CN107129545B - Application of the modification of chitosan in terms of CIK cell culture and prepare the purposes of commercially available culture medium, culture vessel - Google Patents
Application of the modification of chitosan in terms of CIK cell culture and prepare the purposes of commercially available culture medium, culture vessel Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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Abstract
The application in terms of CIK cell culture that the invention discloses modification of chitosan and the purposes for preparing commercially available culture medium, culture vessel, modification of chitosan is prepared by the following method: the dissolution of ionic liquid water is first made to the ionic liquid aqueous solution of 8-12mmol/L, then chitosan is added into the ionic liquid aqueous solution, it is uniformly mixed to being completely dissolved, chitosan concentration is 30-50g/L, again by the solution under stirring condition irradiation reaction, irradiation dose is 40-60kGy, it finally washed, filtered with ethanol precipitation, collect filter cake, filtration cakes torrefaction is ground to obtain the final product.It is demonstrated experimentally that modification of chitosan provided by the invention can be improved the amplification vigor of CIK cell and kill tumor activity, it can be used for reducing cell therapy cost and promote therapeutic effect, be used to prepare commercially available culture medium, culture vessel, wide market.
Description
Technical field
The invention belongs to cellular immunity fields, are related to CIK cell, and in particular to modification of chitosan is in CIK cell culture side
The application in face and the purposes for preparing commercially available culture medium, culture vessel.
Background technique
Chitosan (chitosan) is also known as chitosan, is chitin (chitin) warp being widely present by nature
Cross what deacetylation obtained, chemical name is Chitosan (1-4) -2- amino-B-D glucose.From 1859, Frenchman
After Rouget obtains chitosan first, the biological functionality and compatibility of this natural polymer, blood compatibility, safety,
The excellent performances such as microbic resolvability are by all trades and professions extensive concern, in medicine, food, chemical industry, cosmetics, water process, metal
It extracts and the application study of the numerous areas such as recycling, biochemical and biomedical engineering achieves major progress.For patient, shell is poly-
Sugared reducing blood lipid, hypoglycemic effect existing research report.
Biological therapy is the 4th big tumor treatment model after 3 kinds of operation, chemotherapy and radiotherapy traditional modes, preceding 3 kinds of moulds
Formula or toxic side effect are larger, or cannot remove internal residual tumor cell, and most of tumor patient is led because of relapsed or stubborn eventually
It is lethal to die.Cellular immunotherapy technology occupies an important position in the biological therapy of tumour, shows good application prospect, and
It is increasingly becoming one of the important means of oncotherapy.Cytokine induced kill cell (cytokine-inducedkiller
Cell, CIK) be discovered in recent years a kind of very promising adoptive immunity cell, have proliferation rapidly, kill tumor activity wide spectrum
And the advantages that efficient, toxic side effect is small, have become the main force of knubble biological immunization therapy.The main effects of CIK cell
Cell is CD3+CD56+ phenotype T lymphocyte, has the feature of NK cell and T cell concurrently.Functionally, on the one hand CIK possesses T
The powerful anti-tumor activity of lymphocyte, is proliferated fast the characteristics of on the other hand possessing the non-MHC of NK cell restrictive killing tumour
Speed influences slightly normal hematopoiesis.It the amplification vigor of CIK cell and kills tumor activity and is directly related to treatment cost and effect.
Have not yet to see the application of chitosan and its derived product in terms of CIK cell culture amplification.
Summary of the invention
The present invention is intended to provide a kind of application of modification of chitosan and the modification of chitosan in terms of CIK cell culture
And the purposes of commercially available culture medium, culture vessel is prepared, which can be improved the amplification vigor of CIK cell and kills tumor
Activity reduces cell therapy cost and promotes therapeutic effect.
The present invention is achieved by following technical solution:
A kind of ionic liquid radiation modification chitosan, is prepared by the following method: being irradiated with ionic liquid to chitosan
Modified, the chemical structural formula of the cation of the ionic liquid is as follows:
Wherein, R is hexyl or octyl.
Preferably, the anion of the ionic liquid is bromide ion or chloride ion, and the chemical structural formula of ionic liquid is as follows:
Preferably, the modification of chitosan is prepared by the following method: 8- first is made in the dissolution of ionic liquid water
Then the ionic liquid aqueous solution of 12mmol/L adds chitosan into the ionic liquid aqueous solution, until it is equal to be completely dissolved mixing
It is even, chitosan concentration 30-50g/L, then by the solution under stirring condition irradiation reaction, irradiation dose 40-60kGy, most
It washed, filtered with ethanol precipitation afterwards, collect filter cake, filtration cakes torrefaction is ground to obtain the final product.
Application of the above-mentioned modification of chitosan in terms of CIK cell culture.
A kind of culture medium for CIK cell culture, the above-mentioned modification of chitosan containing effective concentration.
A kind of culture vessel for CIK cell culture, culture vessel inner wall are coated with the coated layer of effective thickness, coating
The material of layer is above-mentioned modification of chitosan.
Preferably, it is filled and is sealed with argon gas inside the culture vessel.
The invention has the advantages that
The present invention provides a kind of modification of chitosan, experiments have shown that the amplification of CIK cell can be improved in the modification of chitosan
Vigor and tumor activity is killed, can be used for reducing cell therapy cost and promote therapeutic effect, is used to prepare commercially available culture medium, training
Support vessel.
Detailed description of the invention
Fig. 1 is the chemical structural formula of each ionic liquid.
Specific embodiment
Technical solution in order to better explain the present invention, is further described combined with specific embodiments below.In embodiment
Non- special emphasis experimental material is routine experiment material, belongs to the scope that those skilled in the art are easily obtained.
Embodiment 1: the preparation of modification of chitosan
Different according to the ionic liquid type of use, experiment is divided into 4 groups of operation repetitives, i.e. hexyl pyridinium bromide group, hexyl
Pyridinium chloride group, octyl pyridinium bromide group, octyl pyridinium chloride group.Method of modifying is as follows:
The dissolution of above-mentioned ionic liquid water is first made to the ionic liquid aqueous solution of 10mmol/L, then to the ionic liquid
Chitosan is added in aqueous solution, until being completely dissolved uniformly mixed, chitosan concentration 40g/L, then by the solution in stirring condition
Lower irradiation reaction (irradiation bomb Co60), irradiation dose 50kGy, 95% ethyl alcohol is finally added into solution, and (6 times of reactions are molten
Liquid product) it staticly settles, filtering and washing collects filter cake, and filter cake is ground to 80 mesh after 40 DEG C or less low temperature dryings.
Certainly, the water-soluble concentration of ionic liquid can be adjusted in 8-12mmol/L, chitosan concentration can in 30-50g/L,
Irradiation dose can be adjusted in 40-60kGy.Drying temperature does not exceed 40 DEG C, otherwise can change colour.
Each ionic liquid chemical structural formula is as shown in Figure 1.
Embodiment 2: influence of the modification of chitosan to CIK cell amplification vigor and killing tumor activity
One, experimental material
10% fetal calf serum and RPMI-1640 culture solution are purchased from Gibco company, and lymphocyte separation medium is purchased from Tianjin Hao
Foreign biological products science and technology limited Company (TBD), IL-2 are purchased from Beijing Shuanglu Pharmaceutical Co., Ltd., anti-human CD3 monoclonal antibody
Purchased from Gibco company, IFN-γ is purchased from U.S. PEPROTEC company, and IL-1 α is purchased from U.S. PEPROTEC company, CCK-8 reagent
Purchased from the green skies Bioisystech Co., Ltd in Shanghai.
BALB/C nude mice, selection Female nude mice, age of mouse 6-7 weeks, weight 18-23g, in nude mice room under the conditions of SPF
Raising, is provided by Nanjing University's Experimental Animal Center.Gastric cancer cell line MGC-803 sings all biological cell libraries purchased from Shanghai.
Two, experimental method
1, the separation and culture of CIK cell
(1) separation of mononuclearcell: acquisition healthy volunteer's peripheral blood, the PBS 1:1 dilution of pre-cooling are slowly added to drench
Bar cell separating liquid upper layer, 650g, 4 DEG C of centrifugation 20min collect white cellular layer, separate mononuclearcell, RPMI-1640 training
It supports the outstanding cell of base weight and is placed on 37 DEG C, 5%CO22h is incubated in incubator.
(2) culture of CIK cell: the suspension cell in collecting monocytic cell, adjustment cell density to 2.5 × 106/ ml,
It is divided into three groups to be cultivated, grouping and cultural method are as follows:
At conventional group: 0 day, INF- γ (1000U/mL) is added in complete medium (RPMI-1640+10%FBS), puts
Enter 5%CO2, be incubated for for 24 hours in 37 DEG C of incubators after, addition IL-2 (500U/mL), IL-1 α (l00U/mL) and anti-human CD3 monoclonal antibody
(20μg/mL).Every 2-3d carries out changing liquid, and supplements the cell factor of equivalent, during the cultivation process, uses before adding fresh culture
Trypan Blue detects the motility rate of cell, counts the absolute number of cell；Continuous culture 12 days, collects cell；
Contrast groups: at 0 day, be added in the complete medium (RPMI-1640+10%FBS) INF- γ (1000U/mL) and
Unmodified chitosan (30 μ g/mL), is put into 5%CO2, be incubated for for 24 hours in 37 DEG C of incubators after, according to conventional group subsequent processes training
It supports, counts the absolute number of cell, collect cell；
It induces A-D group: at 0 day, INF- γ (1000U/mL) is added in complete medium (RPMI-1640+10%FBS)
The modification shell prepared with above-mentioned hexyl pyridinium bromide group, hexyl pyridinium chloride group, octyl pyridinium bromide group, octyl pyridinium chloride group
Glycan (30 μ g/mL), is put into 5%CO2, be incubated for for 24 hours in 37 DEG C of incubators after, organize subsequent processes culture according to conventional, statistics is thin
The absolute number of born of the same parents collects cell.
2, the Vitro Tumor killing activity of CIK cell
External each group CIK cell is detected using CCK-8 method, tumor activity is killed to gastric cancer cell line MGC-803, with logarithmic growth
The MGC-803 cell of phase as target cell, after adhere-wall culture for 24 hours after, respectively to cultivate 12 days each group CIK cells as effect
Cell is respectively placed in 37 DEG C, 5%CO2, in cell incubator under saturated humidity, after it is adherent for 24 hours after, be 10:1 by effect target ratio
Effector cell is added, laying effect cell controls are compareed with target cell, every group of 3 multiple holes, and effector cell and target cell co-culture for 24 hours
Afterwards, CCK-8 solution is added by 10% volume in every hole, is placed in incubator after continuing to be incubated for culture 4h, enzyme linked immunological picks up survey instrument
It selects wavelength to measure each hole light light absorption value for 450nm, and calculates killing activity (killing rate) according to the following formula:
Killing rate=[1- (experimental group OD value-effector cell organizes OD value)/target cell group OD value] × l00%.
3, the in-vivo tumour killing activity of CIK cell
BALB/C nude mice right axillary is inoculated into 0.2mL 1 × 106After/mLMGC-803 stomach cancer cell, then it is randomly divided into 7
Group: control group, conventional group, contrast groups and induction A-D group, every group 20.Conventional group after 10d, contrast groups and induction A-D group it is every
Nude mice injects 0.2mL 1 × 10 at inoculated tumour cell area7The CIK cell of/mL (is respectively corresponded according to above-mentioned routine
Group, induces A-D group Fiber differentiation 12 days at contrast groups), nude mice of control group injects 0.2mL physiological saline, continuously injects 5d.After 15d
Every group of removing tumor mass is simultaneously weighed, and calculates tumour inhibiting rate (%) according to following formula:
Tumour inhibiting rate=(control group tumor quality-experimental group tumor quality)/control group tumor quality × 100%.
4, statistical analysis
T inspection is carried out using SPSS20.0 statistics software, significant difference is thought in P < 0.05.
Three, experimental result
1, the proliferation and motility rate of each group CIK cell
Each group is all 2.5 × 10 when inoculation6A cell, when culture was to the 12nd day, each group proliferation times are as shown in the table, with
Conventional group is compared, and there was no significant difference for contrast groups (P > 0.05), and induction A-D group proliferation times significantly improve, compared with conventional group
With significant difference (P < 0.05).Trypan Blue detects Cell viability before each fluid infusion, guarantees motility rate about 95%.
| Conventional group | Contrast groups | Induce A group | Induce B group | Induce C group | Induce D group | |
| Proliferation times | 177 | 185 | 276 | 279 | 308 | 302 |
2, external each group CIK cell kills tumor activity to gastric cancer cell line MGC-803
Each group CIK cell carries out killing experiments to MGC-803 cell when taking culture respectively to the 12nd day, is in effect target ratio
When 10:1, each group CIK cell shows significantly to kill tumor activity, and wherein the CIK cell of induction group preparation is than conventional group and right
Tumor activity, and significant difference (P < 0.05) are killed with stronger than a group CIK cell for preparation.
Each group CIK cell is as shown in the table to the killing rate of MGC-803 cell.
3, internal each group CIK cell kills tumor activity to Transplanted Gastric Carcinoma
All experimentss nude mice has tumour to be formed when being inoculated with the 7th day in inoculation position, diameter 0.8-1cm.CIK cell
After intervening 15 days, conventional group, contrast groups, all nude mices of induction group lump reduced, and all nude mices of control group is swollen
Block increases.When dissected discovery, compared with the control group, conventional group, contrast groups, the tumor mass of induction group are smaller and limit to, and it is right
It is big according to the tumor mass of group and have tumor by local infiltration phenomenon.Conventional group, contrast groups, induction group tumour inhibiting rate see the table below.
As can be seen from the above table, induction group has more preferably internal tumour inhibiting rate, significant difference (P than conventional group and contrast groups
< 0.05).
Embodiment 3: the application (preparing commercially available culture medium and coating culture bottle) of modification of chitosan
Culture medium: modification of chitosan prepared by embodiment 1, quality volume are added in existing RPMI-1640 culture solution
Concentration is 30 μ g/mL, naturally it is also possible to be designed to other concentration.
Coated cell culture bottle: in existing Tissue Culture Flask, (or other are possibly used for the container of CIK cell culture, such as
Culture dish) inner surface coating 0.2-0.3mm thickness coated layer, coating layer material be above-mentioned modification of chitosan.It is excellent at one
It selects in embodiment, will be coated in the Tissue Culture Flask of the coated layer and fill full argon gas-sealed storage.If being not filled with full argon gas, 50
± 5 DEG C are placed 30 days, and coated layer color is significantly deepened, and becomes dark brown (significant decrease of CIK cell culture effect) by milky；
It is not in such case if being protected with argon gas.The high-temperature stability of coating culture bottle can be improved in this method, and extension is guaranteed the quality
Phase.
Above-described embodiment is only used for that technical solution of the present invention is explained further, it will be appreciated by those skilled in the art that, appoint
All without departing from the present invention, protection scope of the present invention is not limited to above-mentioned specific embodiment for what simple replacement or modification.
Claims (1)
1. a kind of application of modification of chitosan in terms of CIK cell culture, which is characterized in that the modification of chitosan is by the following method
It is prepared: the dissolution of ionic liquid water being first made to the ionic liquid aqueous solution of 8-12mmol/L, then to the ionic liquid
Chitosan is added in aqueous solution, until being completely dissolved uniformly mixed, chitosan concentration 30-50g/L, then by the solution in stirring bar
Irradiation reaction under part, irradiation dose 40-60kGy finally use ethanol precipitation, washing, suction filtration, collect filter cake, and filtration cakes torrefaction is ground
It grinds to obtain the final product；The ionic liquid is one of following ionic liquid:
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| CN104072634A (en) * | 2014-07-16 | 2014-10-01 | 中国科学院海洋研究所 | Preparation method for chitin oligosaccharides |
| CN105969730A (en) * | 2016-07-25 | 2016-09-28 | 中国农业科学院哈尔滨兽医研究所 | Preparation method of pig cytokine-induced killer (CIK) cells |
| CN106520695A (en) * | 2016-12-02 | 2017-03-22 | 中国计量大学 | Additive containing shen-fu polysaccharide and promoting CIK cell proliferation and differentiation, culture medium, culture method and application |
| CN106635986A (en) * | 2016-12-02 | 2017-05-10 | 中国计量大学 | Ginseng polysaccharide capable of promoting multiplication and differentiation of CIK (Cytokine Induced Killer) cells, culture medium, culture method and application |
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| CN104072634A (en) * | 2014-07-16 | 2014-10-01 | 中国科学院海洋研究所 | Preparation method for chitin oligosaccharides |
| CN105969730A (en) * | 2016-07-25 | 2016-09-28 | 中国农业科学院哈尔滨兽医研究所 | Preparation method of pig cytokine-induced killer (CIK) cells |
| CN106520695A (en) * | 2016-12-02 | 2017-03-22 | 中国计量大学 | Additive containing shen-fu polysaccharide and promoting CIK cell proliferation and differentiation, culture medium, culture method and application |
| CN106635986A (en) * | 2016-12-02 | 2017-05-10 | 中国计量大学 | Ginseng polysaccharide capable of promoting multiplication and differentiation of CIK (Cytokine Induced Killer) cells, culture medium, culture method and application |
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