CN106860478A - A kind of universal Tumor vaccine combination and spray and preparation method thereof - Google Patents
A kind of universal Tumor vaccine combination and spray and preparation method thereof Download PDFInfo
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- CN106860478A CN106860478A CN201710055013.2A CN201710055013A CN106860478A CN 106860478 A CN106860478 A CN 106860478A CN 201710055013 A CN201710055013 A CN 201710055013A CN 106860478 A CN106860478 A CN 106860478A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/12—Aerosols; Foams
Abstract
The present invention relates to a kind of method for preparing universal Tumor vaccine combination, it is comprised the following steps：Screened with anti-tumor capacity person from health population according to anti-tumor capacity index；From described peripheral blood is gathered with anti-tumor capacity person；Isolated monocyte and blood plasma from the peripheral blood；The protein in the blood plasma is extracted, protein crude extract administration I is obtained；The monocyte is trained one or more immunocyte culture；Protein crude extract administration II is extracted from the immunocyte culture；The protein crude extract administration I and protein crude extract administration II is mixed, is freezed, the i.e. described universal Tumor vaccine of the powder for obtaining.Present invention also offers the universal Tumor vaccine combination and atomization spray that are prepared by methods described.Various antitumor cell factors are included in universal Tumor vaccine combination of the invention and atomization spray, can be very good to play a part of prevention and health care to tumor susceptibility crowd.
Description
Technical field
The present invention relates to anti-tumor vaccine field, more specifically it relates to a kind of universal Tumor vaccine combination and spray and
Its preparation method.
Background technology
Cancer seriously jeopardizes the health and lives of the mankind, annual complete according to the data display that international cancer research institution announces
The people of ball about 8,000,000 dies from cancer.Analysis detecting data can find that cancer has provincialism and familial phenomenon.Such as
The 32 tumour registration offices in the 2003-2007 whole nations are mainly distributed on coastal area, including 7 cancer high incidence areas, i.e. river
Su Qidong, Jiashan of Zhejiang, Changle of Fujian Province, Henan Linzhou City, Sichuan Yanting, Fusui, Linchu, shantung etc..Cancer family refers to one
The incidence of disease of malignant tumour is (about 20%) high in family, and age of onset is more early, generally presses autosomal dominant pattern heredity, and
Some tumour (such as gland cancer) incidences of disease are very high.Be summarized as These characteristics " cancer family syndrome " by Lynch.Once reported one
Individual cancer family, through five investigation between more than 70 years (1895 start), some offsprings reached for the 7th generation, in 842 descendants
95 cancer patients are found altogether, wherein suffering from the people of adenocarcinoma of colon 48 and the people person of adenocarcinoma of endometrium 18 occupies the majority.There are 13 people in this 95 people
Tumour for multiple, 19 people's carcinogenesis in 40 years old before；72 people have one of parents to suffer from cancer, male and female each 47 in 95 patients
With 48 people, close to 1:1, meet autosomal dominant inheritance.Familial carcinoma refers to that multiple members suffer from same type in a family
Tumour, for example, 12%~25% colorectal cancer patients have intestinal cancer family history.Many kinds of tumor such as breast cancer, intestinal cancer, stomach cancer
Deng, typically distribute, but a part of patient has obvious family history.Additionally, the incidence of disease is generally high in the first degree relative of patient
In 3~4 times of population.Although the mode of inheritance of this kind of cancer also not it is clear that but show some tumour Familial aggregation phenomenons, or
Family member increases to the neurological susceptibility of these tumours.Such as nasopharyngeal carcinoma has obvious Familial aggregation phenomenon, many Nasopharyngeal Carcinoma Patients
There is family to suffer from carninomatosis history.Nasopharyngeal carcinoma has family's occurence tendency vertically and horizontally.
It is not agnate due to gene and epigenetics difference, and habits and customs difference, some tumour meetings
It is occurred frequently in some races.This is difference of the cancer in not agnate incidence.
Equally, relative to tumour, some areas, part family also show powerful anti-tumor capacity, Phenomenon of longevity
Same to have regional and familial, Changshou village is also much in coastal area, and indivedual family in-group anti-tumor capacities are strong,
Average life span existing picture more long there is also.
Generation one side and environmental pollution, the habits and customs close relation of tumour, the tumour of another aspect China are prevented
Control work and also there is a problem of controlling again light anti-always, the education of disease prevention and health protection of tumour fails development extensively, or exist only in and cry out
In the slogan stage, there is no practical action.The public is inadequate for the prevention and health care understanding of tumour, or does not know how to carry out the pre- of tumour
Anti- health care.As can be seen here, the refinement to the prevention and health care measure of tumour will turn into the emphasis for the treatment of and prevention of tumour work.
Accordingly, it would be desirable to a kind of new universal Tumor vaccine.
The content of the invention
Be solve problem above, the invention provides a kind of method for preparing universal Tumor vaccine combination, it include with
Lower step：
S1：Screened with anti-tumor capacity person from health population according to anti-tumor capacity index；
S2：From described peripheral blood is gathered with anti-tumor capacity person；
S3：Isolated monocyte and blood plasma from the peripheral blood；
S4：The protein in the blood plasma is extracted, protein crude extract administration I is obtained；
S5：The monocyte is trained one or more immunocyte culture；
S6：Protein crude extract administration II is extracted from the immunocyte culture；
S7：The protein crude extract administration I and protein crude extract administration II is mixed, is freezed, the i.e. described universal Tumor of the powder for obtaining
Vaccine combination.
Further, the index with anti-tumor capacity person is screened in S1 to be included：Height, body weight, blood pressure, pulse frequency,
Cardio-pulmonary function, blood routine, routine urinalysis, blood sugar, hepatic and renal function, rabat, electrocardiogram, color ultrasound, past medical history, family history, tumor susceptibility
Genetic test and immunologic function are detected.
Further, the protein in the blood plasma is extracted in S4 by saturated ammonium sulphate method.
Further, in S5, the immunocyte is one or more combination in DC, CIK and NK cell.
Further, S6 is comprised the following steps：
S6.1：From the immunocyte culture isolating immune cells, cell free broth is left stand-by；
S6.2：Crush the immunocyte, collected after centrifugation supernatant, by the supernatant and the cell free broth
Mixing, obtains cell crude extract；
S6.3：Protein is extracted from the cell crude extract by saturated ammonium sulphate method, protein crude extract administration is obtained
II。
Further, it is anti-to addition in the mixture of the protein crude extract administration I and protein crude extract administration II before freezing in S7
Then bad hematic acid is freezed again as protein protective agent.
The invention also discloses a kind of universal Tumor vaccine combination, it passes through the above method and prepares.
The invention also discloses a kind of universal Tumor vaccine atomization spray, it is by above-mentioned universal Tumor vaccine combination
Be dissolved in pharmaceutically acceptable solvent after killing bacterium and virus treated, obtain the universal Tumor vaccine atomization spray
Agent.
Further, the pharmaceutically acceptable solvent is sterilized water.
Further, by using cobalt60Irradiate to kill bacterium and the virus in the universal Tumor vaccine combination.
The crowd of the great anti-tumor capacity of the health immunocompetence of itself is strong, contains with anti-tumor capacity in blood
The factor, antineoplastic cell factor is in expression status high, extracts its blood plasma always equally containing the cell factor of expression high, in addition,
The immunocyte immunologic function extracted from their blood is strong, immunocyte secrete in incubation in vitro various kinds of cell because
Son.Therefore, various antitumor cell factors, such as IL-1 are included in universal Tumor vaccine combination of the invention and atomization spray
Family (IL-1 α, IL-1 β, IL-1Ra etc.), IL-2, IL-12 family (IL-12, IL-23, IL-27 and IL-35 etc.), IL-17 family
The active ingredients such as race (IL-17A, IL-17F etc.), IL-18, TNF-α, GM-CSF, IFN-γ.Various cell factors are antitumor
During play an important role.Anti-tumor factor in blood plasma and Process of in vitro secretion with antitumor action
The factor can be very good to play a part of prevention and health care to tumor susceptibility crowd collectively as the reagent of atomization spray.
Specific embodiment
Principle of the invention and feature are described below in conjunction with example, example is served only for explaining the present invention, and
It is non-for limiting the scope of the present invention.
1. the screening with anti-tumor capacity person
Filtered out from healthy population with anti-tumor capacity person, as shown in table 1, (score selection one-level screening scheme ＞
85 points) people as peripheral blood donor.
Table 1 has the screening of anti-tumor capacity person
2. the preparation of universal Tumor vaccine combination
It is prepared by the following method universal Tumor vaccine combination
1) volunteer is selected from the colony of great anti-tumor capacity, the periphery containing PBMC is gathered with haemocyte list milling machine
Blood 100ml；
2) the above-mentioned peripheral blood 2000r/min containing PBMC is centrifuged 10-20min, separates mononuclearcell and blood plasma；
3) blood plasma, plus normal saline dilution are taken, saturated ammonium sulfate solution to ammonium sulfate end is slowly added dropwise while stirring
Concentration 50%, 4 DEG C stand 6h or overnight, and precipitation is dissolved in physiological saline, surpassed by 4000r/min centrifugation 15-30min, precipitation and separation
Salt concentration is filtered, protein crude extract administration I is obtained final product；
4) mononuclearcell is taken, DC, CIK cell is conventionally cultivated respectively；
5) DC, CIK cell are collected, remaining cell free broth is stand-by；By ultrasonication or the method for multigelation
Above-mentioned cell is cracked, then 1500 × g centrifugations 10min, collects supernatant, and the supernatant is mixed with the cell free broth,
Obtain cell crude extract；The cell crude extract is carried out into ammonium sulfate precipitation, desalination of then dialysing obtains final product protein crude extract administration II；
6) ascorbic acid of 0.03mg/ml is added after mixing protein crude extract administration 1 and protein crude extract administration 2 as protein protection
Agent, is then made freeze-dried powder, that is, obtain the universal Tumor vaccine combination.
In above method, DC cells are cultivated by following steps, and prepare tumour Universal load material：
1) inducible factor is added to induce PMBC to be divided into DC cells in PBMC cultures, cell culture must be at thousand grades
Ultra-clean workplace is carried out, and the operation for being possible to open should be carried out in hundred grades of ultra-clean workbench.It is right during culture
Cell product carries out strict quality control, it is ensured that pollution-free.
2) quality control：Cellular morphology is observed and immunophenotype detection, it is ensured that induce function and phenotype more
Ripe immunocyte；And make sterility test, tested without thermal source and mycoplasma, it is ensured that the security of cell；
3) using tumour Universal load material impact DC, make DC ripe, carry antitumor signal.
3. atomization spray is prepared
1) in 10 DEG C with cobalt60Irradiation universal Tumor vaccine combination dry powder, carries out viral inactivation treatment, by inactivation after it is dry
Powder packing is preserved, cobalt60Irradiation dose is 15-30kGy；
2) freeze-dried powder after packing is dissolved with distilled water, is made spray solution, that is, obtain the atomization spray, sprayed with atomization
Agent device carries out atomization absorption.
Resulting tumor vaccine composition and the atomization various antitumor cell factors of spray, such as IL-1 families (IL-1
α, IL-1 β, IL-1Ra etc.), IL-2, IL-12 family (IL-12, IL-23, IL-27 and IL-35 etc.), IL-17 families (IL-
17A, IL-17F etc.), IL-18, TNF-α, GM-CSF, the active ingredient such as IFN-γ.Various cell factors are in antineoplastic process
In play an important role.The factor with antitumor action of anti-tumor factor and the Process of in vitro secretion in blood plasma is total to
With the reagent as atomization spray, can be very good to play a part of prevention and health care to tumor susceptibility crowd.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all it is of the invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (10)
1. a kind of method for preparing universal Tumor vaccine combination, it is characterised in that comprise the following steps：
S1：Screened with anti-tumor capacity person from health population according to anti-tumor capacity index；
S2：From described peripheral blood is gathered with anti-tumor capacity person；
S3：Isolated monocyte and blood plasma from the peripheral blood；
S4：The protein in the blood plasma is extracted, protein crude extract administration I is obtained；
S5：The monocyte is trained one or more immunocyte culture；
S6：Protein crude extract administration II is extracted from the immunocyte culture；
S7：The protein crude extract administration I and protein crude extract administration II is mixed, is freezed, the i.e. described universal Tumor vaccine of the powder for obtaining
Composition.
2. method according to claim 1, it is characterised in that the index bag with anti-tumor capacity person is screened in S1
Include：Height, body weight, blood pressure, pulse frequency, cardio-pulmonary function, blood routine, routine urinalysis, blood sugar, hepatic and renal function, rabat, electrocardiogram, color ultrasound,
Past medical history, family history, tumor susceptibility gene detection and immunologic function detection.
3. method according to claim 1, it is characterised in that extract the blood in S4 by saturated ammonium sulphate method
Protein in slurry.
4. method according to claim 1, it is characterised in that in S5, during the immunocyte is DC, CIK and NK cell
One or more combination.
5. method according to claim 1, it is characterised in that S6 is comprised the following steps：
S6.1：From the immunocyte culture isolating immune cells, cell free broth is left stand-by；
S6.2：The immunocyte is crushed, collected after centrifugation supernatant mixes the supernatant with the cell free broth
Close, obtain cell crude extract；
S6.3：Protein is extracted from the cell crude extract by saturated ammonium sulphate method, protein crude extract administration II is obtained.
6. the method according to any one of claim 1-5, it is characterised in that in S7, it is thick to the albumen before freezing
Ascorbic acid is added in the mixture of extract I and protein crude extract administration II as protein protective agent, is then freezed again.
7. a kind of universal Tumor vaccine combination, it is characterised in that prepared by the method any one of claim 1-6
Obtain.
8. a kind of universal Tumor vaccine is atomized spray, it is characterised in that by the universal Tumor vaccine group described in claim 7
Compound be dissolved in pharmaceutically acceptable solvent after killing bacterium and virus treated, obtains the universal Tumor vaccine atomization
Spray.
9. universal Tumor vaccine according to claim 8 is atomized spray, it is characterised in that the pharmaceutically acceptable solvent
It is sterilized water.
10. universal Tumor vaccine according to claim 8 or claim 9 is atomized spray, it is characterised in that by using cobalt60Irradiation
To kill bacterium and the virus in the universal Tumor vaccine combination.
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| CN201710055013.2A CN106860478A (en) | 2017-01-24 | 2017-01-24 | A kind of universal Tumor vaccine combination and spray and preparation method thereof |
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| CN201710055013.2A CN106860478A (en) | 2017-01-24 | 2017-01-24 | A kind of universal Tumor vaccine combination and spray and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228473A (en) * | 2020-02-14 | 2020-06-05 | 杭州憶盛医疗科技有限公司 | Anti-tumor novel composite vaccine for treating fibrosarcoma |
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| CN102641298A (en) * | 2012-05-15 | 2012-08-22 | 祁岩超 | Effector cell combination for preventing and treating tumors and preparation method thereof |
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| CN1994463A (en) * | 2005-12-28 | 2007-07-11 | 韩苏 | Preparation and use of immune formulation |
| CN102641298A (en) * | 2012-05-15 | 2012-08-22 | 祁岩超 | Effector cell combination for preventing and treating tumors and preparation method thereof |
| CN102758259A (en) * | 2012-07-30 | 2012-10-31 | 济南赛尔生物科技有限公司 | Method for constructing human peripheral blood immune cell bank |
| CN102861107A (en) * | 2012-09-18 | 2013-01-09 | 中国人民解放军军事医学科学院野战输血研究所 | DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition |
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Cited By (1)
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|---|---|---|---|---|
| CN111228473A (en) * | 2020-02-14 | 2020-06-05 | 杭州憶盛医疗科技有限公司 | Anti-tumor novel composite vaccine for treating fibrosarcoma |
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