CN101837123B - TCV and preparation method thereof - Google Patents
TCV and preparation method thereof Download PDFInfo
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- CN101837123B CN101837123B CN201010184853.7A CN201010184853A CN101837123B CN 101837123 B CN101837123 B CN 101837123B CN 201010184853 A CN201010184853 A CN 201010184853A CN 101837123 B CN101837123 B CN 101837123B
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Abstract
The invention belongs to tumor biotherapy field, be specifically related to a kind of TCV and preparation method thereof. Technical problem to be solved by this invention is to provide a kind of vaccine of effectively preventing tumour. The technical scheme of technical solution problem of the present invention is to provide a kind of TCV. This TCV is to pass through inactivation treatment to losing differentiation, Reproductive activity, but still the tumour cell that contains vascular endothelial growth factor expression carrier that can express VEGF is as main active.
Description
Technical field
The invention belongs to tumor biotherapy field, be specifically related to a kind of TCV and preparation method thereof.
Background technology
As everyone knows, a little less than tumour antigen immunogenicity, host is difficult to produce specific immune response, and tumour cell is able to escape machineThe immunosurveillance of body is the unmanageable one of the main reasons of propagation that causes tumour cell. Macrophage is distributed widely in bodyEach tissue, is the important auxiliary cell of immune response initial period, is also the first that body is processed exotic antigen and immunity of organismBarrier. But how to start phagocyte effectively to obtain tumour antigen, a large amount of tumour antigen epi-positions is and is handed to macrophage tableFace is also and passs T cell, thereby the immune response of excitating organism effectively produces the prevention effect to tumour, is that this area is longSince phase, want the problem solving. The development of tumor vaccine is the important hot subject of world today's immunotherapy of tumors. Have at presentThe research report of a lot of tumor vaccines obtains result in zoopery, but still has a lot of problems. Mainly that current majority grindsStudy carefully certain the single antigen molecule in tumour cell, and corresponding gene target spot is as the research object of vaccine, but swollen in recent yearsThe generation of knurl has been realized due to the variation that is not term single gene, but is caused by gene or the protein molecular variation of multiple target spots. This area need to be developed the vaccine of effectively preventing tumour at present.
VEGF (vascularendothelialgrowthfactor, VEGF) is that current known endothelial cell is the strongestMitosis stimulating factor, can stimulate vascular endothelial cell proliferation, promote the formation of tumour angiogenic, be conducive to the life of tumourLong, most study in tumor angiogenesis and anti-angiogenic generation treatment. But VEGF can also be by monocyte tableThe interaction of the acceptor FLT-1 of face has chemotaxis to monokaryon, but current this function to VEGF and sending out in tumourRaw developing meaning research is less.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of vaccine of effectively preventing tumour. Technical solution problem of the present inventionTechnical scheme is to provide a kind of TCV.
This TCV with through inactivation treatment to losing differentiation, Reproductive activity, but still can express VEGFThe tumour cell that contains vascular endothelial growth factor expression carrier be main active.
Inactivation treatment described in above-mentioned TCV is irradiation inactivated.
Tumour cell described in above-mentioned TCV is lung carcinoma cell.
Endothelial growth factor expression carrier described in above-mentioned TCV be can VEGF expression plasmidVEGF-pSectag2。
The present invention also provides the method for preparing above-mentioned TCV. Said method comprises the following steps: by VEGF eucaryonExpression vector proceeds in tumour cell, has screened the strain of VEGF stably express; By Co 60 deactivation place for the strain of VEGF stably expressReason to its cannot break up, hyperplasia, but state that can also expression-secretion VEGF.
It is above-mentioned that to prepare the tumour cell described in TCV method be lung carcinoma cell.
Above-mentioned prepare the endothelial growth factor expression carrier described in TCV method be can VEGF expression plasmidVEGF-pSectag2。
Above-mentionedly prepare the dose irradiation 10～30 that the Co 60 deactivation condition described in TCV method is 10～30GraysMinute.
Above-mentionedly prepare the dose irradiation 20 minutes that the Co 60 deactivation condition described in TCV method is 20Grays.
Further, the tumour cell concentration while carrying out above-mentioned Co 60 deactivation is 1 × 106～100×106Individual/ml.
The present invention also provides the purposes of above-mentioned TCV in the medicine of the anti-curing oncoma of preparation.
Technology path: tumor vaccine of the present invention can utilize its VEGF secreting in vivo to monocytic chemotaxis, will be singleIt is macrophage that nucleus is attracted to tumour cell activation around, and to engulf tumour cell and to offer tumour antigen, induction body producesRaw specific immune response, to reach the object of anti-curing oncoma.
The more current dendritic cell vaccine of technical solution of the present invention has obvious advantage: though BMDC is at present knownStrong antigen presenting cell, but its phagocytic function is very weak or nothing, and adopt after cultured and amplified in vitro DC cell, uses tumour more at presentAntigenic Peptide, tumour antigen or merge as tumor vaccine with tumour cell, macrophage has powerful phagocytic function simultaneouslyAnd antigen presentation, therefore utilize the chemotactic activity of VEGF to attract the immunity of activated mononuclear phagocyte to killing tumor cellsPart, can effectively engulf tumour cell and process tumour antigen, and a large amount of tumour antigen epi-positions is and is handed to Macrophage SurfaceAnd be and pass T cell, thereby the immune response of excitating organism effectively.
Vaccine preparation technology of the present invention is applicable to the clinical preparation with tumor vaccine. Its basic fundamental route has two kinds: the one, byVEGF gene imports the tumor cell line of external built system, makes it cross VEGF expression, constructs the each system of human body and turns VEGGenetic tumour clone is made universal tumor vaccine; The 2nd, get the tumor tissues of postoperative patient, separate primary tumor cell, use glandVirus or lentivirus mediated import VEGF gene, make it cross VEGF expression. VEGF crosses the tumour cell of expression and penetrates through rLine irradiates after deactivation, and the dose irradiation of 20Grays left and right obtains tumor vaccine of the present invention about 20 minutes. R radiation exposure goes outRequirement for through Go60 inactivation treatment are to its activity of not breaking up, growing, but can also survive, and have secrete cytokinesActivity, every kind of tumour cell is according to the situation of himself, and irradiation time and exposure intensity are all different, the cell when irradiationConcentration also can adjust. Tumor vaccine of the present invention is injected in patient body to (subcutaneous, abdominal cavity or vein), can strengthen body to swollenOncocyte immune response, effectively removes the tumour cell of body remnants, the recurrence of anti-curing oncoma and transfer.
In an example of the present invention, with VEGF eukaryotic expression vector transfection tumour cell, the VEGF's of screening high expressedTumor cell clone, irradiates and dedifferentes through Co60 machine r, and dosage is for making it to lose multiplication capacity, but it is certain to retain tumour cellActivity, has secretion of VEGF ability, using the tumour cell of the modification of this routine processes as knurl seedling, in LL/2 lung cancer in miceIn model, obtain good prevention effect.
The treatment of tumour comprises operation, radiotherapy, chemotherapy and four aspects of biological therapy. Utilize clinically this vaccine that these four kinds are controlledTreatment means combine: surgical removal tumor tissues, separate the tumor vaccine that preparation is modified, and select different radiation and chemotherapy sidesCase, even selects lower than routine dose scheme, little on body immune system impact, and the growth of tumour is had to certain tumor-inhibiting action,Be aided with again tumor vaccine of the present invention, produce special immune response to activate body, recurrence and transfer that can effectively anti-curing oncoma.
Beneficial effect of the present invention is that tumor vaccine of the present invention can attract the position of activated mononuclear phagocyte to killing tumor cells,Can effectively engulf tumour cell and process tumour antigen, a large amount of tumour antigen epi-positions is and is handed to Macrophage Surface and presentsGive T cell, produce special immune response thereby effectively activate body, generation, recurrence and transfer that can effectively anti-curing oncoma.For this area provides a kind of new effective tumor vaccine.
Brief description of the drawings
Fig. 1 tumor prevention test time m-gross tumor volume curve map. Result shows that VEGF crosses expression LL/2 tumour cellImmunized mice, can effectively protect mouse to avoid the attack of parental generation LL/2 tumour cell.
The tumour photo of Fig. 2 tumor prevention test, upper row is LL/2MV group, and middle row is LL/2c-p group, and lower row is control group.
Detailed description of the invention
The present invention is specifically described by detailed description of the invention below in conjunction with accompanying drawing.
The preparation of embodiment mono-tumor vaccine of the present invention
Taking mouse LL/2 lung carcinoma cell as model is as example: taking LL/2 mice lung cancer as animal model, by true mouse VEGF164Nuclear expression gene imports LL/2 and carries in tumour cell, has screened VEGF and has stablized high expressed strain (LL/2MV); With the high table of VEGFThe LL/2 tumor cell line reaching, through Go60 inactivation treatment are to its activity of not breaking up, growing, but can also survive, and have secretionThe activity of cell factor. By after the tumor cell inoculation C57 mouse of this deactivation, then give mouse inoculation parental generation LL/2 tumour cell,Observe the protective effect of the TCV parental generation LL/2 tumour cell of this deactivation.
Specific operation process:
1. transfection and the LL/2 cell line that screens high expressed mVEGF1640
LL/2 is incubated to the DMEM matrix (containing amikacin 100u/ml) containing 10% inactivated fetal bovine serum, the life of taking the logarithmLong LL/2 cell 1 × 106Individual in six orifice plates cultivations, only add 2 holes and cultivate. Next day, by the mVEGF-pSectag2 matter of 2ug(sequence of hVEGFmRNA is shown in GeneBank sequence number: GI:3719221 to grain; MVEGF Protein G eneBank sequenceNumber: GI:346974Wei; The structure of carrier is referring to YQ, HuangMJ, YangL, etal., 2001, ImmunogenetherapyoftumorswithvaccinebasedonXenopushomologousvascularendothelialgrowthfactorasamodelAntigen.PNAS98:11545-11550.) or 2ug empty carrier pSectag2 plasmid, according to operational manual, with 5ul lipidBody mixes, transfection LL/2 cell, and transfection is after 48 hours, with Zeocin (bleomycin) screening of 100ug/ml, until sieveSelect the time to reach one month. Filter out the serum of 5 Zeocin resistance clones, with the detection of mVEGF detection kit, select oneThe resistance ZeocinLL/2 clone of high expressed mVEGF, they are 10 years old624 hours endocrine mVEGF of individual tumour cell reach 80.4ng;And parental generation LL/2 only has 1.0ng, empty carrier transfection LL/2 is 1.2ng. The LL/2 of this Expression of Plant Height mVEGF1640 is calledLL/2MV, the LL/2 of empty carrier transfection is LL/2C-P. External doubling time, form and the parental generation of LL/2MV and LL/2C-PLL/2 is consistent, not obviously difference.
Embodiment bis-uses tumor vaccine of the present invention to carry out the test for the treatment of and prevention of tumour
Get 15 8 week age female C57 mouse, be divided at random 3 groups and make prophylactic tria. By the LL/2MV of logarithmic growth andThe tumour cell of LL/2C-P is collected, and uses equally serum-free matrix centrifuge washing 3 times, with blood count chi counting, adjusts cellNumber is 20 × 106Individual/ml, uses C060 machines were through the dose irradiation of 20Grays 20 minutes. By this method process LL/2MV andLL/2C-P (is every injected in mice 2 × 10 respectively at mouse back hypodermic injection 100ul6The individual tumour cell through radioactive ray processing)Each 5 mouse, 100ul days serum matrix of another 5 back hypodermic injections compares. 15 days, interval, every group according to above methodInject again 1 time. 15 days, interval again, regathers the parental generation LL/2 tumour cell centrifuge washing 3 times of logarithmic growth, uses serum-freeIt is 4 × 10 that matrix is adjusted tumour cell concentration6Individual/ml, gives these the 3 groups of right armpit subcutaneous vaccination of mouse 50ul (2 × 105An individual/mouse),Observe the growing state of parental generation LL/2 tumour cell, same until lay one's hand on and when hypodermic tumour enclosed mass, within every 3 days, measure tumour longitudinal and transverseFootpath, until there is mice with tumor knurl body excessive, while being at death's door, all puts to death mice with tumor, and collects respectively the mice serum of 3 groups,Claim tumor tissue weight, and fixing tumor tissues, through FFPE, HE dyeing is done in section.
Experimental result: tumor protection test finds that the LL/2MV tumour cell protection mouse of radioactive ray deactivation is subject to parental generation LL/2 swollenOncocyte is attacked. After inoculation parent LL/2 tumour cell 18 days, control group and LL/2C-P group all can be laid one's hand on and 3 × 4mm2Left and rightEnclosed mass, and LL/2MV group is not laid one's hand on and. Two groups of tumor tissues of control group and LL/2C-P, are the growth of carrying out property, 2 weeks afterwards5 mouse of rear control group all grow to 11 × 11.5mm2, 5 mouse of LL/2L-P group all grow and reach 12 × 10.5mm2AndLL/2MV group has 2 to grow to 6 × 6.5mm2, have 3 not lay one's hand on and (Fig. 1). 33 days eyes of inoculation parental generation LL/2 tumour cellBall is got blood and is put to death mice with tumor, claims tumor tissue weight, and LL/2MV group tumor tissue weight is starkly lower than LL/2MV group (Fig. 2).Result shows that tumor vaccine of the present invention has obvious effect, and has good security.
Claims (10)
1. TCV, is characterized in that: to pass through inactivation treatment to losing differentiation, Reproductive activity, but still can express bloodThe tumour cell that contains vascular endothelial growth factor expression carrier of endothelial tube growth factor is as main active.
2. TCV according to claim 1, is characterized in that: described inactivation treatment is irradiation inactivated.
3. TCV according to claim 1, is characterized in that: described tumour cell is lung carcinoma cell.
4. TCV according to claim 1, is characterized in that: described endothelial growth factor expression carrier isPlasmid VEGF-pSectag2 that can VEGF expression.
5. the method for preparing TCV described in claim 1～4 any one, is characterized in that comprising the following steps: willVEGF carrier for expression of eukaryon proceeds in tumour cell, has screened the strain of VEGF stably express; By VEGF stably express strain cobalt60 inactivation treatment to its cannot break up, hyperplasia, but state that can also expression-secretion VEGF.
6. method according to claim 5, is characterized in that: described tumour cell is lung carcinoma cell.
7. method according to claim 5, is characterized in that: described endothelial growth factor expression carrier is for expressingThe plasmid VEGF-pSectag2 of VEGF.
8. method according to claim 5, is characterized in that: described Co 60 deactivation condition is 10～30GraysDose irradiation 10～30 minutes.
9. method according to claim 8, is characterized in that: the dosage that described Co 60 deactivation condition is 20GraysIrradiate 20 minutes.
10. the purposes of TCV in the medicine of the anti-curing oncoma of preparation described in claim 1～4 any one.
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| CN109125740B (en) * | 2017-06-28 | 2022-04-05 | 成都威斯克生物医药有限公司 | Novel tumor vaccine and application thereof |
| CN110269931B (en) * | 2018-03-16 | 2023-05-09 | 中国科学院上海药物研究所 | Preparation method of hydrogel tumor vaccine, hydrogel tumor vaccine prepared by preparation method and application of hydrogel tumor vaccine |
| CN110090298A (en) * | 2019-05-09 | 2019-08-06 | 武汉大学 | A kind of cell membrane tumor vaccine and preparation method and application |
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