CN104830793A - DC cell based on LMP-1 antigen, targeting immune cell population, preparation method and applications thereof - Google Patents
DC cell based on LMP-1 antigen, targeting immune cell population, preparation method and applications thereof Download PDFInfo
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Abstract
The invention discloses a DC cell based on LMP-1 antigen, a targeting immune cell population, a preparation method and applications thereof. The preparation method of DC cell comprises the following steps: subjecting mononuclear cells to first induced differentiation culture to obtain immature DC cells; transfecting the immature DC cells by LMP-1 antigen mRNA to obtain transfected immature DC cells; subjecting the transfected immature DC cell to second induced differentiation culture to obtain mature DC cells; wherein the mononuclear cells are separated from peripheral blood mononuclear cells of a sample. Through the provided preparation method, DC cells can be obtained efficiently and safely. Moreover, the provided preparation method has the advantages of low cost and high efficiency, and can effectively solve the problem of cell toxicity. The DC cells can be applied to cell immunotherapy or used to trigger the lymph cells from the same patient to generate targeting immune cell population rich in CTL cells so as to achieve a better effect in tumor immunotherapy.
Description
Technical field
The present invention relates to biological technical field, particularly, the present invention relates to DC cell, targeted immune cell mass and its production and use.
Background technology
Malignant tumour is the main fatal disease types of the mankind, and mortality ratio remains high.It is the first that Ministry of Health's recent statistics data show that malignant tumour occupies China resident top ten cause of the death.Chinese Incidence patient 2,680,000 people in 2010, dead 1,970,000 people; The current whole nation has malignant tumor patient more than 600 ten thousand people.Medical expense every year for malignant tumor patient accounts for 20% of Health Expenditure, is the maximum medical burden of the whole society.Current treatment means for reaching healing, to extend the effect of malignant tumor patient lifetime and the targets such as quality of making the life better still very limited, even the malignant tumour early diagnosis especially for some type is also difficult to obtain satisfied curative effect, the malignant tumour as transitivity, general or progressive stage is then more difficult to cure.
In current ideas of cancer therapy, the topical therapeutics such as operation and radiotherapy have good curative effect to limitation tumour, then mainly rely on chemotherapy or immunotherapy to carry out systemic treatment for the MRD after general, transitivity or topical therapeutic.Tumor invasion is the result of multi-step, polygene effect, and tumour cell shows growth, differentiation and apoptosis out of control.Clinically the diversity of patient symptom and idiogenetics heterogeneous, and the appearance of tumour distant metastasis focus, makes oncotherapy with the viewpoint of systemic disease, must adopt the comprehensive therapeutic plan of individuation.Namely according to the feature of each tumour patient, adopt topical therapeutic in conjunction with whole body therapeutic; Not only to eliminate the tumour of local lesion, also will control the Preventive growth of tumour and tumour to the invasion and attack of important organ, really effectively could improve curative ratio and the lifetime of tumour patient.The treatment of usual tumor by local focus can take operation and Physiotherapy (comprise radio-frequency (RF) ablation, thermotherapy, radiotherapy, freezing, ultrasonicly involve laser therapy etc.) method elimination, but for the not detectable tumour minimal disease of iconography (as the residual after transfer and relapse stove, topical therapeutic, the cancer cells in blood), above topical therapeutic means are then helpless, must be solved by systemic treatment means.Because the transfer of tumour, recurrence, diffusion are more fatal, therefore systemic treatment seems particularly important and urgent.Current systemic treatment has the treatment of traditional chemotherapy, molecular targeted agents, biotherapy (comprising cellular replacement therapy and immune cell therapy) and gene therapy etc.Although classic chemotherapy has made some progress in some oncotherapy, prolongation tumour patient aspect lifetime is still contributed not quite.And tumour cell is to the natural drug resistance of chemotherapeutic and Acquired lung infection and chemotherapeutic toxicity, makes chemotherapeutical development receive serious obstruction.Clinical practice for many years proves that it is not the strong event of whole body therapeutic.
Immune cell therapy is that recent two decades grows up, and immune cell therapy mainly comprises CIK and DC cell.Due to its therapeutic domain wide (can be used for noumenal tumour and leukemia), more effective to tumour minimal disease (comprising the cancer cells in transfer, recurrence stove, blood) especially, have no side effect, and be applicable to tumour each stage (as Radiotherapy chemotherapy in late period can not use), and be equally applicable to virus infection; Therefore it is a strong event of whole body therapeutic.Immune cell therapy has significant effect to raising quality of life, prolongation lifetime, and praise highly extremely both at home and abroad, be one of best means of current whole body therapeutic, clinical potentials is huge.
But the preparation method at present for the immunocyte carrying out immune cell therapy still haves much room for improvement.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is to propose a kind of safe, efficient method preparing DC cell and targeted immune cell mass.
It should be noted that, the present invention completes based on the following discovery of contriver:
Along with the development of biotherapy technology, immunotherapy is carried out to malignant tumour and becomes study hotspot, immunotherapy is the brand-new pattern of one of oncotherapy, and especially cell-mediated adoptive immunotherapy comparatively maturation has entered the clinical application stage, and achieves obvious result for the treatment of.Cell-mediated adoptive immunotherapy has become one of important means of assisting therapy after tumour patient Radiotherapy chemotherapy, its for promoting reconstruction of patients immune system, eliminate residual and bone marrow purging all has good result.People kill and wound (LAK) cell, tumor-infiltrated lymph (TIL) cell, cytokine-induced killer cell (CIK) cell etc. by research lymphokine-activation, show CIK cell be a kind of novel, efficient, there is non-principal histocompatibility complex (MHC) the restricted immune effector cell that wide spectrum kills tumor activity, in immunotherapy of tumors, manifest huge using value.Dendritic cell (DC) is main antigen presenting cell powerful in human body, antigen-specific cellular poison lymphocyte (CTL) can be induced react and the propagation affecting β cell (cell of Regular Insulin can be produced in the pancreas islet of pancreas) by direct or indirect mode, activation humoral immunoresponse(HI).By CIK cell and DC combination therapy malignant tumour, contribute to the T cell Immune anergy removing tumour patient, have the effect of synergistic antitumor.The treatment (CIK/DC cell therapy) of cytokine induced kill cell associating dendritic cell to malignant tumour that increasing test and clinical practice show demonstrates good therapeutic action, has huge development potentiality and application prospect.
Wherein, CTL cell is the T cell subgroup of CD8+, is a kind of specific T cell, has direct killing effect to some virus, tumour cell etc., forms body disease-resistant poison, antineoplastic important defence line with natural killer cell.CTL kill mechanism has: 1. discharge pore-forming protein, granzyme, cracking target cell.2. the apoptosis of target cell is mediated by FasL.
Antigen load technology main Types based on DC has: tumor antigen peptide impacts DC; Tumour cell antigen impacts DC; Tumour cell RNA impacts DC; The immunotherapy of genetic modification DC.Contriver finds; tumor-antigen peptide load DC is that application tumor-antigen peptide impacts sensitization DC in vitro; then feedback or immunization are to lotus knurl host; body can be obviously induced to produce Peptide-specific CTL; produce protective immunological reaction; dc Antigens all is at present developed as vaccine, produces antibody and CTL in body, does not prepare CTL cell in vitro.The immune response evoked in vivo due to antigen likely produces the possible of systemic font immunity, and contriver utilizes this technology to prepare CTL cell and combined immune cell mass in vitro, can solve current problem.In addition, current useful various carrier is as antigen, the viral vector Antigens such as rAAv prepare CTL technology, ACTL technology is exactly one of them, but due to the integration characteristic of virus, cause the genome toxicity problem of DC cell to can't resolve always, so contriver utilizes the carrier of mRNA antigen technical substitution virus type or DNA class, viral vector has been exceeded from security, simultaneously industrialization angle, mRNA in-vitro transcription more can realize industrialization, simple to operate, and Quality Control and quality inspection can stdn.
In other words, the present invention utilizes mRNA-DC-CTL technology to prepare targeted immune cell mass, particularly: by the peripheral blood lymphocytes (Monocytes) of the mRNA transfection patient of VAA determinant, through cytokine induction, monocyte transformation is the DC cell with powerful antigen presentation function.By the DC cell obtained through this technology, stimulate the T lymphocyte of patient in vitro, produce the cytotoxic T lymphocyte (CytotoxicT lymphocytes, CTL) effectively killing and wounding virus.The CTL produced has virus antigen specificity, i.e. targeting, and the virus only for certain or several specific virus related antigen positive has lethal effect, to the cell of antigen negative without any effect.
And then, according to an aspect of the present invention, the invention provides a kind of method preparing DC cell.According to embodiments of the invention, the method comprises the following steps: monocyte is carried out the first differentiation-inducing cultivation, to obtain immature DC cell; Utilize immature DC cell described in LMP-1 antigen mRNA transfection, to obtain the immature DC cell through transfection; And the described immature DC cell through transfection is carried out the second differentiation-inducing cultivation, to obtain ripe DC cell, wherein, described monocyte is separated to obtain from the peripheral blood mononuclear cell of sample.
Contriver is surprised to find, utilize method of the present invention can prepare DC cell (i.e. dendritic cell) safely and efficiently, and method cost of the present invention is low, efficiency is high and can effectively solve cytotoxicity problem, safety non-toxic after the DC cell feedback patient obtained, immune cell therapy can be effective to, and the lymphocyte that this DC cell can also be effective to stimulate same patient to originate produces the targeted immune cell mass being rich in a large amount of CTL cell, and then better effects if after the immunotherapy infected for tumour and nerpes vinrus hominis.
In addition, the method for the DC of preparation cell according to the above embodiment of the present invention can also have following additional technical characteristic:
According to embodiments of the invention, described LMP-1 antigen mRNA is obtained by following steps: preparation LMP-1 antigen cDNA plasmid, and the nucleotide sequence of described LMP-1 antigen cDNA is as shown in SEQ ID NO:1; Described LMP-1 antigen cDNA plasmid is carried out linearization process, to obtain the LMP-1 antigen cDNA plasmid through linearization process; And the described LMP-1 antigen cDNA plasmid through linearization process is carried out in-vitro transcription, to obtain LMP-1 antigen mRNA.Thereby, it is possible to effectively prepare LMP-1 antigen mRNA.
According to embodiments of the invention, with pcDNA3.1 carrier for skeleton carrier, prepare described LMP-1 antigen cDNA plasmid.Thus, preparation efficiency is high, effective.
According to embodiments of the invention, described pcDNA3.1 carrier has BamhI, XbaI and Bst1107I restriction enzyme site.Thereby, it is possible to effectively carry out plasmid clone and follow-up linearization process.
According to embodiments of the invention, carry out described linearization process based on restriction enzyme site Bst1107I.
According to embodiments of the invention, T7Message Machine test kit is utilized to carry out described in-vitro transcription.Thus, transcriptional efficiency is high, effective.
According to embodiments of the invention, after carrying out described in-vitro transcription, comprise the step of edulcoration purification further.Thus, the carrying out of follow-up LMP-1 antigen mRNA transfection is conducive to.
According to embodiments of the invention, utilization electricity turns or PEI method carries out described transfection.Thus, transfection efficiency is high, effective.
According to embodiments of the invention, utilize PEI method to carry out described transfection, wherein, PEI is 3:1 with the mixing quality ratio of described LMP-1 antigen mRNA.Thus, transfection is given prominence to.
According to embodiments of the invention, DC substratum is utilized to carry out described first differentiation-inducing cultivation and described second differentiation-inducing cultivation.Thereby, it is possible to effectively induction obtains DC cell.
According to embodiments of the invention, described DC substratum comprises: serum-free cell culture medium; The CTL4 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, wherein, described serum origin is in described sample.Thus, culture effect is good.According to embodiments of the invention, described serum free medium is the serum free medium of Takara.According to other embodiments of the present invention, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin.Thus, culture effect is given prominence to.
According to embodiments of the invention, carry out described first differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.According to embodiments of the invention, carry out described second differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.Thus, culture effect is good.
According to a further aspect in the invention, present invention also offers a kind of method preparing targeted immune cell mass.According to embodiments of the invention, the method comprises the following steps: the peripheral blood mononuclear cell providing sample; The peripheral blood mononuclear cell of described sample is separated into lymphocyte and monocyte; Described lymphocyte is carried out activation culture, to obtain the lymphocyte through activation culture; Monocyte is carried out the first differentiation-inducing cultivation, to obtain immature DC cell; Utilize immature DC cell described in LMP-1 antigen mRNA transfection, to obtain the immature DC cell through transfection; The described immature DC cell through transfection is carried out the second differentiation-inducing cultivation, to obtain ripe DC cell; And the DC cell of described maturation and the described lymphocyte through activation culture are carried out mixed culture, to obtain targeted immune cell mass.
Contriver is surprised to find, method of the present invention is utilized can effectively to prepare targeted immune cell mass, this targeted immune cell mass not only has the powerful CTL cell killing and wounding specific virus, also has the immunocytes such as CIK, NK, can be effective to immune cell therapy.In addition, method cost of the present invention is low, efficiency is high, and can effectively solve cytotoxicity problem, safety non-toxic after the targeted immune cell mass feedback patient of acquisition.
According to embodiments of the invention, described LMP-1 antigen mRNA is obtained by following steps: preparation LMP-1 antigen cDNA plasmid, and the nucleotide sequence of described LMP-1 antigen cDNA is as shown in SEQ ID NO:1; Described LMP-1 antigen cDNA plasmid is carried out linearization process, to obtain the LMP-1 antigen cDNA plasmid through linearization process; And the described LMP-1 antigen cDNA plasmid through linearization process is carried out in-vitro transcription, to obtain LMP-1 antigen mRNA.Thereby, it is possible to effectively prepare LMP-1 antigen mRNA.
According to embodiments of the invention, with pcDNA3.1 carrier for skeleton carrier, prepare described LMP-1 antigen cDNA plasmid.Thus, preparation efficiency is high, effective.
According to embodiments of the invention, described pcDNA3.1 carrier has BamhI, XbaI and Bst1107I restriction enzyme site.Thereby, it is possible to effectively carry out plasmid clone and follow-up linearization process.
According to embodiments of the invention, carry out described linearization process based on restriction enzyme site Bst1107I.
According to embodiments of the invention, T7Message Machine test kit is utilized to carry out described in-vitro transcription.Thereby, it is possible to effectively carry out plasmid clone and follow-up linearization process.
According to embodiments of the invention, after carrying out described in-vitro transcription, comprise the step of edulcoration purification further.Thus, the carrying out of follow-up LMP-1 antigen mRNA transfection is conducive to.
According to embodiments of the invention, utilization electricity turns or PEI method carries out described transfection.Thus, transfection efficiency is high, effective.
According to embodiments of the invention, utilize PEI method to carry out described transfection, wherein, PEI is 3:1 with the mixing quality ratio of described LMP-1 antigen mRNA.Thus, transfection is given prominence to.
According to embodiments of the invention, described activation culture comprises: utilize CTL1 cell culture medium to carry out the first activation culture 24 hours to described lymphocyte; CTL2 cell culture medium is utilized to carry out the second activation culture 2-3 days to the lymphocyte through the first activation culture; And utilize CTL3 cell culture medium to carry out the 3rd activation culture 4-5 days to the lymphocyte through the second activation culture, every equivalent fluid infusion in 2-3 days once.Thereby, it is possible to effectively make lymphocyte activation, be conducive to the acquisition of follow-up targeted immune cell mass.
According to embodiments of the invention, described CTL1 cell culture medium comprises: serum-free cell culture medium; The CTL1 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, described CTL2 cell culture medium comprises: serum-free cell culture medium; The CTL2 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, described CTL3 cell culture medium comprises: serum-free cell culture medium; The CTL3 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, wherein, described serum origin is in described sample.Thus, activation culture is effective.According to embodiments of the invention, described serum free medium is the serum free medium of Takara.According to other embodiments of the present invention, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin.Thus, activation culture is remarkably productive, and lymphocyte activation is effective, is conducive to the acquisition of follow-up targeted immune cell mass.
According to embodiments of the invention, DC substratum is utilized to carry out described first differentiation-inducing cultivation and described second differentiation-inducing cultivation.Thereby, it is possible to effectively induction obtains DC cell.
According to embodiments of the invention, described DC substratum comprises: serum-free cell culture medium; The CTL4 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, wherein, described serum origin is in described sample.Thus, culture effect is good.According to embodiments of the invention, described serum free medium is the serum free medium of Takara.According to other embodiments of the present invention, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin.Thus, culture effect is given prominence to.
According to embodiments of the invention, carry out described first differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.According to embodiments of the invention, carry out described second differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.Thus, culture effect is good.
According to embodiments of the invention, CTL3 cell culture medium is utilized to carry out described mixed culture 6-8 days, preferably 7 days.Thereby, it is possible to effectively obtain targeted immune cell mass.
According to embodiments of the invention, described CTL3 cell culture medium comprises: serum-free cell culture medium; The CTL3 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, wherein, described serum origin is in described sample.Thus, culture effect is good.According to embodiments of the invention, described serum free medium is the serum free medium of Takara.According to other embodiments of the present invention, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin.Thereby, it is possible to prepare targeted immune cell mass efficiently.
According to embodiments of the invention, be that 1:20 carries out described mixed culture according to the DC cell of described maturation and the described lymphocytic volume ratio through activation culture.Thus, mixed culture is effective, can prepare targeted immune cell mass efficiently.
In addition, according to other embodiments of the present invention, the method preparing targeted immune cell mass of the present invention can also comprise the following steps:
The first step: extract nerpes vinrus hominis's infected patient vein peripheral blood (50-100 milliliter) or adopt blood cell separator directly to isolate peripheral blood mononuclear cell (PBMC).
Second step: through cultivating, PBMC is separated into lymphocyte and monocyte, lymphocyte continues to cultivate, for subsequent use; Utilize LMP-1 antigen mRNA transfection monocyte, and utilize the dendritic cell (DC) of cytokine induction maturation to produce.
3rd step: by the DC cell of maturation and lymphocyte mixed culture, becomes cytotoxic T lymphocyte that have antigen-specific, that kill and wound antigen positive virus by lymphocyte induction, obtains targeted immune cell mass.
According to another aspect of the invention, present invention also offers a kind of targeted immune cell mass.According to embodiments of the invention, it is prepared by the foregoing method preparing targeted immune cell mass.Contriver finds, targeted immune cell mass of the present invention not only has the powerful CTL cell killing and wounding specific tumors cell, also have the immunocytes such as CIK, NK, and cost is low, safety non-toxic, can be effective to immune cell therapy.
In accordance with a further aspect of the present invention, the DC cell that present invention also offers targeted immune cell mass and utilize the method for the foregoing DC of preparation cell to prepare is preparing the purposes in medicine, described medicine is used for the treatment of LMP-1 to be infected, or suppresses the transfer of LMP-1 positive tumor, diffusion, recurrence.
According to embodiments of the invention, described LMP-1 infects for nerpes vinrus hominis infects.
It should be noted that, contriver finds, because mRNA is without the need to promotor, can not unconfinedly increase, can not enter on nucleus Insertion Into Host Cell karyomit(e), without hereditary genotoxicity, the features such as the security that tool is very high, thus the present invention intends preparing DC cell and targeted immune cell mass (the targeted immune cell cluster for LMP-1) by mRNA-DC-CTL method, for use in the immunotherapy that nerpes vinrus hominis infects.Method of the present invention breaches the medicine of stimulation DC in the past can not the limitation of conventional preparation.Further, utilize mRNA transfection DC in vitro and produce belonging to for the first time of specificity Mutiple Targets CTL cell.
Particularly, method of the present invention have the following advantages one of at least:
1, LMP-1mRNA antigen is constructed first in the world.
2, LMP-1 antigen mRNA transfection DC cell is utilized to carry out external evoked acquisition LMP-1 specific target immunocyte group (being rich in CTL cell) in the world first.
3, target immunocyte group of the present invention comprises: CTL cell (CD8+, CD3+CD8+CD28+), CIK cell (CD3+CD56+), NK cell (CD3-CD56+), make it not only have the powerful CTL cell killing and wounding specific virus, also have the immunocytes such as CIK, NK, the overall immunity of patient can be promoted, the obvious symptom improving patient, can help that immune response is more complete, usefulness is higher.
4, the whole process that prepared by target immunocyte group only needs 12-14 days.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows according to one embodiment of the invention, the result of plasmid enzyme restriction qualification in LMP-1 antigen cDNA plasmid construction process;
Fig. 2 shows according to one embodiment of the invention, the result figure of effect after GFP detection mRNA transfection;
Fig. 3 shows according to one embodiment of the invention, the statistic analysis result that the flow cytomery result based on targeted immune cell mass obtains.
Embodiment
It should be noted that, term " first ", " second " only for describing object, and can not be interpreted as instruction or hint relative importance or imply the quantity indicating indicated technical characteristic.Thus, be limited with " first ", the feature of " second " can express or impliedly comprise one or more these features.Further, in describing the invention, except as otherwise noted, the implication of " multiple " is two or more.
Below in conjunction with embodiment, the solution of the present invention is made an explanation.It will be understood to those of skill in the art that the following examples only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition in embodiment, (such as show with reference to J. Pehanorm Brooker etc. according to the technology described by the document in this area or condition, " Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be and by the conventional products of commercial acquisition, such as, can be able to purchase from Illumina company.
Embodiment 1:
One, material and equipment
1, laboratory rank: GLP
2, plant and instrument:
Negative 80 degree of Ultralow Temperature Freezers (the U.S. water chestnut of middle section, DW-HL388); General refrigerator (Haier, double door ,-20 DEG C and 4 DEG C); Mold incubator (Shanghai is won fast, MJX-160B-Z); Constant water bath box (SSW-420-2S); CO
2incubator (HF240); Low speed centrifuge (Town in Shanghai booth, TDL-40C); Inverted biologic microscope (section believes, model: BLD-1); Biohazard Safety Equipment (Suzhou purifies, double); Electric pipettor (German Pulan moral) 1-10ml; Micropipette rifle (big dragon) 1 overlaps, 4; Program temperature reduction box (Thermo); Liquid nitrogen container (Jin Feng, YDS-120-216); PCR instrument; Gel electrophoresis imaging system; Electrophoresis apparatus; Thermostat;
3, cell cultures consumptive material:
Cell culture bags (Takara company); 75cm
2culturing bottle, 175cm
2culturing bottle, 1.8ml cryopreservation tube, 1.5ml EP pipe, 50ml centrifuge tube, 15ml centrifuge tube, all from Coning company of the U.S.; 0.22um syringe needle filter (U.S. company BD); 100um cell screen cloth (U.S. company BD); 30ml syringe (Shanghai Medical apparatus).
4, cell culture reagent:
Serum-free cell culture medium (Takara, article No.: GT-T561); Australia's foetal calf serum (FBS QUALIFIEDAUSTRALIA ORIGIN), Gibco, article No.: 10099-141;
Lymphocyte separation medium Ficoll liquid (GE, article No.: 17-1440-02);
The dual anti-solution of penicillin/streptomycin (PENICILLIN STREPTOMYCIN SOL, PS), Gibco, article No.: 15140-122.
5, the preparation of substratum:
5.1 reagent:
The CTL1 factor (comprising IFN-γ, 1000U/ml): 1ml, preserves for-20 DEG C for a long time, preserves 2 weeks for 4 DEG C;
The CTL2 factor (comprises IL-1,1000U/ml; IL-2,1000U/ml; CD3 antibody, 100ng/ml; CD28 antibody, 100ng/ml): 1ml, preserves for a long time for-20 DEG C, preserves 2 weeks for 4 DEG C;
The CTL3 factor (comprises IL-2,1000U/ml; IL-7,20ng/ml; IL-15,20ng/ml): 1ml, preserves for a long time for-20 DEG C, preserves 2 weeks for 4 DEG C;
The CTL4 factor (comprises GM-CSF, 1000U/ml; IL-4,500U/ml; TNF-α, 500U/ml; IL-1,500U/ml; IL-6,500U/ml): 1ml, preserves for a long time for-20 DEG C, preserves 2 weeks for 4 DEG C;
Tumour antigen mRNA transfection mixture reagent A: 100ul, preserves for a long time, once uses up after dissolving for-80 DEG C.
5.2 compound methods:
Basic medium: add the gentamicin of 2% in serum-free cell culture medium, wherein use after 0.22um syringe needle frit.
CTL1 substratum: the 1 pipe CTL1 factor taken out from-20 degree, normal temperature melts, and is added in 20ml basic medium, and adds the serum of the client of 10%, wherein use after 0.22um syringe needle frit.
CTL2 substratum: the 1 pipe CTL2 factor taken out from-20 degree, normal temperature melts, and is added in 20ml basic medium, wherein uses after 0.22um syringe needle frit.
CTL3 substratum: the 1 pipe CTL3 factor taken out from-20 degree, normal temperature melts, and is added in 500ml basic medium, wherein uses after 0.22um syringe needle frit, whole culturing process 3 pipes.
DC substratum: the 1 pipe CTL4 factor taken out from-20 degree, normal temperature melts, and is added in 50ml basic medium, and adds the serum of the client of 10%, wherein use after 0.22um syringe needle frit.
Two, method
1 builds LMP-1 antigen cDNA plasmid
First, LMP-1 antigen gene is carried out synthetic, and obtain the cDNA of gene.
The cDNA sequence of LMP-1 antigen gene is as follows:
ATGGGGCGTTTGACCTCCGGCGTCGGGACGGCGGCCCTGCTAGTTGTCGCGGTGGGACTCCGCGTCGTCTGCGCCAAATACGCCTTAGCAGACCCCTCGCTTAAGATGGCCGATCCCAATCGATTTCGCGGGAAGAACCTTCCGGTTTTGGACCAGCTGACCGACCCCCCCGGGGTGAAGCGTGTTTACCACATTCAGCCGAGCCTGGAGGACCCGTTCCAGCCCCCCAGCATCCCGATCACTGTGTACTACGCAGTGCTGGAACGTGCCTGCCGCAGCGTGCTCCTACATGCCCCATCGGAGGCCCCCCAGATCGTGCGCGGGGCTTCGGACGAGGCCCGAAAGCACACGTACAACCTGACCATCGCCTGGTATCGCATGGGAGACAATTGCGCTATCCCCATCACGGTTATGGAATACACCGAGTGCCCCTACAACAAGTCGTTGGGGGTCTGCCCCATCCGAACGCAGCCCCGCTGGAGCTACTATGACAGCTTTAGCGCCGTCAGCGAGGATAACCTGGGATTCCTGATGCACGCCCCCGCCTTCGAGACCGCGGGTACGTACCTGCGGCTAGTGAAGATAAACGACTGGACGGAGATCACACAATTTATCCTGGAGCACCGGGCCCGCGCCTCCTGCAAGTACGCTCTCCCCCTGCGCATCCCCCCGGCAGCGTGCCTCACCTCGAAGGCCTACCAACAGGGCGTGACGGTCGACAGCATCGGGATGCTACCCCGCTTTATCCCCGAAAACCAGCGCACCGTCGCCCTATACAGCTTAAAAATCGCCGGGTGGCACGGCCCCAAGCCCCCGTACACCAGCACCCTGCTGCCGCCGGAGCTGTCCGACACCACCAACGCCACGCAACCCGAACTCGTTCCGGAAGACCCCGAGGACTCGGCCCTCTTAGAGGATCCCGCCGGGACGGTGTCTTCGCAGATCCCCCCAAACTGGCACATCCCGTCGATCCAGGACGTCGCGCCGCACCACGCCCCCGCCGCCCCCAGCAACCCGGGCCTGATCATCGGCGCGCTGGCCGGCAGTACCCTGGCGGTGCTGGTCATCGGCGGTATTGCGTTTTGGGTACGCCGCCGCGCTCAGATGGCCCCCAAGCGCCTACGTCTCCCCCACATCCGGGATGACGACGCGCCCCCCTCGCACCAGCCATTGTTTTACTAGA(SEQ ID NO：1)
Then, with pcDNA3.1 carrier for skeleton carrier, by enzymatic cleavage methods, the LMP-1 antigen gene cDNA sequence shown in the SEQ ID NO:1 of above-mentioned synthetic is cloned on carrier, prepares LMP-1 antigen cDNA plasmid.
Wherein, Fig. 1 is shown in by plasmid enzyme restriction picture.As shown in Figure 1, left side swimming lane is: DNA marker, from top to bottom, and band order: 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp, 3000bp, 5000bp, 10000bp; Right lanes is: A040-1 plasmid, is by carrying out digestions acquisition to the BamHI/SalI restriction enzyme site of pcDNA3.1 carrier.
Cut qualification through order-checking and enzyme, the LMP-1 gene order of synthetic is correct, and contriver successfully builds and obtains LMP-1 antigen cDNA plasmid.
2 in-vitro transcription
2.1 linearization process
Get the LMP-1 antigen cDNA plasmid built, the linearizing restriction enzyme site Bst1107I (distance terminator codon is distant, about 2KB) based on the suggestion of pcDNA3.1 carrier carries out linearization process.
2.2 in-vitro transcription
Utilize T7Message Machine test kit (Ambion company), the LMP-1 antigen cDNA plasmid (namely as the DNA profiling of in-vitro transcription) through linearization process is carried out in-vitro transcription, specific as follows:
First, in the plastic centrifuge tube of a RNase-free, at room temperature add following ingredients in order, preparation feedback system (20 microlitre):
Then, wrap the mouth of pipe with sealed membrane, 60 DEG C of water-bath 30min, the RNase inhibition in reaction system is played one's part to the full, to remove the RNase in template.
1 microlitre t7 rna polymerase (note: this is the consumption of 20 microlitre reaction systems is added after the cooling of question response system, to other reaction systems, the consumption of each composition can increase and decrease in proportion), 37 DEG C of insulations 2 hours (noting: extending soaking time can not significantly improve output).
Then, 70 DEG C of heating, 10 minutes deactivation t7 rna polymerases.
2.3 remove DNA profiling
In above-mentioned in-vitro transcription system, add 1 microlitre RNase-free DNase (3-5U/uL), and be incubated 15-30 minute at 37 DEG C, then moisturizing is to 100uL;
Then, with the saturated phenol-chloroform extracting of isopyknic Tris once;
Next, add the ethanol of 3M NaAc and the 2 times volume of 0.1 times of volume, the centrifugal 15-30 minute of maximum speed after mixing, add the ethanol of 1mL 70% after abandoning supernatant, after vibration, centrifugal 2 minutes of maximum speed, abandons supernatant, dry, the RNA of gained precipitation and in-vitro transcription gained.
Get 1-3 microlitre product electrophoresis detection transcription effects, detect qualified after the RNA obtained is placed in-80 DEG C of preservations.
3 targeted immune cell mass preparations
3.1. the separation of peripheral blood mononuclear cell
The cell obtained is separated in the 100ml peripheral blood that simplexvirus patient from the 1 routine LMP-1 positive is gathered, transfer to centrifugal (1800rpm in 50ml centrifuge tube, 10min), suck supernatant, getting the liquid after supernatant is slowly added on lymphocyte separation medium Ficoll liquid (GE), volume ratio is 1:1, centrifugal (2000rpm, 20min);
Then, the white flock cell collecting interface adds PBS, blows and beats mixing gently, centrifugal (1500rpm, 10min);
Next, repeated centrifugation washing totally 3 times;
Then, collect cell and blow and beat mixing gently, add basic medium 20ml and blow and beat evenly, be transferred to 75cm
2in culturing bottle, in 37 DEG C, 5%CO
22h cultivated by incubator.
3.2 lymphocytes are separated with monocytic
Take out 75cm
2culturing bottle, gently by 75cm
2culturing bottle straightens vertical, draws 18ml nutrient solution in new 175cm
2in bottle, for use in lymphocytic activation culture;
For former bottle (75cm
2culturing bottle) in remaining 2ml raffinate, monocyte will be used for be induced to differentiate into DC cell.
3.3 lymphocytic cultivations
For cultivating lymphocytic 175cm
2in bottle, add 20ml CTL1 cell culture medium, after 24h, add 20mlCTL2 culture medium culturing again, then every 2 ~ 3d CTL3 substratum equivalent fluid infusion, whether qualifiedly carry out active survival rate test checking.
3.4. monocytic process
A, at the surplus former bottle (75cm having 2ml raffinate
2culturing bottle) in add 15ml DC substratum and continue cultivation 4 days, within every 2 ~ 3 days half, change liquid once, monocyte to be induced to differentiate into immature DC cell.
B, PEI infection protocol carries out antigen gene mRNA transfection: the 5th day, immature DC cell (centrifugal 1500rpm is collected with 50ml centrifuge tube, 10min), after discarding supernatant, lower confluent monolayer cells is transferred to 1.5ml centrifuge tube, then reagent A (namely LMP-1mRNA and PEI mixes the rear mixture obtained according to mass ratio 1:3) the fully mixing bottom cell of 100ul is directly added, build centrifuge tube, be placed in cell culture incubator and hatch 30 minutes, take out turned upside down several times every 10 minutes.
Wherein, PEI (1ug/ul) Polysciences (CAT#23966-2 is mixed with liquid storage) obtains according to following steps preparation: first, will be heated to about 80 DEG C dissolve PEI, cool to room temperature without endotoxic sterilized water; Then, adjusted to ph to 7.0, sterilizes with the frit of 0.22um, is stored in-20 DEG C after packing, and working fluid can save backup at 4 DEG C.
GFP detects the effect after mRNA transfection, and as shown in Figure 2, wherein, left figure is picture before DC transfection to result, and right figure is picture after DC transfection, and transfection efficiency is up to 80% as shown in Figure 2.
C, take out centrifuge tube afterwards, with liquid-transfering gun cell suspension taken out and be added to culturing bottle (75cm
2) in, and add DC substratum, continue cultivation 3 days, to obtain ripe DC cell.
3.5. Dual culture
After 8th day, the DC cell of maturation is mixed according to volume ratio 1:20 with the aforementioned lymphocyte through activation culture, with CTL3 substratum Dual culture seven days, the generation of induction CTL cell and homicidal wound sexual cell thereof.
Dual culture, after seven days, carries out flow cytometer detection to cell, and then convection type detected result carries out statistical study.Wherein, Fig. 3 shows the statistic analysis result that the flow cytomery result based on targeted immune cell mass obtains, the per-cent namely in targeted immune cell mass shared by various immunocyte.As shown in Figure 3, not only containing a large amount of CTL cells in the immunocyte group that the present embodiment prepares, and also have the panimmunity such as NK and CIK cell cell: CTL cell (CD8+, CD3+CD8+CD28+), CIK cell (CD3+CD56+), NK cell (CD3-CD56+), namely this immunocyte group not only has the powerful CTL cell killing and wounding specific tumors cell, also has the immunocytes such as CIK, NK.
Thus, contriver finds, transfected dendritic cell in vitro can activated T lymphocytes effectively, produces CTL, and enormous amount, reaches 10
9more than individual.Guarantee clinical efficacy is good.And traditional DC cell attempts activated T lymphocytes in vivo, produce CTL, but the immunity system microenvironment of the weakness of patient makes DC cell be difficult to activated T cell generation CTL in vivo, quantity is few, causes unsatisfactory curative effect, and without specificity, targeting.
Embodiment 2: clinical treatment
CTL cell embodiment 1 prepared feeds back patient, and observes feedback effect, specific as follows:
The simplexvirus patient 20 of the LMP-1 positive is divided into 2 groups (A group 10 and B groups 10), wherein, for A group, according to the method in embodiment 1 after the 14th day and the 15th day obtain enough CTL cells, feed back to patient, each feedback cell concentration is 1 ~ 2*10
9; B group does not feed back cell (in contrast).It is for 2 times a course for the treatment of that blood sampling one-time continuous feeds back.After 3 courses for the treatment of terminate, observe.
Found that: A group and B group have obvious difference, all patients of A group all feel that after feedback health sense tired out alleviates, and appetite increases, pain relief, and body weight increases, and wherein has 6 patient's herpesvirus infection symptoms to alleviate; And the every symptom of B group is not all improved.Can obviously find out, after feeding back CTL cell, patients symptomatic is obviously alleviated.
Thus, the CTL cell that known the present invention prepares can promote the overall immunity of patient, obviously improves the symptom of patient, can help that immune response is more complete, usefulness is higher.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. prepare a method for DC cell, it is characterized in that, comprise the following steps:
Monocyte is carried out the first differentiation-inducing cultivation, to obtain immature DC cell;
Utilize immature DC cell described in LMP-1 antigen mRNA transfection, to obtain the immature DC cell through transfection; And
The described immature DC cell through transfection is carried out the second differentiation-inducing cultivation, to obtain ripe DC cell,
Wherein, described monocyte is separated to obtain from the peripheral blood mononuclear cell of sample.
2. method according to claim 1, is characterized in that, described LMP-1 antigen mRNA is obtained by following steps:
Preparation LMP-1 antigen cDNA plasmid, the nucleotide sequence of described LMP-1 antigen cDNA is as shown in SEQ ID NO:1;
Described LMP-1 antigen cDNA plasmid is carried out linearization process, to obtain the LMP-1 antigen cDNA plasmid through linearization process; And
The described LMP-1 antigen cDNA plasmid through linearization process is carried out in-vitro transcription, to obtain LMP-1 antigen mRNA.
3. method according to claim 2, is characterized in that, with pcDNA3.1 carrier for skeleton carrier, prepares described LMP-1 antigen cDNA plasmid,
Optionally, described pcDNA3.1 carrier has BamhI, XbaI and Bst1107I restriction enzyme site,
Optionally, carry out described linearization process based on restriction enzyme site Bst1107I,
Optionally, T7Message Machine test kit is utilized to carry out described in-vitro transcription,
Optionally, after carrying out described in-vitro transcription, comprise the step of edulcoration purification further.
4. method according to claim 1, is characterized in that, utilization electricity turns or PEI method carries out described transfection,
Optionally, utilize PEI method to carry out described transfection, wherein, PEI is 3:1 with the mixing quality ratio of described LMP-1 antigen mRNA,
Optionally, DC substratum is utilized to carry out described first differentiation-inducing cultivation and described second differentiation-inducing cultivation,
Optionally, described DC substratum comprises:
Serum-free cell culture medium;
The CTL4 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Wherein, described serum origin in described sample,
Optionally, described serum free medium is the serum free medium of Takara,
Optionally, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin,
Optionally, carry out described first differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once,
Optionally, carry out described second differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.
5. prepare a method for targeted immune cell mass, it is characterized in that, comprise the following steps:
The peripheral blood mononuclear cell of sample is provided;
The peripheral blood mononuclear cell of described sample is separated into lymphocyte and monocyte;
Described lymphocyte is carried out activation culture, to obtain the lymphocyte through activation culture;
Monocyte is carried out the first differentiation-inducing cultivation, to obtain immature DC cell;
Utilize immature DC cell described in LMP-1 antigen mRNA transfection, to obtain the immature DC cell through transfection;
The described immature DC cell through transfection is carried out the second differentiation-inducing cultivation, to obtain ripe DC cell; And
The DC cell of described maturation and the described lymphocyte through activation culture are carried out mixed culture, to obtain targeted immune cell mass.
6. method according to claim 5, is characterized in that, described LMP-1 antigen mRNA is obtained by following steps:
Preparation LMP-1 antigen cDNA plasmid, the nucleotide sequence of described LMP-1 antigen cDNA is as shown in SEQ ID NO:1;
Described LMP-1 antigen cDNA plasmid is carried out linearization process, to obtain the LMP-1 antigen cDNA plasmid through linearization process; And
The described LMP-1 antigen cDNA plasmid through linearization process is carried out in-vitro transcription, to obtain LMP-1 antigen mRNA,
Optionally, with pcDNA3.1 carrier for skeleton carrier, prepare described LMP-1 antigen cDNA plasmid,
Optionally, described pcDNA3.1 carrier has BamhI, XbaI and Bst1107I restriction enzyme site,
Optionally, carry out described linearization process based on restriction enzyme site Bst1107I,
Optionally, T7Message Machine test kit is utilized to carry out described in-vitro transcription,
Optionally, after carrying out described in-vitro transcription, comprise the step of edulcoration purification further.
7. method according to claim 5, is characterized in that, utilization electricity turns or PEI method carries out described transfection,
Optionally, utilize PEI method to carry out described transfection, wherein, PEI is 3:1 with the mixing quality ratio of described LMP-1 antigen mRNA,
Optionally, described activation culture comprises:
CTL1 cell culture medium is utilized to carry out the first activation culture 24 hours to described lymphocyte;
CTL2 cell culture medium is utilized to carry out the second activation culture 2-3 days to the lymphocyte through the first activation culture; And
CTL3 cell culture medium is utilized to carry out the 3rd activation culture 4-5 days to the lymphocyte through the second activation culture, every equivalent fluid infusion in 2-3 days once,
Optionally, described CTL1 cell culture medium comprises:
Serum-free cell culture medium;
The CTL1 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Described CTL2 cell culture medium comprises:
Serum-free cell culture medium;
The CTL2 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Described CTL3 cell culture medium comprises:
Serum-free cell culture medium;
The CTL3 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Wherein, described serum origin in described sample,
Optionally, described serum free medium is the serum free medium of Takara,
Optionally, the microbiotic of 0.5 volume % ~ 5 volume % is added with in described serum free medium, preferred gentamicin.
8. method according to claim 5, is characterized in that, utilizes DC substratum to carry out described first differentiation-inducing cultivation and described second differentiation-inducing cultivation,
Optionally, described DC substratum comprises:
Serum-free cell culture medium;
The CTL4 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %, wherein, described serum origin in described sample,
Optionally, described serum free medium is the serum free medium of Takara,
Optionally, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin,
Optionally, carry out described first differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once,
Optionally, carry out described second differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once,
Optionally, CTL3 cell culture medium is utilized to carry out described mixed culture 6-8 days, preferably 7 days,
Optionally, described CTL3 cell culture medium comprises:
Serum-free cell culture medium;
The CTL3 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Wherein, described serum origin in described sample,
Optionally, described serum free medium is the serum free medium of Takara,
Optionally, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin,
Optionally, be that 1:20 carries out described mixed culture according to the DC cell of described maturation and the described lymphocytic volume ratio through activation culture.
9. a targeted immune cell mass, it is prepared by the method described in any one of claim 5-8.
10. targeted immune cell mass according to claim 9, and the DC cell utilizing the method preparing DC cell described in any one of claim 1-4 to prepare is preparing the purposes in medicine, described medicine is used for the treatment of LMP-1 to be infected, or suppresses the transfer of LMP-1 positive tumor, diffusion, recurrence
Optionally, described LMP-1 infects for nerpes vinrus hominis infects.
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