CN106267174A - The preparation method of M1 macrophage tumor vaccin - Google Patents

The preparation method of M1 macrophage tumor vaccin Download PDF

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Publication number
CN106267174A
CN106267174A CN201610634205.4A CN201610634205A CN106267174A CN 106267174 A CN106267174 A CN 106267174A CN 201610634205 A CN201610634205 A CN 201610634205A CN 106267174 A CN106267174 A CN 106267174A
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tumor
cell
macrophage
normal saline
vaccin
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袁小林
杨真
张青
袁儒非
崔益芬
孙秀艳
李纯
关庆琳
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Dalian University
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Dalian University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens

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Abstract

The present invention relates to the preparation method of M1 macrophage tumor vaccin, belong to immunotherapy of tumors technical field.The present invention comprises and prepares tumor cell lysis liquid, carries out the differentiation culture of macrophage, and carries out the M1 polarization induction of macrophage.The invention have the benefit that M1 type macrophage is the main cleaning cell of body pathogenic microorganism and slough, and there is antigen presentation, play a crucial role in the induction and process of immune regulation of specific immune response.Use necrotic tumor cells to stimulate M1 macrophage and the M1 macrophage tumor vaccin prepared, tumor cell will can be overcome to express drawback not enough, that antigenicity is weak because of surface antigen, there is good induction specificity antineoplastic immunity reagentia.

Description

The preparation method of M1 macrophage tumor vaccin
Technical field
The present invention relates to immunotherapy of tumors technical field, the preparation method of particularly M1 macrophage tumor vaccin.
Background technology
Specificity active immunotherapy treatment tumor has certain curative effect and for many institutes confirms how to prepare The tumor vaccine of high antigen presentation is the key improving this therapy curative effect.At present, the tumor vaccine being in basis and clinical investigation phase is main There are the autologous tumor cell tumor vaccine of modification, single protein ingredient tumor vaccine or heat shock protein peptide complex tumor vaccine and dendritic cell Tumor vaccine etc., such as patent 93112060.8 etc..The autologous tumor cell tumor vaccine modified is to use suitable chemical factors thin to tumor Born of the same parents carry out processing to promote allosteric or the coupling of cell cortex protein, or make tumor thin by the means such as virus transfection and transgenic The foreign body increase of born of the same parents, the tumor vaccine prepared to improve the immunogenicity of tumor cell, though tumor cell is after treatment necessarily The antigenicity of tumor cell can be increased in degree, but method of modifying the most fundamentally changes tumor cell surface MHC1 quasi-molecule Expressing and reduce, tomour specific or heterogenetic antigen can not express the drawback at cell surface, autologous modification tumor cell tumor vaccine Immunoreactive fall out effect is not strong, and clinical efficacy is the most very good.
Single protein ingredient tumor vaccine (tumor vaccine as prepared with MAGE1, MAGE2 of extracting from melanoma cell), These tumor vaccine can not overcome the heterogeneity of tumor, uses the treatment of such tumor vaccine that immune system only can be made to be somebody's turn to do expressing in solid tumor Antigen tumor cell produces and kills, and will not kill the tumor cell not expressing this antigen in same tumor, and single albumen becomes The clinical efficacy dividing tumor vaccine is the most undesirable.
Heat shock protein peptide complex tumor vaccine is to utilize isolated heat shock protein peptide from tumor cell to be combined system Standby tumor vaccine, the tumor vaccine of this protein ingredient can overcome the restriction of MHCI quasi-molecule to can be used in allogeneic, and energy Enough overcoming the heterogeneity of tumor, experimentation shows that this tumor vaccine has a preferable tumor-inhibiting action, but being prepared as of this tumor vaccine This is the highest, tumor vaccine cell quantity is few, is not easy to clinical practice application.
Dendritic cell tumor vaccine (DC Cell vaccine) is the tumor vaccine of hot research the most both at home and abroad, DC glucagonoma Seedling is to utilize dendritic cell and tumor thin or tumor cell content mixed culture and the Tumor vaccine prepared, these tumor Seedlings Though it is weak and cannot be used for allochthonous drawback that tumor vaccine can overcome tumor-cell antigen, but heat shock protein peptide complex and The separation and Extraction of DC precursor is more complicated, and costly, is unsuitable for clinical practice.This invention is to have for setting up one Imitate and be easy to the method for preparing tumour vaccinum of clinical practice.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, the present invention to provide a kind of M1 macrophage tumor vaccin Preparation method.The present invention solves it and technical problem is that and take techniques below scheme to realize: the system of M1 macrophage tumor vaccin Preparation Method, comprises the steps of inducing tumor cell necrosis and prepares lysate, carrying out the differentiation of Monocyte-macrophages Cultivating, the M1 polarization induction and the tumor cell lysis liquid that carry out macrophage stimulate M1 macrophage.
Preferably, described tumor cell lysis liquid step of preparing comprises the steps of grinding tumor tissues and carries out once It is filtrated to get filtrate；Described filtrate is joined in the test tube equipped with Ficoll-Hypaque lymphocyte separation medium, 2000rpm Centrifugal 20min, draws mononuclearcell layer and obtains tumor cell, be moved in another test tube by described tumor cell；Physiology salt Tumor cell described in aqueous suspension, 1000rpm is centrifuged 10min, abandons supernatant, in triplicate；With normal saline by described tumor cell Furnishing 4 × 107C/mL, 37 DEG C, 5%CO2Under the conditions of place 48 hours prepared cell suspension；Described cell suspension is placed in-80 DEG C Refrigerator 25min, takes out and puts into rapidly 10min in 37 DEG C of water-baths, 3 times repeatedly；Described cell suspension 1000rpm is centrifuged 5min, Take supernatant, be dispensed in centrifuge tube ,-20 DEG C of preservations.
Preferably, the differentiation culture step of described macrophage comprises the steps of and heparin anti-coagulating is injected centrifuge tube In, at 4 DEG C, 1000rpm is centrifuged 5min, abandons upper plasma and obtains hemocyte；Hank ' s liquid or physiology is added in described hemocyte Blood cell suspension is made in saline mixing, and described blood cell suspension is slowly added into the centrifuge tube equipped with Ficoll-Hypaque In, at 4 DEG C, 2000rpm is centrifuged 20min；Take PBMC and move it in centrifuge tube, adding Hank ' s liquid or normal saline, 4 At DEG C, 1000rpm is centrifuged 5min, abandons supernatant, washes cell 3 times with Hank ' s liquid or normal saline；Add anteserum-less substrate suspension institute State cell, cell suspension is added in culture bottle, 37 DEG C, 5%CO2Under the conditions of cultivate 2 hours；Jiggling culture bottle, suction is abandoned Non-adherent cell.
Preferably, the M1 polarization induction step of described macrophage comprises the steps of the serum-free added containing GM-CSF Pei Ji, 37 DEG C, 5%CO2Under the conditions of cultivate 9 days；Add and contain IFN-γ and the anteserum-less substrate of tumor cell lysis liquid, 37 DEG C, 5%CO2Under the conditions of cultivate 3 days；Jiggle culture bottle, inhale and abandon Pei Ji；Use normal saline flushing attached cell, scrape patch Parietal cell, goes to, in centrifuge tube, add saline, and at 4 DEG C, 1000rpm is centrifuged 5min, supernatant discarded；Normal saline is washed cell and is laid equal stress on Outstanding cell, adds CpG, filters, is loaded by cell suspension in cryopreservation tube, seals adhesive label.
Advantages of the present invention and good effect be: M1 type macrophage is the main of body pathogenic microorganism and slough Cleaning cell, and there is antigen presentation, play a crucial role in the induction and process of immune regulation of specific immune response. Use necrotic tumor cells lysate to stimulate M1 macrophage and the M1 macrophage tumor vaccin prepared, tumor can be overcome Cell expresses drawback not enough, that antigenicity is weak because of surface antigen, has good induction specificity antineoplastic immunity reagentia.
Accompanying drawing explanation
Fig. 1 is M1 type macrophage degree CD68, the expression rate of CD14 molecule；
Fig. 2 is M1 type macrophage degree CD68, the expression rate of MHC II molecule；
Fig. 3 is M1 type macrophage degree CD80, the expression rate of CD86 molecule；
Fig. 4 is the handling process of experimental mice；
Fig. 5 is the tumor formation rate of each group of mice；
Fig. 6 is the gross tumor volume of each group of mice；
Fig. 7 is the tumor weight of each group of mice；
Fig. 8 is the tumor splenocyte tumor killing rate of each group of mice.
Detailed description of the invention
Below in conjunction with the accompanying drawings, by specific embodiment, the invention will be further described.Following example are descriptive , it not determinate, it is impossible to limit protection scope of the present invention with this.
Experiment equipment
Low temperature normal speed centrifuge (U.S., Beckman), CO2Cell culture incubator (Japan, Sanyo), pipettor (U.S., Thermo), measuring pipette (U.S., Kimble), 25cm2Culture bottle (U.S., Corning), 15ml centrifuge tube (U.S., Corning), 50ml centrifuge tube (U.S., Corning), cell scraper (U.S., Corning), piston (China, Shandong prestige High medical high polymer limited company), culture dish (U.S., Corning), cellular filter (U.S., Corning), be inverted aobvious Micro mirror (Japan, Nikon), cryopreservation tube (U.S., Corning).
Experiment reagent
Ficoll-Hypaque cell separation liquid (Hao ocean, Tianjin biological product science and technology limited Company)), sodium chloride note Penetrate liquid (Shijiazhuang four medicine company), NaCl (examination of traditional Chinese medicines Lu), KCl (examination of traditional Chinese medicines Lu), MgSO4·7H2(it is limited that Tianjin causes remote chemistry to O Company), MgCl2·6H2O (Tianjin Zhi Yuan Chemical Co., Ltd.), CaCl2(Tianjin Bo Di Chemical Co., Ltd.), Na2HPO4· 12H2O (Tianjin recovery development in science and technology company limited), KH2PO4(Tianjin Zhi Yuan Chemical Co., Ltd.), glucose (traditional Chinese medicines Lu Examination), phenol red (upper the graceful bio tech ltd of Hypon), heparin sodium (Shanghai No.1 Bio-Chemical Pharmacetical Industry Co., Ltd), IFN-γ (north Fourth Ring, capital Biology Pharmacy Co., Ltd), GM-CSF (Beijing Shuanglu Pharmaceutical Co., Ltd.), anteserum-less substrate (Thermo), CpG(Peprotech)。
The preparation of tumor cell lysis liquid: fresh tumor tissue (in vitro 0.5 hour) is cut into 1mm3Square, after washing, Using piston tumor mass to be ground in culture dish, 200 mesh filter screens filter, and filtrate joins the Ficoll-equipped with dilution In Hypaque test tube [density of Ficoll-Hypaque is 1.077 ± 0.02): normal saline=3:1], 2000rpm is centrifuged 20min, draws mononuclearcell layer, is moved into by tumor cell in another test tube.Normal saline suspension cell, 1000rpm from Heart 10min, abandons supernatant, in triplicate.With normal saline by cell furnishing 4 × 107C/mL, 37 DEG C, 5%CO2Under the conditions of place 48 Hour, cell suspension is placed in-80 DEG C of refrigerator 25min, takes out and put into rapidly 10min in 37 DEG C of water-baths, 3 times repeatedly.1000rpm Centrifugal 5min, takes supernatant, is dispensed in centrifuge tube ,-20 DEG C of preservations.
Configuration Hank ' s liquid: 1. solution A: NaCl 160g, KCl8g, MgSO4·7H2O 2g、MgCl2·6H2O 2g adds To 800ml tri-distilled water, dissolve.CaCl22.8g is dissolved in 100ml tri-distilled water, joins in above-mentioned 800ml solution, supply after dissolving Tri-distilled water is to 1000ml.2. second liquid: Na2HPO4·12H2O 3.04g、KH2PO41.2g, glucose 20g add 800ml distilled water, Dissolve.Add 0.4% phenol red solution 100ml, above-mentioned two liquid mixing, add tri-distilled water to 1000ml.By solution A 1 part, second during use Liquid 1 part, tri-distilled water 18 parts filtration, 8 pounds of sterilizings in 20min minute, 4 DEG C of preservations.
The differentiation culture of macrophage is with M1 polarization induction: injected by 20ml heparin anti-coagulating in 50ml centrifuge tube, at 4 DEG C Lower 1000rpm is centrifuged 5min.Upper plasma is abandoned in shifting, adds Hank ' s liquid or normal saline to 20ml, mixing.Blood cell suspension is delayed Slowly joining in the centrifuge tube equipped with 4ml Ficoll-Hypaque (density: 1.077 ± 0.001), often pipe 10ml, at 4 DEG C 2000rpm is centrifuged 20min, draws precipitation (PBMC layer) cell and moves it in 15ml centrifuge tube.Add Hank ' s liquid or physiology Saline is to 15ml, and at 4 DEG C, 1000rpm is centrifuged 5min, abandons supernatant.Hank ' s liquid or normal saline wash cell 3 times.Add 16ml Anteserum-less substrate suspension PBMC cell, joins 25cm by cell suspension2In culture bottle, every bottle of 8ml cell suspension, 37 DEG C, 5% CO2Under the conditions of cultivate 2 hours.Jiggle culture bottle, inhale and abandon non-adherent cell.Add the serum-free containing 100ng/mlGM-CSF Pei Ji, every bottle of 12ml, 37 DEG C, 5%CO2Under the conditions of cultivate 9 days.Add containing 100IU/ml IFN-γ and 100 μ l/ml tumors thin The anteserum-less substrate of cellular lysate liquid (preparation method such as method 1), every bottle of 4ml, 37 DEG C, 5%CO2Under the conditions of cultivate 3 days.Draw 2ml supernatant is used for mycoplasma inspection (RT-PCR method), then jiggles culture bottle, inhales and abandons Pei Ji.Use normal saline flushing Attached cell 3 times, scrapes attached cell with cell scraper, goes in centrifuge tube, adds normal saline to 50mL.At 4 DEG C 1000rpm is centrifuged 5min, supernatant discarded.Normal saline washes cell 3 times.1ml normal saline re-suspended cell, adds 4mg/ml's 0.1mlCpG, 200 mesh filter screens filter, are encased in by cell suspension in 2mL cryopreservation tube, and sealing, adhesive label, for notch graft Kind.
Measure of merit: in Fig. 5 to Fig. 8, A is physiological saline group；B is macrophage inoculation group；C is macrophage Tumor vaccination group.A, B and C group mice respectively at left hind subcutaneous vaccination normal saline, without tumor stimulate huge bite thin Born of the same parents and macrophage tumor vaccin (downright bad H22Tumor cell stimulates M1 macrophage to obtain), 0.1ml/ only (inoculate with C group by B group Macrophage concentration be 2 × 106/ ml, normal saline dilution), 1 times a week, continuous 2 times, after being spaced 2 weeks, four groups of mices are respectively In the well-grown H of left fore subcutaneous vaccination22Tumor cell, only (concentration of cell suspension is 2 × 10 to 0.1ml/6/ ml, physiology salt Water dilutes), tumor cell inoculation puts to death whole mice after 4 weeks, and calculates four groups of mice tumor formation rates, electronic balance weighing tumor respectively Weight, the length (L) of vernier caliper measurement tumor nodule and width (W), calculate tumor volume.Note (: volume=0.5 × L × W2)。 After result display macrophage tumor vaccin inoculation group tumor cell attack, tumor tumor formation rate, tumor volume and tumor weight are significantly lower than Non-stimulating expression of macrophage inoculation group and normal saline inoculation group (P < 0.05).The splenocyte pair of macrophage tumor vaccin inoculation group The killing rate of tumor cell, apparently higher than non-stimulating expression of macrophage inoculation group and normal saline inoculation group (P < 0.05), shows huge biting Cell tumour vaccine can induce specificity antineoplastic immunity reaction in vivo.

Claims (4)

  1. The preparation method of 1.M1 macrophage tumor vaccin, it is characterised in that comprise the steps of inducing tumor cell downright bad and Preparing lysate, carry out the differentiation culture of Monocyte-macrophages, the M1 polarization induction and the tumor that carry out macrophage are thin Cellular lysate liquid stimulates M1 macrophage.
  2. The preparation method of M1 macrophage tumor vaccin the most according to claim 1, it is characterised in that described induced tumor Necrocytosis and to prepare lysate step specific as follows:
    Grind tumor tissues and be once filtrated to get filtrate；
    Described filtrate being joined in the test tube equipped with Ficoll-Hypaque lymphocyte separation medium, 2000rpm is centrifuged 20min, draws mononuclearcell layer and obtains tumor cell, be moved in another test tube by described tumor cell；
    With the normal saline described tumor cell of suspension, 1000rpm is centrifuged 10min, abandons supernatant, in triplicate；
    With normal saline by described tumor cell furnishing 4 × 107C/mL, 37 DEG C, 5%CO2Under the conditions of place 48 hours prepare necrosis Cell suspension；
    Described cell suspension is placed in-80 DEG C of refrigerator 25min, takes out and put into rapidly 10min in 37 DEG C of water-baths, 3 times repeatedly；
    Described cell suspension 1000rpm is centrifuged 5min, takes supernatant, be dispensed in centrifuge tube ,-20 DEG C of preservations.
  3. The preparation method of M1 macrophage tumor vaccin the most according to claim 1, it is characterised in that described macrophage Differentiation culture step comprise the steps of
    Being injected by heparin anti-coagulating in centrifuge tube, at 4 DEG C, 1000rpm is centrifuged 5min, removes upper plasma and obtains hemocyte；
    In described hemocyte, add Hank ' s liquid or normal saline and blood cell suspension is made in mixing, described blood cell suspension is delayed Slowly joining equipped with in the centrifuge tube of Ficoll-Hypaque, at 4 DEG C, 2000rpm is centrifuged 20min；
    Taking PBMC and move it in centrifuge tube, adding Hank ' s liquid or normal saline, at 4 DEG C, 1000rpm is centrifuged 5min, abandons Clearly, cell is washed 3 times with Hank ' s liquid or normal saline；
    Add the anteserum-less substrate described cell of suspension, cell suspension is added in culture bottle, 37 DEG C, 5%CO2Under the conditions of cultivate 2 little Time；Jiggle culture bottle, inhale and abandon non-adherent cell.
  4. The preparation method of M1 macrophage tumor vaccin the most according to claim 1, it is characterised in that described macrophage M1 polarization induction step comprise the steps of
    Inhale to abandon and the culture bottle of non-adherent cell adds the anteserum-less substrate containing GM-CSF, 37 DEG C, 5%CO2Under the conditions of cultivate 9 days；
    Add containing IFN-γ and the anteserum-less substrate of tumor cell lysis liquid, 37 DEG C, 5%CO2Under the conditions of cultivate 3 days；
    Jiggle culture bottle, inhale and abandon Pei Ji；
    Use normal saline flushing attached cell, scrape attached cell, go to, in centrifuge tube, add saline, at 4 DEG C 1000rpm from Heart 5min, supernatant discarded；
    Normal saline washes cell re-suspended cell, adds CpG, filters, is loaded by cell suspension in cryopreservation tube, seals label adhering Sign.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109609452A (en) * 2018-12-27 2019-04-12 青岛麦迪赛斯生物科技有限公司 A kind of efficient macrophages in vitro preparation method
WO2020061084A1 (en) * 2018-09-17 2020-03-26 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center Target-primed macrophages and therapeutic uses thereof
CN114134122A (en) * 2021-12-06 2022-03-04 大连大学 Macrophage-mediated mesenchymal stem cell directed differentiation culture method

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CN1413731A (en) * 2002-09-17 2003-04-30 袁小林 Macrophage tumor vaccin and its preparation method
CN1609199A (en) * 2004-11-17 2005-04-27 哈尔滨医科大学 Macrophage in vitro inducing culture method
CN103958546A (en) * 2011-10-21 2014-07-30 特朗斯吉有限公司 Modulation of macrophage activation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020061084A1 (en) * 2018-09-17 2020-03-26 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center Target-primed macrophages and therapeutic uses thereof
CN109609452A (en) * 2018-12-27 2019-04-12 青岛麦迪赛斯生物科技有限公司 A kind of efficient macrophages in vitro preparation method
CN114134122A (en) * 2021-12-06 2022-03-04 大连大学 Macrophage-mediated mesenchymal stem cell directed differentiation culture method
CN114134122B (en) * 2021-12-06 2024-05-28 大连大学 Macrophage-mediated mesenchymal stem cell directional differentiation culture method

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Application publication date: 20170104