrix represents glioma stem cell.The ratio of dendritic cell tumor vaccine and neuroglial cytoma is 1:1, and neuroglial cytoma separately carries out radiation exposure with the dosage of 5Gy and 10Gy respectively.Result shows, no matter be negative at CD133 or that CD133 is positive neuroglial cytoma, after radiation exposure, the number of neuroglial cytoma all declines, particularly separately impose the group of dendritic cell tumor vaccine, the situation that neuroglial cytoma number declines is more obvious.But the number of glioma stem cell, does not but have because of radiation exposure or imposes dendron shape tumor vaccine and decline, and the dendron shape tumor vaccine of display comparative example cannot effectively kill glioma stem cell.
With reference to figure 3, (A) part is the displaing micro photo figure of dendritic cell tumor vaccine for glioma stem cell toxic action of comparative example, (B) part is the displaing micro photo figure of dendritic cell tumor vaccine of the present invention for glioma stem cell toxic action, result shows, impose the group of the dendritic cell tumor vaccine of comparative example, most glioma stem cell is still attached on Tissue Culture Dish, intercellular boundary line is obviously uninfluenced, but impose the group of dendron shape tumor vaccine of the present invention, cell becomes more greatly in vesicle shape, and have storehouse and floating group to produce, cell becomes unhealthy and ongoing death.
Another reference diagram 4 and Fig. 5, Fig. 4 is the displaing micro photo figure of dendritic cell tumor vaccine of the present invention to glioma stem cell toxic action, and (A), (B) and (C) part are respectively the microphotograph imposing after dendritic cell tumor vaccine of the present invention 18th, 90 and 120 hour.Fig. 5 be dendritic cell tumor vaccine of the present invention and neuroglial cytoma respectively with the ratio of 1:1,3:1 and 10:1 to carrying out toxic action, in the quantification figure of the 18th and the 120th hour.Shown by Fig. 4 result, namely dendritic cell tumor vaccine of the present invention had toxic action to glioma stem cell at the 18th hour, and its effect is more obvious in the 90th hour and the 120th hour, cell encystation blister and dead gradually.And show in the result of Fig. 5, dendritic cell tumor vaccine of the present invention not only has toxic action for neuroglial cytoma, also for glioma stem cell, there is toxic action, effect particularly at the 120th hour is quite remarkable, and for the toxic action of neuroglial cytoma and glioma stem cell, be also proportionate with the ratio of dendritic cell tumor vaccine and neuroglial cytoma.
According to above-mentioned, the preparation method of dendritic cell tumor vaccine of the present invention is not limited to glioma, also can be used for the preparation that various tumor tissue in vitro carries out dendritic cell tumor vaccine, and obtained dendritic cell tumor vaccine not only has good poisoning tumor cell ability, more for cancer stem cell, there is specific toxic action, be that the tumor vaccine of target does not have toxic action for cancer stem cell with tumor cell to solve, and in the rear relapse rate for the treatment of and the high problem of the rate of transform.And dendritic cell tumor vaccine of the present invention is because autologous tumor stem cell and autologous dendritic cell obtain, low for the immunologic rejection for the treatment of in the future, comprehensive and the specificity of immunity is also more better than the tumor vaccine of non-used autologous dendritic cell and tumor antigen, for having the tumor therapeuticing method of prospect.
The present invention with embodiment disclose as above, but itself and be not used to limit the present invention, any those skilled in the art, without departing from the spirit and scope of the present invention, can do various amendment and change, therefore protection scope of the present invention is as the criterion with claims.
Claims (10)
1. a preparation method for dendritic cell tumor vaccine, comprises the following step:
A () carries out primary separation to a tumor tissues and cultivation obtains tumor cell;
B () has the cancer stem cell of specific cells surface antigen from the screening of this tumor cell with flow cytometer;
C this cancer stem cell that step (b) obtains by () irradiates lonizing radiation;
(d) isolating dendritic cells;
E (), by this dendritic cell this cancer stem cell co-cultivation with (c) step gained, obtains the dendritic cell tumor vaccine mixing this dendritic cell and this cancer stem cell.
2. the preparation method of dendritic cell tumor vaccine according to claim 1, wherein this tumor tissues is glioblastoma multiforme.
3. the preparation method of dendritic cell tumor vaccine according to claim 1, wherein this cancer stem cell is glioma stem cell.
4. the preparation method of dendritic cell tumor vaccine according to claim 1, wherein this specific cells surface antigen is CD133 and CD15.
5. the preparation method of dendritic cell tumor vaccine according to claim 1, wherein this dose radiation is 100Gy.
6. a dendritic cell tumor vaccine is that a dendritic cell and a cancer stem cell are obtained by mixing.
7. dendritic cell tumor vaccine according to claim 6, wherein this dendritic cell is autologous dendritic cell.
8. dendritic cell tumor vaccine according to claim 6, wherein this cancer stem cell is autologous cancer stem cell.
9. dendritic cell tumor vaccine according to claim 6, wherein this cancer stem cell is CD133 positive cell.
10. dendritic cell tumor vaccine according to claim 6, wherein this cancer stem cell is glioma stem cell.
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| CN109954133A (en) * | 2017-12-22 | 2019-07-02 | 上海市浦东医院（复旦大学附属浦东医院） | Tumor vaccine and preparation method thereof |
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Application publication date: 20160302 |