CN105288603A - Tumor vaccine for treating liver cancer and preparing method thereof - Google Patents
Tumor vaccine for treating liver cancer and preparing method thereof Download PDFInfo
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Classifications
-
- 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
Abstract
The invention discloses a tumor vaccine for treating liver cancer and a preparing method thereof. The tumor vaccine with the improved treating effect comprises exosome globules excreted by liver cancer cells. The liver cancer cells are treated in the mode that Cephaloziellins J and SAHA are combined and medicine is added, and the molar concentration ratio of the Cephaloziellins J to the SAHA is 0.8-1.2:1. According to the tumor vaccine and the preparing method, it is creatively used that the liver cancer cells are treated through the Cephaloziellins J, the SAHA and the medicine, and the result shows that the inhibiting effect can be remarkably improved through exosomes excreted by the treated liver cancer cells and solubility immune molecules of the exosomes. The treating effect of the exosome tumor vaccine is remarkably improved, and important clinical application value is achieved.
Description
Technical field
The invention belongs to tumor vaccine field, be specifically related to the Hepatoma Vaccine that a kind of therapeutic effect improves, and the preparation method of this Hepatoma Vaccine.
Background technology
In China's primary hepatocarcinoma, more than 90% is hepatocarcinoma (HCC), and be secondly cholangiocellular carcinoma, men and women's sickness rate ratio is 2 ~ 5:1.Tumor cell has antigenicity, and body can be caused to produce immunne response, and this is the theoretical basis of immunotherapy of tumors.In recent years, due to the development of molecular immunology, cytobiology and biotechnology, make the clinical practice of tumor vaccine, monoclonal antibody, cytokine, immunologically competent cell infusion and gene transfer technique etc. become possibility, make the immunization therapy of hepatocarcinoma research have great development.Especially Hepatoma Vaccine receives much concern, and has become one of study hotspot of liver cancer immunity treatment.Use the Hepatoma Vaccine built based on hepatoma carcinoma cell, hepatocellular carcinoma antigen, dendritic cell and nucleic acid, by the immune system of excitating organism, induction body produces for the immunne response of liver cancer-specific antigen, reaches and kills the tumor cell of expressing hepatocellular carcinoma antigen and the object producing Graft Versus Tumor.Found by clinical trial, Hepatoma Vaccine can reduce hepatocarcinoma relapse and metastasis, improve life in patients and extend the time-to-live, become the pith of hepatocarcinoma Comprehensive Treatment.
Although a lot of to the correlational study of tumor vaccine both at home and abroad, but that up to the present makes a breakthrough is rare, the subject matter existed has: DNA being imported to specific cells, to carry out expressing this technology still immature, and use the safety issue of foreign DNA not yet to solve; Because tumor cell presents height heterogeneity, the multiple oncocytes belonging to same type tumor can express different antigen, the T cell activated by a kind of tumor antigen can only kill and wound the tumor cell of a part, and the oncocyte of not expressing this antigen then can not be killed and wounded; Although tumour-cell vaccine can comprise most tumor antigen, current research shows the limited use of its activated T cell, it can be used as the effect of vaccine unsatisfactory.Therefore, how to provide a kind of tumor vaccine, there is not use safety sex chromosome mosaicism and to most tumor antigen effectively, can become and have problem to be solved.
Exosomes be a class originate from endocytosis system and be discharged in extracellular, the duplicature vesicle of diameter between 40-100nm.Exosomes can be secreted by the various kinds of cell comprising dendritic cell, tumor cell etc., containing a large amount of and its source and the closely-related protein of function and lipid components, as the important carrier of intercellular trafficking information, participates in multiple pathophysiological process.The exosomes in tumor cell source contains the important immune molecule such as HLA-G, hot body gram albumen, and show antitumor action by number of ways, and it is as a kind of novel tumor vaccine, comparatively DC vaccine has obvious advantage.
But, but some related experiment are had to show in recent years, the exosomes in some tumors source can suppress or even destroy the immunocyte played a role in tumor, such as lower the expression of some NK receptors, have influence on the activation of some inherent immunity cells in tumour immunity, what also have significantly can suppress IL-2 thus the propagation of suppression Human Lymphocytes, thus in the immunization therapy of tumor, plays some negative effects.These exosomes originated by tumor may be exactly the key factor that tumor tissues escape body immune system is removed, and bring a lot of difficulty and challenge to the immunization therapy of tumor.Therefore, how to improve the immunostimulatory potency of tumor cell source exosomes, and the immunosuppression capability reducing it there is great practical significance in the immunization therapy of tumor.
Summary of the invention
One is the object of the present invention is to provide not have inhibiting Hepatoma Vaccine to the proliferative function of lymphocyte.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
The Hepatoma Vaccine that a kind of therapeutic effect improves, comprise the exosomes corpusculum of secretion of hepatoma, described hepatoma carcinoma cell is through CephaloziellinsJ and SAHA associating agent-feeding treatment, and the ratio of the molar concentration of CephaloziellinsJ and SAHA is 0.8 ~ 1.2:1.
Further, the ratio of the molar concentration of CephaloziellinsJ and SAHA is 1:1.
Further, the Hepatoma Vaccine that described therapeutic effect improves also comprises adjuvant.
Further, described adjuvant is for being aluminium adjuvant.
The preparation method of described Hepatoma Vaccine, comprises the steps: that the DMEI culture fluid of hepatoma carcinoma cell containing hyclone is at CO
2cultivate in incubator, cell is monolayer adherence growth, within every 3 ~ 4 days, goes down to posterity 1 time, inoculates until Growth of Cells to during logarithmic (log) phase; After inoculation 24h, with CephaloziellinsJ and SAHA agent-feeding treatment cell, after continuing to cultivate 24h, collect culture supernatant, cryopreservation; By the hepatoma carcinoma cell culture supernatant centrifugal segregation cell collected, get supernatant; Centrifugal segregation cell debris again, collects supernatant, ultrafiltration concentration, centrifugally obtains concentrated solution, move in centrifuge tube, ultracentrifugation under cryogenic conditions by the concentrated solution of separation and purification, and gained precipitation is namely containing exosomes corpusculum.
Further, described preparation method comprises the steps: the DMEI culture fluid of hepatoma carcinoma cell containing 100ml/L hyclone, at 37 DEG C of 50ml/LCO
2cultivate in incubator, cell be monolayer adherence growth, within every 3 ~ 4 days, go down to posterity 1 time, until Growth of Cells to during logarithmic (log) phase by 3 × 10
6/ 100ml inoculates; With 1 × 10 after inoculation 24h
-6the CephaloziellinsJ and 1 × 10 of mol/L
-6the SAHA process cell of mol/L, collects culture supernatant, 4 DEG C of preservations after continuing to cultivate 24h; The centrifugal 10min of hepatoma carcinoma cell culture supernatant 300g collected is removed cell, gets supernatant; Cell debris is removed again with the centrifugal 30min of 1500g, collect supernatant, by 100kUMWCOCentriplus centrifugal ultrafiltration pipe ultrafiltration concentration, concentrated solution is obtained with the centrifugal 30min of 1500g, the concentrated solution of separation and purification is moved in centrifuge tube, use level angle with 100kg ultracentrifugation 60min under 4 DEG C of conditions, gained precipitation is namely containing exosomes corpusculum.
Described Hepatoma Vaccine is applied in the medicine of the anti-hepatocarcinoma of preparation.
Advantage of the present invention: nanoscale vesicles exosomes and the soluble immune molecule thereof of known undressed secretion of hepatoma have significant inhibitory action to the proliferative function of lymphocyte.The present invention creatively uses CephaloziellinsJ and SAHA agent-feeding treatment hepatoma carcinoma cell, and result shows, the exosomes of the secretion of hepatoma after process and soluble immune molecule thereof then can significantly improve aforesaid inhibitory action.Invention significantly improves exosomes tumor vaccine therapy effect, there is important clinical value.
Detailed description of the invention
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, such as, condition described in textbook and experiment guide, or according to the condition that manufacturer advises.The chemical constitution of CephaloziellinsJ and preparation method are see document: SecondaryMetabolitesfromtheChineseLiverwortCephaloziella kiaeri, J.Nat.Prod., 2013,76,1700-1708.
Embodiment 1: cell culture
Experiment material: H22-H8D8 cell strain, 10% hyclone, mycillin mixed liquor (Beijing company of Tag U.S.).
Experimental technique: H22-H8D8 cell strain is incubated in the DMEI culture fluid containing 10% hyclone, penicillin 100IU/mL, streptomycin 100 × μ g/mL, 37 DEG C of 5%CO
2cultivate in incubator, in time growing to logarithmic (log) phase, by 3 × 10
6/ 100ml inoculates.
Embodiment 2: drug treating
Experiment material: CephaloziellinsJ makes by oneself, the same document of preparation method (SecondaryMetabolitesfromtheChineseLiverwortCephaloziella kiaeri, J.Nat.Prod., 2013,76,1700-1708), through being accredited as CephaloziellinsJ; SAHA is purchased from sigma company.
Experiment grouping: cell culture fluid volume often organizes strict conformance, blank group, exosomes matched group (not dosing group), exosomes experimental group 1 (dosing CephaloziellinsJ), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (CephaloziellinsJ and SAHA associating dosing).
Experimental technique: matched group, does not add medicine after inoculation, collects supernatant 150ml in contrast after 24h; Dosing group 1, by concentration 1 × 10 after inoculation 24h
-6the CephaloziellinsJ process of mol/L, collects culture supernatant 150ml after dosing 24h; Dosing group 2, by concentration 1 × 10 after inoculation 24h
-6the SAHA process of mol/L, collects culture supernatant 150ml after dosing 24h; Dosing group 3, with 1 × 10 after inoculation 24h
-6the CephaloziellinsJ and 1 × 10 of mol/L
-6the SAHA process of mol/L, collects culture supernatant 150ml after dosing 24h, and 4 DEG C of preservations.
Embodiment 3: drug treating
Experiment material: CephaloziellinsJ makes by oneself, the same document of preparation method (SecondaryMetabolitesfromtheChineseLiverwortCephaloziella kiaeri, J.Nat.Prod., 2013,76,1700-1708), through being accredited as CephaloziellinsJ; SAHA is purchased from sigma company.
Experiment grouping: cell culture fluid volume often organizes strict conformance, blank group, exosomes matched group (not dosing group), exosomes experimental group 1 (dosing CephaloziellinsJ), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (CephaloziellinsJ and SAHA associating dosing).
Experimental technique: matched group, does not add medicine after inoculation, collects supernatant 150ml in contrast after 24h; Dosing group 1, by concentration 1 × 10 after inoculation 24h
-6the CephaloziellinsJ process of mol/L, collects culture supernatant 150ml after dosing 24h; Dosing group 2, by concentration 1 × 10 after inoculation 24h
-6the SAHA process of mol/L, collects culture supernatant 150ml after dosing 24h; Dosing group 3, with 0.8 × 10 after inoculation 24h
-6the CephaloziellinsJ and 1 × 10 of mol/L
-6the SAHA process of mol/L, collects culture supernatant 150ml after dosing 24h, and 4 DEG C of preservations.
Embodiment 4: drug treating
Experiment material: CephaloziellinsJ makes by oneself, the same document of preparation method (SecondaryMetabolitesfromtheChineseLiverwortCephaloziella kiaeri, J.Nat.Prod., 2013,76,1700-1708), through being accredited as CephaloziellinsJ; SAHA is purchased from sigma company.
Experiment grouping: cell culture fluid volume often organizes strict conformance, blank group, exosomes matched group (not dosing group), exosomes experimental group 1 (dosing CephaloziellinsJ), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (CephaloziellinsJ and SAHA associating dosing).
Experimental technique: matched group, does not add medicine after inoculation, collects supernatant 150ml in contrast after 24h; Dosing group 1, by concentration 1 × 10 after inoculation 24h
-6the CephaloziellinsJ process of mol/L, collects culture supernatant 150ml after dosing 24h; Dosing group 2, by concentration 1 × 10 after inoculation 24h
- 6the SAHA process of mol/L, collects culture supernatant 150ml after dosing 24h; Dosing group 3, with 1.2 × 10 after inoculation 24h
-6the CephaloziellinsJ and 1 × 10 of mol/L
-6the SAHA process of mol/L, collects culture supernatant 150ml after dosing 24h, and 4 DEG C of preservations.
The isolation and purification of embodiment 5:exosomes
Experimental apparatus: HMAC-CP7OG low temperature Ultracentrifuge, 100KUMWC0MilliporeAmicon high-recovery high flow rate cross-flow ultrafiltration centrifuge tube (Millipore company).
Experimental technique: the experimental group collect embodiment 2 ~ 4 and cellular control unit culture supernatant remove cell with the centrifugal 10min of 300g, get supernatant; Cell debris is removed with the centrifugal 30min of 1500g, collect supernatant, by 100kUMWCOCentriplus centrifugal ultrafiltration pipe ultrafiltration concentration, 6ml concentrated solution is obtained with the centrifugal 30min of 1500g, the concentrated solution of separation and purification is moved in the centrifuge tube of 1.5ml, at 4 DEG C, namely contains exosomes corpusculum with level angle with 100kg ultracentrifugation 60min gained precipitation.
The Electronic Speculum qualification of embodiment 6:exosomes
Drip 20-30 μ 1exosomes suspension on load sample copper mesh, room temperature leaves standstill lmin filter paper and blots liquid from side, drip 20ml/L Salkowski's solution (pH6.8) about 30 μ l on copper mesh, room temperature negative staining lmin filter paper blots negative staining liquid, observes and take a picture under room temperature under drying about 10min transmission electron microscope.Result shows, and the exosomes corpusculum diameter of H22-H8D8 emiocytosis under Electronic Speculum is the film microcapsule structure of 30-80nm, rounded or oval, and intracavity is low electron density composition.
Embodiment 7:H3-TdR incorporation methods detects PBMC cell proliferative condition
Experiment material: H3-TdR (Shanghai nuclear research institute), mycillin mixed liquor (Beijing Tag is beautiful), hyclone
Experimental apparatus: β-liquid scintillation counter measurement (FJ-2107G type)
Experiment grouping: blank group (only adding phytohemagglutinin and PBMC cell), exosomes matched group (not dosing group), exosomes experimental group 1 (dosing CephaloziellinsJ), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (CephaloziellinsJ and SAHA associating dosing), supernatant matched group (not dosing group) after exosomes extracts, supernatant experimental group 1 (dosing CephaloziellinsJ) after exosomes extracts, supernatant experimental group 2 (dosing SAHA) after exosomes extracts, supernatant experimental group 3 (CephaloziellinsJ and SAHA associating dosing) after exosomes extracts, often organize three wells.
Experimental technique: the PBMC cell of separator well adds the fresh RPMI1640 culture medium of 5ml containing 10% hyclone, after cell counting, dilution is 5000/50 μ l, according to previous ready-made grouping, add in 96 orifice plates with 50 μ l/ holes, add the mixed liquor of penicillin 100IU/mL, streptomycin 100 × μ g/mL simultaneously with the ratio of 1:1000, then add 50 μ l samples, finally add 100 μ 1IPHA.Put into 37 DEG C of 50%CO
2incubator spends the night, second day can 10 × inverted microscope under to observe PBMC cell be bulk propagation, H3-TdR is added in culture plate, every hole 3.7 × 10
4after individual Bq continues to cultivate 6h, go culture medium, wash 3 times with 1 × PBS, the NaOH broken cell film of 1mol/L, adds scintillation solution and anti-quencher, with β-liquid scintillation counter measurement, and record cpm value (minute counting).Result is as shown in table 1.
The supernatant of table 1 after the exosomes before and after combination medicine process and extraction is on the impact of PBMC cell proliferation
| Group | Group number | Cpm value (X ± S) |
| Blank | 3 | 24096±2207.62 |
| Exosomes matched group | 3 | 5023±25.34 |
| Exosomes experimental group 1 | 3 | 13472±774.28 |
| Exosomes experimental group 2 | 3 | 13689±767.74 |
| Exosomes experimental group 3 | 3 | 23998±2041.31 |
| Supernatant matched group after exosomes extracts | 3 | 4966±415.45 |
| Supernatant experimental group 1 after exosomes extracts | 3 | 14926±5504.52 |
| Supernatant experimental group 2 after exosomes extracts | 3 | 14714±5721.52 |
| Supernatant experimental group 3 after exosomes extracts | 3 | 23839±5202.46 |
Result shows: compared with blank group, after exosomes and the PBMC co-culture of cells of drug treating, have appreciable impact to lymphopoiesis, obviously reduces (P<0.05), through drug treating, obviously can improve the suppression of exosomes to the proliferative function of lymphocyte, the exosomes (exosomes experimental group 3) wherein obtained after pharmaceutical composition process is minimum to the suppression of the proliferative function of lymphocyte, exosomes (exosomes experimental group 1 and exosomes experimental group 2) is obtained to lymphopoietic suppression after being significantly less than drug alone process, there is significant difference (P<0.05) in PBMC propagation value and exosomes matched group comparative statistics, compare without significant difference (P>0.05) with blank group.At experimental group, after the supernatant of drug treating after exosomes extracts and PBMC co-culture of cells, on cell proliferation is almost without impact, compare with blank group without remarkable significant difference (P>0.05), supernatant without drug treating then has a significant impact the proliferative function of lymphocyte, and statistically all there were significant differences compared with blank group or supernatant experimental group (P<0.05).Dosing group 3 in embodiment 3,4 and embodiment 2 can prepare the similar vaccine of therapeutic activity.
Embodiment 8: tumor inhibition
Experiment material: healthy kunming mouse, H22-H8D8 liver cancer mouse, murine hepatocarcinoma cell (H22-H8D8), DMEI culture medium (purchased from GIBCO), hyclone.
Experimental technique:
(1) set up mice with tumor model: H22-H8D8 liver cancer mouse ascites is got by sterile working, Trypan Blue, oncocyte counting under light microscopic, oncocyte 9000 of living, adjustment cell concentration is 1 × 10
7individual/ml, in every only healthy kunming mouse right axil subcutaneous vaccination 0.2ml, makes solid type mice with tumor model.
(2) tumor vaccine is prepared: carry out murine hepatocarcinoma cell (H22-H8D8) according to described embodiment 1 and cultivate; Cultured cells is divided into 4 groups, first group of not dosing, second group of dosing CephaloziellinsJ, the 3rd group of dosing SAHA, the 4th group of CephaloziellinsJ and SAHA associating dosing, concrete agent-feeding treatment is with reference to embodiment 2; The each group of cell culture supernatant collected is prepared exosomes tumor vaccine with reference to embodiment 5.
(3) grouping and treatment: healthy kunming mouse 30 is divided into 5 groups at random, often organizes each 6.Blank group: tumor cell inoculation same day, in mice lower limb root subcutaneous injection normal saline; Exosomes matched group: tumor cell inoculation same day, in tumor vaccine (10 μ g/ only) prepared by mice lower limb root subcutaneous injection first group; Exosomes experimental group 1: tumor cell inoculation same day, in tumor vaccine (10 μ g/ only) prepared by mice lower limb root subcutaneous injection second group; Exosomes experimental group 2: tumor cell inoculation same day, in mice lower limb root subcutaneous injection the 3rd group of tumor vaccine prepared (10 μ g/ only); Exosomes experimental group 3: tumor cell inoculation same day, in mice lower limb root subcutaneous injection the 4th group of tumor vaccine prepared (10 μ g/ only).Repeated 1 time at interval of 1 day, totally 3 times.Within after all mouse inoculation tumors the 15th day, put to death, get tumor body, weigh, survey gross tumor volume, calculate tumor-like hyperplasia, gross tumor volume suppression ratio.
(4) take tumor weight and calculate tumor control rate
After sacrifice, take out tumor tumor body respectively, peel off totally, wipe clean blood stains with filter paper, electronic balance takes tumor weight, calculates tumour inhibiting rate: tumour inhibiting rate (%)=(matched group tumor size-treatment group tumors size)/matched group tumor size × 100%.
Table 2 tumor vaccine is to the inhibitory action of transplanted human hepatocellular carcinoma
| Experiment grouping | Tumor is average |
| Blank group | 3.34±0.32 |
| Exosomes matched group | 2.72±0.18 |
| Exosomes experimental group 1 | 1.74±0.25 |
| Exosomes experimental group 2 | 1.65±0.27 |
| Exosomes experimental group 3 | 0.85±0.19 |
After adopting exosomes tumor vaccine therapy, Growth of Transplanted Hepatocarcinoma in Mice is suppressed, exosomes experimental group 1, exosomes experimental group 2, exosomes experimental group 3 tumor quality is respectively (1.74 ± 0.25) g, (1.65 ± 0.27) g, (0.85 ± 0.19) g, with blank group (3.34 ± 0.32) g, exosomes matched group (2.72 ± 0.18) g is than significant difference (P<0.05), tumor vaccine inhibitory action prepared by CephaloziellinsJ and SAHA associating dosing is the strongest, tumor quality is (0.85 ± 0.19) g, inhibitory rate 74.55%.Dosing group 3 in embodiment 3,4 and embodiment 2 can prepare the similar vaccine of therapeutic activity.
Embodiment 9: Hepatoma Vaccine adjuvant is selected
Adjuvant is made a general reference a class and can is combined with antigen by the mode of physics or chemistry and be strengthened the material of its specific immunity specifically.The most widely used adjuvant is aluminium adjuvant in the world now.Aluminium adjuvant vaccine mainly contains 2 kinds of preparation methoies: one is added in antigenic solution by aluminium adjuvant to form aluminate albumen precipitation; Another kind is added in previously prepared aluminium hydroxide, aluminum phosphate, aluminium hydroxide and aluminum phosphate mixture or aluminium oxide by antigenic solution to form adsorptivity vaccine.Aluminium phosphate adjuvant and aluminum hydroxide adjuvant are positive charge under at physiological ph, and electronegative protein is attracted in the grid structure of aluminum salt, for improving the therapeutic effect of Hepatoma Vaccine in the preparation of Hepatoma Vaccine of the present invention.
Liposome can also be selected as the adjuvant of Hepatoma Vaccine of the present invention, improve the therapeutic effect of Hepatoma Vaccine.
The present invention creatively uses CephaloziellinsJ and SAHA agent-feeding treatment hepatoma carcinoma cell, and result shows, the exosomes of the secretion of hepatoma after process and soluble immune molecule thereof then can significantly improve aforesaid inhibitory action.Invention significantly improves exosomes tumor vaccine therapy effect, there is important clinical value.
The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (7)
1. the Hepatoma Vaccine of a therapeutic effect improvement, it is characterized in that: the exosomes corpusculum comprising secretion of hepatoma, described hepatoma carcinoma cell is through CephaloziellinsJ and SAHA associating agent-feeding treatment, and the ratio of the molar concentration of CephaloziellinsJ and SAHA is 0.8 ~ 1.2:1.
2. the Hepatoma Vaccine of therapeutic effect improvement according to claim 1, is characterized in that: the ratio of the molar concentration of CephaloziellinsJ and SAHA is 1:1.
3. the Hepatoma Vaccine of therapeutic effect improvement according to claim 1 and 2, is characterized in that: also comprise adjuvant.
4. the Hepatoma Vaccine of therapeutic effect improvement according to claim 3, is characterized in that: described adjuvant is for being aluminium adjuvant.
5. the preparation method of the Hepatoma Vaccine described in claim 1 or 2, is characterized in that comprising the steps: that the DMEI culture fluid of hepatoma carcinoma cell containing hyclone is at CO
2cultivate in incubator, cell is monolayer adherence growth, within every 3 ~ 4 days, goes down to posterity 1 time, inoculates until Growth of Cells to during logarithmic (log) phase; After inoculation 24h, with CephaloziellinsJ and SAHA agent-feeding treatment cell, after continuing to cultivate 24h, collect culture supernatant, cryopreservation; By the hepatoma carcinoma cell culture supernatant centrifugal segregation cell collected, get supernatant; Centrifugal segregation cell debris again, collects supernatant, ultrafiltration concentration, centrifugally obtains concentrated solution, move in centrifuge tube, ultracentrifugation under cryogenic conditions by the concentrated solution of separation and purification, and gained precipitation is namely containing exosomes corpusculum.
6. preparation method according to claim 5, is characterized in that comprising the steps: the DMEI culture fluid of hepatoma carcinoma cell containing 100ml/L hyclone, at 37 DEG C of 50ml/LCO
2cultivate in incubator, cell be monolayer adherence growth, within every 3 ~ 4 days, go down to posterity 1 time, until Growth of Cells to during logarithmic (log) phase by 3 × 10
6/ 100ml inoculates; With 1 × 10 after inoculation 24h
-6the CephaloziellinsJ and 1 × 10 of mol/L
-6the SAHA process cell of mol/L, collects culture supernatant, 4 DEG C of preservations after continuing to cultivate 24h; The centrifugal 10min of hepatoma carcinoma cell culture supernatant 300g collected is removed cell, gets supernatant; Cell debris is removed again with the centrifugal 30min of 1500g, collect supernatant, by 100kUMWCOCentriplus centrifugal ultrafiltration pipe ultrafiltration concentration, concentrated solution is obtained with the centrifugal 30min of 1500g, the concentrated solution of separation and purification is moved in centrifuge tube, use level angle with 100kg ultracentrifugation 60min under 4 DEG C of conditions, gained precipitation is namely containing exosomes corpusculum.
7. the Hepatoma Vaccine described in claim 1 or 2 is applied in the medicine of the anti-hepatocarcinoma of preparation.
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| CN201680035580.5A CN107708727A (en) | 2015-10-28 | 2016-08-19 | It is a kind of to be used to treat tumor vaccine of liver cancer and preparation method thereof |
| PCT/CN2016/096036 WO2017071380A1 (en) | 2015-10-28 | 2016-08-19 | Tumor vaccine for use in treating liver cancer and preparation method for vaccine |
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| CN110604813A (en) * | 2018-06-14 | 2019-12-24 | 深圳市人民医院 | Application method of tumor cell-derived exosome antigen in DC vaccine |
| CN117547600B (en) * | 2023-11-15 | 2024-04-30 | 华中科技大学同济医学院附属协和医院 | Preparation and application of liposome vaccine targeting HDAC |
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| CN101948453A (en) * | 2006-05-23 | 2011-01-19 | 山东绿叶制药有限公司 | Novel NEO-clerodane typed diterpene compound and application thereof |
| CN103816535A (en) * | 2014-03-04 | 2014-05-28 | 肖文华 | Tumour vaccine and preparation method thereof |
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| CN105078966A (en) * | 2015-08-28 | 2015-11-25 | 林天样 | Application of cephaloziellin A in preparing medicines in preparing medicine for treating gastric cancer |
| CN105288603A (en) * | 2015-10-28 | 2016-02-03 | 杨廷稳 | Tumor vaccine for treating liver cancer and preparing method thereof |
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| CN101948453A (en) * | 2006-05-23 | 2011-01-19 | 山东绿叶制药有限公司 | Novel NEO-clerodane typed diterpene compound and application thereof |
| CN103816535A (en) * | 2014-03-04 | 2014-05-28 | 肖文华 | Tumour vaccine and preparation method thereof |
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| RUI-IUAN等人: "Secondary Metabolites from the Chinese Liverwort Cephaloziella kiaeri", 《JOURNAL OF NATURAL PRODUCTS》 * |
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