CN101314035B - Uses of biological polyoses microcapsule - Google Patents
Uses of biological polyoses microcapsule Download PDFInfo
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- CN101314035B CN101314035B CN2008100398348A CN200810039834A CN101314035B CN 101314035 B CN101314035 B CN 101314035B CN 2008100398348 A CN2008100398348 A CN 2008100398348A CN 200810039834 A CN200810039834 A CN 200810039834A CN 101314035 B CN101314035 B CN 101314035B
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Abstract
The invention discloses an application of a biological polysaccharide microcapsule, which is used to couple with antigen or antigenic determinant and prepare a substance which can promote the T lymphocyte proliferation or improve the capability of T lymphocyte to kill specificities of cells expressing the antigen or the antigenic determinant.
Description
Technical field
The invention belongs to field of immunology, more specifically, the present invention relates to a kind of biological polyoses microcapsule and promote the T lymphopoiesis or improve the T lymphocyte the purposes in the material of the cell-specific kill capability of expressing described antigen or antigenic determinant in preparation.
Background technology
To be that the function found so far is the strongest, unique knownly can activate the lymphocytic antigen presenting cell of initial T to dendritic cell.Dendritic cell picked-up tumor associated antigen, submission is given the T lymphocyte and is activated corresponding C D8 after the processing
+And CD4
+The T lymphocyte, to induce body cell immunity and humoral immune function, the antitumor action of the immunity of taking the initiative.This framework is the generally accepted theoretical basis of present tumor biotherapy research field, promptly utilizes dendritic cell vaccine to carry out antineoplaston.
The key of dendritic cell tumor vaccine success is to improve the immunogenicity of dendritic cell to the tumor associated antigen of less immunogenic.For this reason, those skilled in the art have carried out many-sided trial, as utilize the antigen of various ways such as external synthetic tumor antigen polypeptide, the full cell extract of tumor and tumor antigen nucleic acid to stimulate dendritic cell, but problem such as these method ubiquity efficient are low, poor specificity, side effect are many.Generally speaking, the T lymphocyte need be accepted the dendritic cell repetitious stimulation could produce certain tumor-killing effect, easily produces problems such as immunologic tolerance, and classical way also is difficult to solve.Therefore, this area presses for finds a kind of immunogenic effective way that improves antigenic protein, to overcome the difficult problem that prior art exists.
In the inventor's work in advance, it is good to have prepared a kind of biocompatibility, the biological polyoses microcapsule that particle diameter is little (referring to patent ZL200310108684.9 and patent application 200610024132.3), this microcapsule can forever be preserved in alcoholic solution, also can be suspended in the solution and preserve, time was 1 year, still kept stable, and flocculation or decomposing phenomenon did not occur.Because this microcapsule is a kind of degradable material and stable performance, and using value is preferably arranged, therefore be necessary it is carried out further R and D, in the hope of finding new purposes.
Summary of the invention
The object of the present invention is to provide a kind of purposes of biological polyoses microcapsule, be used for and antigen or antigenic determinant coupling mutually, preparation promotes the T lymphopoiesis or improves the material of T lymphocyte to the cell-specific kill capability of expressing described antigen or antigenic determinant
Another object of the present invention is to provide the lymphocytic propagation of a kind of T of promotion or improve the material of T lymphocyte the cell-specific kill capability of expressing described antigen or antigenic determinant, described material contains biological polyoses microcapsule, and mutually link coupled with it antigen or antigenic determinant.
In a first aspect of the present invention, a kind of purposes of biological polyoses microcapsule is provided, be used for and one or more antigen or antigenic determinant coupling mutually, preparation promotes the T lymphopoiesis or improves the material of T lymphocyte to the cell-specific kill capability of expressing described antigen or antigenic determinant; Wherein, described biological polyoses microcapsule contains the biological polyoses of 50-90% weight, and capsular diameter is between the 50-1000 nanometer.
In another preference, described coupling is a covalent coupling.
In another preference, after described biological polyoses microcapsule and antigen or the antigenic determinant coupling, promote antigenic submission effect.
In another preference, after described biological polyoses microcapsule and antigen or the antigenic determinant coupling, promote the kill capability of T lymphocyte to the cell that carries described antigen or antigenic determinant.
In another preference, described biological polyoses microcapsule is to adopt following method preparation:
A) be that 0.8-3% biological polyoses aqueous solution and 20-1000mM aqueous metal salt add respectively in the organic solvent that is dissolved with surfactant with w/v, add mineral oil, mix, identical up to oil phase with water density, form biological polyoses water/oil suspension and metal saline/oil suspension respectively, wherein said biological polyoses is selected from alginate, Gellan glue, pectin, pectic acid, carrageenan; Described slaine is selected from the water soluble salt that following metal ion forms: K
+, Ca
2+, Ba
2+, Sr
2+, Cu
2+, Fe
2+, Fe
3+, Al
3+Or its combination;
B) biological polyoses water/oil suspension and the metal saline/oil suspension that step a) is obtained used ultrasonic emulsification respectively on ice bath, forms stable biological polyoses water/oil emulsion and metal saline/oil emulsion;
C) at 4-35 ℃ of blend step b) the biological polyoses emulsion that obtains and slaine emulsion 5-60 minute, form reactant mixture;
D), obtain the aqueous phase reactions product to the centrifugal layering of the reactant mixture of step c);
E) product dehydration, surfactant and oil phase substance are removed in washing then, obtain biological polyoses microcapsule.
In another preference, described biological polyoses microcapsule is to adopt following method preparation:
A) be that the adding of 0.8-3% biological polyoses aqueous solution is dissolved with in the organic solvent of surfactant with w/v, add mineral oil, mix, identical up to oil phase with water density, form biological polyoses water/oil suspension;
B) the biological polyoses water/oil suspension that a) obtains is carried out ultrasonic emulsification, form stable biological polyoses water/oil emulsion;
C) dripping volume ratio in biological polyoses water/oil emulsion is the equal solubility acid of 0.5-10% (preferable 1-5%) water oil phase; Add 5-50% (preferable 10-40% then; Better 15-30%) organic solvent fully mixes; Centrifugal removal oil phase; Obtain the aqueous phase reactions product;
D) to c) to add volume ratio in the aqueous phase reactions product that obtains be the equal solubility acid of water oil phase of 0.1-2% (preferable 0.2-1%), carries out ultrasonication; Drip metal salt solution (as 20 ± 20mM in the ultrasonication process; 20 ± 10mM) preferable solution fully mix, and regulate PH6-8 (preferable 7-8); Surfactant and oil phase substance are removed in washing then, obtain biological polyoses microcapsule.
In another preference, the equal solubility acid of described water oil phase is selected from: acetic acid, propanoic acid, butanoic acid, isopropyl acid, isopropylformic acid., malic acid, or other small molecular organic acid.
In another preference, if biological polyoses is alginate, Gellan glue, pectin, pectic acid, described slaine is selected from: CaCl
2, BaCl
2, SrCl
2Or its mixture; Perhaps
In another preference, if biological polyoses is a carrageenan, described slaine metal ion is K
+Ion, concentration are 40-500mM.
In another preference, described surfactant is Span 65, or surfactant is nonionic surfactant, and surfactant concentration is 5-50mg/ml; Described organic solvent is selected from dichloromethane, chloroform.
In another preference, the dehydration in the step e) is carried out with dehydrated alcohol, and washing is to use washed with dichloromethane earlier, uses absolute ethanol washing then.
In another preference, also comprise step:
F) biological polyoses microcapsule that step e) is obtained is put into the buffer of pH4.5-6.2, with the pH that contains water solublity diamine compound, water-soluble carbodiimide, N-hydroxy thiosuccinimide is that the buffer of 4-6.9 mixes, wherein the biological polyoses microcapsule dry weight is 1 gram with the ratio of diamine compound: the 0.1-0.6 mM, 10-50 ℃ of following lucifuge and mixed 10-30 hour, remove metal ion with the metal ion chelation agent solution-treated that is selected from ethylenediaminetetraacetic acid or citric acid then.
In another preference, described antigen is tumor specific antigen.
In another preference, described antigen is selected from: carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), cancer antigens c A125, cancer antigens c a153, carbohydrate antigen Ca242, malignant tumor related substances (TSGF), prostate-specific-antigen PSA, the mucoprotein MUC1 of epidermis.
In another preference, described material promotes the T lymphopoiesis or improves the T lymphocyte to expressing the cell-specific kill capability of described antigen or antigenic determinant by stimulating dendritic cell multiplication.
In a second aspect of the present invention, the material of a kind of T of promotion lymphopoiesis or the raising T lymphocyte cell-specific kill capability to expressing described antigen or antigenic determinant is provided, described material contains biological polyoses microcapsule, and mutually link coupled with it one or more antigens or antigenic determinant; The content of antigen or antigenic determinant is 0.1-30% (preferably 0.2-20% in the described material; 0.5-15% more preferably) weight; Wherein, described biological polyoses microcapsule contains the biological polyoses of 50-90% weight, and capsular diameter is between the 50-1000 nanometer.
In another preference, described biological polyoses microcapsule is a calcium alginate microcapsule.
In a third aspect of the present invention, the purposes of described material is provided, be used to prepare and promote the T lymphopoiesis or improve the compositions of T lymphocyte the cell-specific kill capability of expressing described antigen or antigenic determinant.
In a fourth aspect of the present invention, a kind of compositions is provided, it contains the described T of promotion lymphopoiesis or improves the material of T lymphocyte to the cell-specific kill capability of expressing described antigen or antigenic determinant, and pharmaceutically acceptable carrier.
Description of drawings
Fig. 1. the TEM photo of calcium alginate microcapsule.
Fig. 2. the calcium alginate microcapsule particle size distribution.
Fig. 3. laser confocal microscope is observed the phagocytosis (excitation wavelength: 655nm) of dendritic cell to quantum dot embedding labelling calcium alginate microcapsule.A:30 minute; B:12 hour.
Fig. 4. the flow cytometry that HLA-DR expressed after calcium alginate microcapsule stimulated immature dendritic cell (dash area is contrast, down together).A: do not add stimulus object (negative control); B:10 μ g/ml LPS (positive control); The c:0.6mg/ml calcium alginate microcapsule; D:1.2 mg/ml calcium alginate microcapsule.
Fig. 5. from the stimulation index of body T lymphocyte proliferation assay.
Fig. 6. dendritic cell and stimulus object are cultivated the Flow cytometry that CD86 expresses after 14 hours altogether.A: do not add stimulus object (negative control; B:10 μ g/ml LPS (positive control); The c:0.6mg/ml calcium alginate microcapsule; D:100 μ g/ml recombinant C EA albumen; E:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule mixture; F:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule conjugate.
Fig. 7 .Western Blot detects purification of Recombinant CEA protein.Wherein, 1, the CEA standard substance, 100 μ g/ml; 2, purification of recombinant proteins matter.
Fig. 8. dendritic cell is by different stimulated thing effect IL-12 abduction delivering after 14 hours.1: do not add stimulus object (negative control); 2:10 μ g/ml LPS (positive control); 3:0.6mg/ml calcium alginate microcapsule; 4:100 μ g/ml recombinant C EA albumen; 5:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule mixture; 6:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule conjugate.
Fig. 9. dendritic cell is by different stimulated thing effect TNF-α abduction delivering after 14 hours.1: do not add stimulus object (negative control); 2:10 μ g/ml LPS (positive control); 3:0.6mg/ml calcium alginate microcapsule; 4:100 μ g/ml recombinant C EA albumen; 5:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule mixture; 6:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule conjugate.
Figure 10. allosome T lymphocyte and lymphopoiesis after the dendritic cell of different stimulated is cultivated 5 days altogether.1:T lymphocyte single culture (negative control I); 2: do not add stimulus object (negative control II); 3:0.6mg/ml calcium alginate microcapsule; 4:100 μ g/ml recombinant C EA albumen; 5:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule mixture; 6:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule conjugate.
Figure 11 .T lymphocyte is expressed the experiment of 293 cell killings to non-CEA.1: without the T lymphocyte (negative control I) of the inductive dendritic cell stimulation of stimulus object; 2:10 μ g/ml LPS (positive control); 3:0.6mg/ml calcium alginate microcapsule; 4:100 μ g/ml recombinant C EA albumen; 5:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule mixture; 6:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule conjugate.
Figure 12 .T lymphocyte is expressed the experiment of HCT-8 cell killing to CEA.1: do not add T lymphocyte (negative control I); 2: without the T lymphocyte (negative control II) of dendritic cell stimulation; 3: without the T lymphocyte (negative control III) of the inductive dendritic cell stimulation of stimulus object; 4:10 μ g/ml LPS (positive control); 5:0.6mg/ml calcium alginate microcapsule; 6:100 μ g/ml recombinant C EA albumen; 7:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule mixture; 8:100 μ g/ml recombinant C EA albumen and 0.6mg/ml calcium alginate microcapsule conjugate.
The specific embodiment
The inventor is through deep research, be surprised to find that biological polyoses microcapsule and antigen (or antigenic determinant) mutually after the coupling, can promote the T lymphopoiesis effectively or improve the T lymphocyte, greatly strengthen the immunogenicity of described antigen (particularly tumor antigen) expressing the cell-specific kill capability of described antigen or antigenic determinant.
Biological polyoses microcapsule
Biological polyoses microcapsule of the present invention contains the biological polyoses of 50-90% weight, and the diameter of microcapsule is in (preferably between the 50-900 nanometer, more preferably between the 50-800 nanometer) between the 50-1000 nanometer.Described biological polyoses can be selected from sodium alginate, pectin, pectic acid and carrageenan etc.The preparation method of described biological polyoses microcapsule is known, for example can adopt the method described in formerly the patent ZL200310108684.9 and application number 200610024132.3, the biological polyoses capsule grain diameter that adopts these methods to obtain is approaching, and physicochemical property is basic identical.
The inventor studies show that, described biological polyoses (as calcium alginate) microcapsule can be engulfed by dendritic cell; Dendritic cell surface C D86 and HLA-DR expression raise after described biological polyoses microcapsule stimulates, and show the ability that described biological polyoses microcapsule has stimulates the maturing dendritic cell differentiation.Its mechanism of action that stimulates the maturing dendritic cell differentiation may be to produce autocrine cytokine TNF-α by inducing cell to realize induction.
Biological polyoses microcapsule and antigenic conjugate
Though unloaded biological polyoses microcapsule can stimulate maturing dendritic cell, can't stimulate the T lymphopoiesis.After the process number of research projects, the inventor is surprised to find that, if with a kind of antigen and biological polyoses microcapsule coupling mutually, then can promote the lymphocytic propagation of T greatly.And, can not promote the T lymphopoiesis with antigen and biological polyoses microcapsule are mixed mutually.
Therefore, the invention provides the lymphocytic propagation of a kind of T of promotion and improve the material of T lymphocyte the cell-specific kill capability of expressing described antigen or antigenic determinant, described material contains biological polyoses microcapsule, and mutually link coupled with it antigen or antigenic determinant.Described coupling is covalent coupling preferably.Have free carboxy abundant, that have neither part nor lot in ionomer in the described biological polyoses microcapsule structure, thereby can be used for antigenic covalent coupling load; And its hydrogel character can make the antigen of load be in the water environment all the time, keeps biological conformation and activity thereby be of value to antigen protein.
Multiple described biological polyoses microcapsule and the link coupled method of antigen all be can be applicable to the present invention.As optimal way of the present invention, a kind of method for preparing conjugate can be a NHS/EDAC peptide bond synthetic reaction, the key step of this reaction is as follows: biological polyoses microcapsule was added in the MES buffer ultrasonication 0.5-5 minute, add NHS and EDAC and mix also reaction; Adding antigen then fully reacts; Remove afterwards link coupled floating preteins does not take place, obtain described biological polyoses microcapsule and antigenic conjugate.Qualitative evaluation shows that this method has high coupling efficiency.Usually, the content of described antigen in conjugate is 0.1-30% weight; Preferably 0.2-20% weight; 0.5-15% weight more preferably; As 1%, 2%, 3%, 5%, 6%, 10% weight (dry weight).
" antigen " is meant a kind of material that can stimulate human or animal's body to produce antibody or primed lymphocyte, and it has immunogenicity (antigenicity) and/or reactionogenicity." immunogenicity " is meant the ability that can stimulate body to form specific antibody or primed lymphocyte.Known in the art or unknown many antigens all can be used for the present invention, preferably disease association antigen.Described antigen can be natural antigen, or utilizes the antigen of gene recombination technology preparation.The antigen fragment that contains antigenic determinant also is operable.
Described biological polyoses microcapsule can with the coupling mutually of a kind of antigen, thereby also can with the effect of coupling performance therapeutic alliance mutually of multiple antigen.Many antigen or antigen fragment also are available by covalent bond (as peptide bond) or the coupling antigen polypeptide that forms that is connected in some cases, for example contain the fusion rotein of many antigens or antigen fragment.
As particularly preferred mode of the present invention, described antigen is tumor specific antigen.Tumor specific antigen is meant in tumor and takes place, occurs in the evolution or the antigenic substance of overexpression that the expression of this antigen in tumor cell is much higher than normal cell.Any tumor specific antigen all can be used to the present invention, and is preferred, and described antigen is selected from: carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) etc.Described tumor specific antigen can be well and described biological polyoses microcapsule coupling, the effect of the specificity tumor killing cell (the described tumor specific antigen of this tumor cells expression) that performance is excellent.
Described biological polyoses microcapsule and antigenic conjugate are effective especially for promoting the T lymphopoiesis or improving the T lymphocyte to the cell-specific kill capability of expressing described antigen or antigenic determinant, make immunogenicity of antigens improve greatly.Even as bovine serum albumin or the more weak antigen protein of this antigenicity of carcinoembryonic antigen, it also can promote the lymphocytic propagation of T after coupling mutually significantly with described biological polyoses microcapsule.
In one embodiment of the invention, utilize calcium alginate microcapsule can improve the characteristics of load protein immunization originality, stimulate dendritic cell with the microcapsule behind the coupling recombinant C EA albumen, find that the proteic microcapsule of load recombinant C EA can stimulate maturing dendritic cell, especially dendron shape surface HLA-DR that the proteic microcapsule of load recombinant C EA stimulates and CD86 express simple recombinant C EA albumen and the microcapsule mixture stimulates high, the capsule coupling is described fully to the simple mixing of the immunogenic raising effect of antigen greater than albumen and microcapsule, this is owing to the raising greatly that variation aspect two of the quality and quantities causes submission efficient has taken place after coupling CEA.
In another embodiment of the present invention, carried out T lymphocyte killing experiments, can observe CTL has the specific killing effect to colon cancer cell line HCT-8, shows that calcium alginate microcapsule can significantly increase the ability of the specific killing of CTL.Be that experiment in vitro shows, calcium alginate microcapsule is a kind of effective immunological adjuvant, but coupling recombinant C EA albumen inducing immune cells produces the immunoreation of specific killing CEA tumor cell.
Pharmaceutical composition
The present invention also provides a kind of compositions, and it contains: described material, and pharmaceutically acceptable carrier.Described compositions can be used for promoting the T lymphopoiesis or improves the T lymphocyte to expressing the cell-specific kill capability of described antigen or antigenic determinant.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and does not have excessive bad side reaction (as toxicity), promptly has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.(Mack Pub.Co. can find proving absolutely about pharmaceutically acceptable carrier in N.J.1991) at Remington ' s PharmaceuticalSciences.Acceptable carrier can contain liquid on combination of Chinese medicine is learned, as water, saline, glycerol and sorbitol.In addition, also may there be complementary material in these carriers, as lubricant, fluidizer, wetting agent or emulsifying agent, pH buffer substance and stabilizing agent, as albumin etc.
Described compositions can be made the various dosage forms that are suitable for the mammal administration, described dosage form includes but not limited to: injection, tablet, Emulsion.
In use, be with the promotion of the present invention T lymphopoiesis of safe and effective amount or improve the T lymphocyte material of the cell-specific kill capability of expressing described antigen or antigenic determinant is applied to mammal (as the people), wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 1 microgram/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
I. material and method
1. material
Sodium alginate:
LVCR (NF grade), mean molecule quantity 54,000 is available from international special product (International Specialty Products, ISP) (Hong Kong) company limited.
Dichloromethane, petroleum ether, glacial acetic acid are available from TEDIA company.Ethanol is available from Merck company, sorbitol anhydride tristearate (Span65), and HEPES is available from Sigma company.Calcium chloride and sodium hydroxide are available from Amersco company, and quantum dot (Non-targeted Quantum Dots) is available from Invitrogen company.
Plasmid: pGEM4 contains total length people CEA coded sequence.Available from McGill Cancer Centre.PcGFP contains the GFP albumen coded sequence, available from Invitrogen company.PcDNA-3 is available from Invitrogen company.
Express and use cell: 293 cells are available from Invitrogen company.
CEA standard substance and CEA monoclonal antibody (mouse anti human CEA) are available from Fitzgerald company.Cationic polymer transfection reagent poly aziridine (PEI) is available from Chemicon company, and two anti-(goat anti-mouse IgG) and immunoblotting reaction (Western Blot) developer Super Signal West Pica Trial Kit are available from Pierce company.
Phycoerythrin is available from Hangzhou Ao Weisheng thing Engineering Co., Ltd.BSA (BSA) is available from Trace NZ Desert Biologicals company.2-N-morpholino b acid (MES), N-hydroxy-succinamide (NHS), N-(3-dimethyl aminopropyl)-N '-ethyl-carbodiimide hydrochloride (EDC) are available from Sigma-Aldrich company.
Human peripheral blood single nucleus cell is got the Freshman peripheral blood and is obtained human peripheral blood single nucleus cell through the Ficoll centrifugalize from Shanghai Central Blood Station.
GM-CSF and IL-4 are from R﹠amp; D company; Glutamine is from Sigma company; IL-2 is from Peprotech company.
Dendritic cell growth medium: RPMI-1640 adds 10% hyclone and 2mM glutamine, and other adds 30ng/ml GM-CSF and 20ng/ml IL-4.
T LSM: RPMI-1640 adds 10% hyclone and 2mM glutamine.
T lymphocyte growth culture medium: RPMI-1640 adds 10% hyclone and 2mM glutamine, and other adds 100ng/ml IL-2.
CD14 sorting magnetic bead kit is available from Miltenyi company, and LPS and "diazoresorcinol" are available from Sigma company; TNF-α is available from Peprotech company, and the nylon hair is available from Yuhang Special Chemical Fiber Plant, Shanghai.
The HCT-8 cell: the human colon cancer cell strain, secretion CEA is available from Shanghai Inst. of Life Science, CAS.
HCT-8 cell growth medium: RPMI-1640 adds 10% hyclone and 2mM glutamine.293 cell attachment culture medium: RPMI-1640 add 10% hyclone and 2mM glutamine.
TNF-α ELISA detection kit is available from U-Cytech company, and IL-12 p70 ELISA detection kit is available from eBioscience company.
Mouse anti human CD86, HLA-DR antibody and homotype contrast thereof are available from Catalog company.
2. the preparation of sodium alginate soln
Take by weighing the 1g sodium alginate and be added in the 100ml ultra-pure water, stir 6 hours to dissolving, adding HEPES is 10mM to final concentration, and using 1.5M NaOH regulator solution pH is 7.4.Solution is sub-packed in the sterilization centrifuge tube, puts 4 ℃ of refrigerators.
3. acid precipitation method prepares calcium alginate microcapsule
Take by weighing 240mg Span65 and be dissolved in the 10ml dichloromethane, add the 1ml w/v and be 1% sodium alginate soln (contain 10mM HEPES, pH 7.4), it is identical with oil phase proportion to add the about 9ml adjusting of petroleum ether water again.Ultrasonication 8 minutes (ultrasonic mixed instrument is from the letter Instr Ltd. in Shanghai, model JYD-900, power 300W) forms emulsion.Ultrasonication slowly dripped glacial acetic acid 600 μ l in the time of 5 minutes.Add dichloromethane 5ml, mix back centrifugal 15 minutes of 1600rpm (Beckman Model J-6B centrifuge), syringe is taken out the sub-cloud oil phase, adds dichloromethane 10ml again, fully mixes the back recentrifuge and discards oil phase.
Add volume fraction 0.5% acetic acid solution 15ml, ultrasonication 8 minutes (power 200W) drips 20mM CaCl in the time of 5 minutes
2Solution 4ml.Change liquid over to the 100ml beaker, with NaOH regulator solution pH value to 7.4, stirring at room 30 minutes.Change solution over to centrifuge tube, add dehydrated alcohol 10ml, mixed back 2000rpm centrifugal 15 minutes.Abandon supernatant, ultrasonication is 3 minutes behind the adding dehydrated alcohol 15ml.Washed with dichloromethane was 1 time after identical washing process repeated 3 times, absolute ethanol washing 2 times.Solution adds the 6ml buffer after abandoning supernatant, is stored in 4 ℃ of refrigerators after ultrasonic 3 minutes.
4. the calcium alginate microcapsule of embedded quantum dots preparation
Get the quantum dot solution (Invitrogen) that 10 μ l concentration are 2 μ M, add 10 μ l glycerol, fully mix the back and add 1ml 1% sodium alginate soln (contain 10mM HEPES, pH 7.4), all the other steps prepare with aforementioned calcium alginate microcapsule, preparation back fluorescence microscope.
5.CEA construction of recombinant plasmid and protein expression and purification
(1) structure of expression plasmid rhCEA
Adopt restriction endonuclease EcoR I digested plasmid pGEM4 to obtain the CEA full-length cDNA, connect, make up the novel plasmid pcCEA that contains total length CEA cDNA, make up plasmid and identify through digestion with restriction enzyme and order-checking with carrier pcDNA-3 through identical endonuclease digestion.Use C end primer (5-GAACCTGAGGCTCAGAACACAAC-3) and N end primer (5-TACTAGTGGTGGTGGTGGTGGTGAATCAGAGCAACCCCAACC-3), with novel plasmid pcCEA is that template is carried out pcr amplification, obtain the purified back of segment and be connected the proteic plasmid rhCEA of construction expression recombinant C EA with carrier pcDNA-3.Making up plasmid identifies through digestion with restriction enzyme and order-checking.
(2) expression and purification of rhCEA in 293 cells
Transient transfection and recombinant C EA protein expression: get 10 μ g/ml rhCEA plasmids, 0.1 μ g/ml pcGFP plasmid (GFP is a labelled protein) 6ml, 1ml 60 μ g/ml PEI and mix and add to 60ml after 30 minutes and contain 1 * 10
6In the RPMI-1640 culture fluid of/ml 293 cells, 37 ℃ of 5%CO
2Condition is cultivated and is added 140ml LCHL culture medium continuation cultivation after 4 hours again.Fluorescence microscope transfection effect.Trypan blue method cell counting, cell motility rate are lower than at 90% o'clock to be stopped to cultivate.
Recombinant C EA Protein Detection in the cell culture fluid: get each 15 μ l of 293 cell culture fluids and 100 μ g/ml CEA standard substance, mix back boiling water heating 5 minutes with 15 μ l, 2 * SDS, carry out electrophoresis, albumen goes to cellulose acetate film behind the electrophoresis.Washing TBST Buffer rinsing 3 times after 1 hour adds 1 μ g/ml CEA antibody 3ml in cellulose acetate film 10% skim milk, and room temperature was hatched 2 hours, adds 1 μ g/ml, two anti-5ml after the TBST Buffer rinsing 3 times, and colour developing is taken pictures.
Recombinant C EA protein purification: 293 cell culture fluid 1500rpm get supernatant and mix at 1: 1 with 2 * Binding Buffer after centrifugal 10 minutes.Behind the 2ml His resin dress post, the Binding Buffer balance eluting post of 10 times of column volumes adds supernatant and 2 * Binding Buffer mixed liquor again, the Binding Buffer flush away impurity of 5 times of column volumes behind the slow post excessively.The 500mM imidazoles eluant solution recombinant C EA albumen of 10 times of column volumes of reuse, the recombiant protein of collection eluting.
Purified recombinant C EA albumen uses Western Blot check (Fig. 7), and CEA standard substance size is 180kD, shears the interior sheet of born of the same parents and has no progeny, and recombinant C EA albumen is about 160kD, and the discrepant reason of its molecular weight with standard substance is that degree of glycosylation is different.
6. sodium alginate micro gel capsule and protein covalent coupling
Get 1ml calcium alginate microcapsule alcohol suspension, abandon supernatant after centrifugal.Add 1.1ml 50mMMES (PH6.5) buffer (available from Sigma company), ultrasonication 1 minute (power 200W).Add freshly prepared 0.04M NHS (available from Sigma company) and 0.02 M EDAC solution (available from Sigma company), 300 μ l, fully mix.The lucifuge reaction adds 100 μ g/ml phycoerythrin, 100 μ l or 100 μ g/ml recombinant C EA albumen 1.5ml or 10mg/ml BSA 40 μ l (final concentration phycoerythrin 6.25 μ g/ml, recombinant C EA albumen 50 μ g/ml, BSA 200 μ g/ml) under the room temperature after 2 hours.Reaction is spent the night under the room temperature.After reaction finishes, centrifugally add the equal volume pure water after removing floating preteins.Sonic oscillation 2 minutes disperses the calcium alginate microcapsule of coupling protein, 4 ℃ of preservations again.
The coupling fluorescin is quantitative: get the calcium alginate microcapsule aqueous solution of coupling fluorescin (phycoerythrin), measure 565nm absorbance value OD
565, obtain link coupled fluorescin amount with fluorescin quantitative criterion curve ratio, calculate coupling efficiency: coupling efficiency=coupling fluorescin amount/reaction fluorescin amount.
7. dendritic cell is separated and cultivation with T is lymphocytic
(1) dendritic cell is separated
Get the 10ml human peripheral blood single nucleus cell, 1000rpm abandons supernatant after centrifugal 5 minutes, adds 10mlPBS and cleans 2 times, carries out the cell numeration.Get 5 * 10
7Individual cell, 1000rpm abandons supernatant after centrifugal 5 minutes, adds 400 μ l PBSE (containing 0.5%BSA, the PBS solution of 2mM EDTA) and 100 μ l CD14 sorting magnetic beads, flicks mixing, ice bath 30 minutes.Add 2ml PBSE, centrifugal 5 minutes of 1000rpm abandons supernatant, adds 500 μ l PBSE.
Simultaneously, super-clean bench is installed magnetic separating device and syringe, will treat behind the 500 μ l PBSE rinse syringes that sorting cells adds syringe, 500 μ l PBSE washing, washing process repeats 3 times, and (the CD14 negative cells that washing obtains can add the RPMI-1640 that contains 10% hyclone and cultivate, separate use for the T lymphocyte), magnetic separating device is removed in the washing back, adds 1ml PBSE in the syringe, connect chock plug, go out the cell of collection, the cell numeration obtains CD14
+The precursor dendritic cell.
Cell is with 2 * 10
6The density of/ml is inoculated in six orifice plates, every hole 4 * 10
6Individual cell adds the dendritic cell growth medium, 37 ℃ of 5%CO
2Cultivate.
(2) dendritic cell is cultivated and is stimulated
Dendritic cell is cultivated the 4th day half amount and is changed liquid, and every hole is gently inhaled the 1ml culture fluid and given up, and adds the dendritic cell grown cultures liquid that 1ml contains double somatomedin, 37 ℃ of 5%CO
2Continue to cultivate 3 days, the 6th day once more half amount change liquid.
Dendritic cell is cultivated and can be added 100ng/ml TNF-α or 10 μ g/ml LPS stimulation maturing dendritic cell on the 7th day.Stimulus object adds back 37 ℃ of 5%CO
2Cultivated 14 hours.
The mature dendritic cell featheriness gets final product levitating, and 1500rpm abandons supernatant, cell counting after centrifugal 2 minutes.
(3) the T lymphocyte separates
The T lymphocyte separates use nylon hair post partition method.
Get the nylon hair of the loose no conglomeration of 1g, after cleaning 6 times, the 300ml distilled water added 300ml 0.2MHCl solution stirring 3 hours, re-using the 300ml pure water cleans 6 times, 121 ℃ of steam sterilizations, 60 ℃ are toasted 8 hours to remove residue moisture content, with the loose 5ml glass syringe of filling in of nylon hair, be filled to 3ml scale place.At syringe needle and syringe indirect one aseptic short plastic tube (can use mosquito forceps to clamp this section in the experiment), be nylon hair post with the control flow velocity.
Nylon hair post is fixed in the super-clean bench brandreth, 15ml T LSM flushing nylon hair post, mosquito forceps on the folder when liquid level is higher than nylon hair post cylinder 1ml, 37 ℃ of 5%CO
2Place and made column equilibration in 1 hour.
Get CD14 negative cells and attached cell not behind the isolated dendritic cell, centrifugal 2 minutes of 1500rpm abandons supernatant, adds 3ml T LSM, cell counting.
Nylon hair post is fixing once more, and cell adds in the post, opens mosquito forceps, treats to press from both sides when liquid level arrives cylinder and goes up mosquito forceps, and the liquid of collecting is added in the post once more.37 ℃ of 5%CO
2Cultivated 1 hour.
After nylon hair post is fixing, open mosquito forceps, emit liquid, keep every of about 3 seconds of flow velocity, slowly add 10ml simultaneously and be preheated to 37 ℃ T LSM.Effusive cell is the T lymphocyte.The T lymphocyte is abandoned supernatant through 1500rpm after centrifugal 2 minutes, adds 5ml T LSM, and cell numeration back adds T lymphocyte growth culture fluid, and regulating cell density is 1 * 10
6/ ml, the every hole of six orifice plates adds cell 2ml, 37 ℃ of 5%CO
2Cultivate.
(4) the T lymphocyte is cultivated
The T lymphocyte is cultivated and was changed liquid with the 7th day half amount on the 4th day, and the 1ml culture fluid is gently inhaled in every hole, and 1500rpm abandons supernatant after centrifugal 2 minutes, is transferred to foramen primum, 37 ℃ of 5%CO after adding 1ml contains the T lymphocyte growth culture fluid of double somatomedin
2Continue to cultivate.
(5) cell counting
Cell counting can adopt trypan blue counting and high-throughout "diazoresorcinol" viable count method."diazoresorcinol" viable count method is as follows: the PBS solution of preparation 1.2mM "diazoresorcinol" as storage liquid, during use is diluted to storage liquid 200 μ M behind the filtration sterilization.Every hole adds cell to be measured or as the allogenic cell 100 μ l through trypan blue counting of standard curve, every group of cell set multiple hole more than 3, adds "diazoresorcinol" solution 20 μ l, reads the 605nm absorbance value, 37 ℃ of 5%CO after the vibration evenly in 96 orifice plates
2Cultivate and read the 605nm absorbance value once more after 30 minutes, both differences are relevant with the logarithm of cell density.
8. the calcium alginate microcapsule immune effect is measured
(1) cytophagy experiment
Dendritic cell is cultivated the calcium alginate microcapsule that added the 0.6mg/ml embedded quantum dots on the 6th day, 37 ℃ of 5%CO
2Cultivated respectively 0.5 hour and 12 hours, laser confocal microscope is observed.
(2) Flow cytometry dendritic cell surface antigen
After cultivating the 6th day dendritic cell and different stimulated thing and stimulating dendritic cell results after 14 hours, PBS cleans 2 times, adds mouse anti human CD86 respectively, and HLA-DR and the contrast of corresponding homotype were hatched 30 minutes, and flow cytometer detected after PBS cleaned 3 times.
(3) T lymphocyte proliferation assay
Dendritic cell is cultivated and was added different stimulated thing, 37 ℃ of 5%CO on the 6th day
2Cultivate after 14 hours, twice harvesting of centrifuge washing cultivated altogether with 1: 30,1: 100,1: 300 ratio and T lymphocyte respectively, establishes 3 multiple holes for every group, and other establishes one group of T lymphocyte that does not add dendritic cell as blank.37 ℃ of 5%CO
2Cultivate after 5 days the "diazoresorcinol" method and measure and respectively organize cell density, calculate stimulation index: stimulation index (SI)=cell mixing group cell density/do not add dendritic cell T lymphocyte group cell density.
(4) ELISA detects the expression of cell conditioned medium TNF-α and IL-12
Dendritic cell is cultivated and was added different stimulated thing, 37 ℃ of 5%CO on the 6th day
2Cultivate to gather in the crops after 14 hours and respectively organize cell, ELISA detects the expression of cell conditioned medium TNF-α and IL-12.
(5) T lymphocyte killing experiments
Get 293 cells and the HCT-8 cell adds 96 orifice plates respectively, it is 1 * 10 that every hole adds density
5/ ml cell 100 μ l add not the T lymphocyte of dendritic cell stimulation on the same group, 37 ℃ of 5%CO with 1: 30 ratio
2Cultivated 5 days.The "diazoresorcinol" method is measured and is respectively organized cell density.
II. embodiment
1. ultramicroscope morphologic observation
TEM photo such as Fig. 1 of calcium alginate microcapsule.Calcium alginate microcapsule particle size distribution such as Fig. 2.
Phycoerythrin coupling result shows that the load ratio of protein in calcium alginate microcapsule is 69.3%.
The interaction of embodiment 2. calcium alginate microcapsules and dendritic cell
(1) dendritic cell engulfing to calcium alginate microcapsule
At the calcium alginate microcapsule that in immature dendritic cell, added quantum dot embedding labelling on the 6th day that dendritic cell is cultivated, carry out laser confocal microscope and observe, the result is as shown in Figure 3.After 30 minutes, calcium alginate microcapsule enters in the dendritic cell, and it is discrete to distribute; After 12 hours, capsule obviously is positioned specific region in the cell.
(2) calcium alginate microcapsule is to the influence of maturing dendritic cell differentiation
Cultivating the 6th day adds calcium alginate microcapsule respectively in immature dendritic cell, dosage level is 0.6 and 1.2mg/ml (dry weight), with 10 μ g/ml LPS as positive control, not add any stimulus object group as negative control.
LPS as positive control can promote maturing dendritic cell by Toll sample receptor signal approach, is to use stimulus object the most widely.After LPS stimulated, dendritic cell surface costimulatory molecules CD80, CD86 and MHC-II quasi-molecule raised, and Th1 cytokines TNF-α, IFN-γ and IL-12 secretion showed increased stimulate T lymphopoiesis ability to improve, and activate specific immune response.
Stimulate to cultivate after 14 hours dendritic cell surface antigen CD86 and HLA-DR are done flow cytometry, dash area is the homotype results of comparison.Two kinds of dosage calcium alginate microcapsules all can promote the expression of dendritic cell surface C D86 and HLA-DR (as Fig. 4), and present the dosage correlation effect.When dosage reached for 1.2mg/ml, dendritic cell surface HLA-DR expression rate was 96.5%, and the CD86 expression rate is 88.9%, and both are all near positive control.The high expressed explanation calcium alginate microcapsule of MHC-II quasi-molecule HLA-DR and costimulating factor CD86 has the effect that stimulates maturing dendritic cell.
Utilizing the "diazoresorcinol" method to detect from the lymphocytic propagation result of body T shows, stimulation ratio (dendritic cell: from body T lymphocyte) is 1: 300 o'clock, and the coupled product of 200 μ g/ml BSA and 0.6mg/ml calcium alginate microcapsule and solubility 200 μ g/ml BSA contrast induce dendritic cell T lymphocyte stimulation indices to be respectively 1.41 ± 0.07 and 1.18 ± 0.04; The stimulation ratio is 1: 100 o'clock, and stimulation index is respectively 1.51 ± 0.07 and 1.26 ± 0.04; The stimulation ratio is 1: 30 o'clock, and stimulation index is respectively 1.68 ± 0.05 and 1.51 ± 0.05 (Fig. 5).Therefore, BSA is through the calcium alginate microcapsule load, and its inductive dendritic cell stimulation index is higher than simple BSA (P＜0.01 is all arranged), can improve dendritic cell and stimulate T lymphocyte efficient; By contrast, concentration is that the unloaded calcium alginate microcapsule of 0.6mg/ml is that 1: 300,1: 100,1: 30 stimulation index is respectively 0.97 ± 0.06,0.98 ± 0.05,0.97 ± 0.04 corresponding to the stimulation ratio, and failing stimulates from body T lymphopoiesis (P＞0.05).
The immunological adjuvant effect of embodiment 4. calcium alginate microcapsules
Select recombinant C EA albumen as target protein, detect with its with the calcium alginate microcapsule coupling after the capsular immunological adjuvant effect of formation.CD86 expression such as Fig. 6, unloaded microcapsule, CEA and load have the microcapsule of CEA all to show the stimulation ability, but clearly, the coupling load have the stimulation of the microcapsule of CEA can the unloaded microcapsule of force rate and the unloaded capsule mixture of CEA/ all significantly stronger.The HLA-DR expression draws similar conclusion.
Collection is respectively organized dendritic cell and is cultivated supernatant after 14 hours, and ELISA detects IL-12 and TNF-α concentration in the supernatant, result such as Fig. 8 and Fig. 9.Immature dendritic cell (negative control) the cell conditioned medium IL-12 content 20 ± 3pg/ml that does not add stimulus object, TNF-alpha content 35 ± 4pg/ml; Add LPS (positive control) cell conditioned medium IL-12 content 225 ± 17pg/ml, TNF-alpha content 315 ± 33pg/ml; 0.6mg/ml microcapsule stimulated cells supernatant IL-12 content 31 ± 15pg/ml, TNF-alpha content 152 ± 9pg/ml; 100 μ g/ml recombinant C EA albumen stimulated cells supernatant IL-12 content, 46 ± 16pg/ml, TNF-alpha content 11 ± 3pg/ml; 100 μ g/ml recombinant C EA albumen and microcapsule mixture stimulated cells supernatant IL-12 content 34 ± 20pg/ml, TNF-alpha content 10 ± 3pg/ml; Recombinant C EA albumen and microcapsule conjugate stimulated cells supernatant IL-12 content 29 ± 16pg/ml, TNF-alpha content 125 ± 18pg/ml.No matter promptly do not compare with adding stimulus object (negative control), be free recombinant C EA albumen or calcium alginate microcapsule, or both mix or coupling all can't improve the ability that dendritic cell is secreted IL-12; But, calcium alginate microcapsule can significantly be induced and be improved dendritic cell TNF secretion-α, and in its coupling can also keep after the recombinant C EA albumen, the prompting calcium alginate microcapsule induces maturing dendritic cell to realize by inducing the latter to express TNF-α.
Be the immunological adjuvant effect of calcium alginate microcapsule after the further check recombinant C EA load, designed the different stimulated thing and induced the experiment that dendritic cell and allosome T lymphocyte are cultivated altogether behind the maturing dendritic cell that the result as shown in figure 10.T lymphocyte single culture is after 5 days, and cell density is 9.3 ± 3.2 * 10
5/ ml, not adding stimulus object dendritic cell and T lymphocyte, to cultivate back T lymphocyte density altogether be 13.4 ± 0.4 * 10
5/ ml; Microcapsule group cell density is 14.2 ± 3.1 * 10
5/ ml; Recombinant C EA protein groups cell density is 21.1 ± 2.9 * 10
5/ ml; Mixture group cell density is 19.3 ± 3.1 * 10
5/ ml; Conjugate group cell density is 25.0 ± 1.9 * 10
5/ m.Compare with recombinant C EA protein mixture with capsule, conjugate stimulates allosome T lymphopoiesis ability stronger.
Stimulate the effect of specific CTL killing tumor cell for the check calcium alginate microcapsule, the human embryo kidney (HEK) 293 cell action effect cells that the inventor selects human colon cancer cell strain HCT-8 and no CEA to express: the HCT-8 cell has adhesiveness, adherent growth form class epithelioid cells is considered to the main secretory cell of CEA.With initial concentration 1 * 10
5/ ml with not on the same group the T lymphocyte that stimulates of dendritic cell (the T lymphocyte: the effector lymphocyte) cultivate altogether, carry out T lymphocyte killing experiments, result such as Figure 11 and Figure 12 showed with 1: 30.
In the T lymphocyte killing experiments of 293 cells, T lymphocyte group (negative control) cell density that not adding the inductive dendritic cell of stimulus object stimulates is 7.9 ± 1.1 * 10
6/ ml; LPS group (positive control) cell density is 7.9 ± 1.7 * 10
6/ ml; Microcapsule group cell density is 8.8 ± 1.0 * 10
6/ ml; Recombinant C EA protein groups cell density is 7.9 ± 0.5 * 10
6/ ml; Mixture group cell density is 8.8 ± 0.6 * 10
6/ ml; Conjugate group cell density is 7.7 ± 1.2 * 10
6/ ml.Each organizes cell density does not have marked difference, i.e. recombinant C EA albumen or calcium alginate microcapsule or both mixture, and stimulus object induces the immunoreation of stimulation that 293 cells are not all had significant fragmentation effect (P＞0.05).
In the T lymphocyte killing experiments of HCT-8 cell, HCT-8 group (negative control I) cell density is 2.2 ± 0.7 * 10
5/ ml; Pure T lymphocyte group (negative control II) cell density is 2.1 ± 0.5 * 10
5/ ml; Immature dendritic cell group (negative control III) cell density is 2.0 ± 0.4 * 10
5/ ml; LPS group (positive control) cell density is 0.5 ± 0.1 * 10
5/ ml; Microcapsule group cell density is 1.8 ± 0.5 * 10
5/ ml; Recombinant C EA protein groups cell density is 1.6 ± 0.5 * 10
5/ ml; Mixture group cell density is 1.5 ± 0.3 * 10
5/ ml; Conjugate group cell density is 0.4 ± 0.1 * 10
5/ ml.Different with 293 cell killing experimental results, though simple T lymphocyte and the inductive T lymphocyte of immaturity dendron shape do not have obvious lethal effect (P＞0.05) to the HCT-8 cell, LPS can significantly increase the fragmentation effect of T lymphocyte to the HCT-8 cell.Simultaneously, but the mixture specificity of simple recombinant C EA albumen or recombinant C EA albumen and calcium alginate microcapsule improves the lymphocytic fragmentation effect of T, and this fragmentation effect is more remarkable after recombinant C EA albumen and calcium alginate microcapsule coupling.Be that the lymphocytic specific killing effect of the raising T more much higher than both mixture (p＜0.05) is arranged after recombinant C EA albumen and the calcium alginate microcapsule coupling.
The antigenicity and the immunological adjuvant effect of embodiment 5 alginic acid barium microcapsules
Method described in the employing ZL200310108684.9 has prepared alginic acid barium microcapsule.
Adopt as described above similarly method, the coded sequence of alpha-fetoprotein (AFP) is building up among the carrier pcDNA-3, the recombinant vector that obtains is transformed 293 cellular expressions obtain AFP albumen, and purification.
Adopt similar as described above NHS/EDAC peptide bond building-up reactions method,, obtain coupled product AFP albumen and the coupling mutually of alginic acid barium microcapsule.
Check the antigenicity of the proteic alginic acid barium of this coupling AFP microcapsule, and with unloaded calcium alginate microcapsule and simple AFP albumen relatively, find the inductive dendritic cell stimulation index of coupling AFP proteic alginic acid barium microcapsule apparently higher than simple AFP (P＜0.01), can improve dendritic cell and stimulate T lymphocyte efficient; By contrast, unloaded alginic acid barium microcapsule fails to stimulate from body T lymphopoiesis (P＞0.05).
Check the stimulation specific CTL of the proteic alginic acid barium of this coupling AFP microcapsule to kill and wound the effect of carrying the antigenic tumor cell of AFP, and with unloaded calcium alginate microcapsule and simple AFP albumen relatively, found that the proteic alginic acid barium of coupling AFP microcapsule can significantly improve the lymphocytic specific killing effect of T.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (1)
1. the purposes of a biological polyoses microcapsule is used for and carcinoembryonic antigen coupling mutually, and preparation promotes the T lymphopoiesis or improves the T lymphocyte to expressing the material of described antigenic cell-specific kill capability by inducing maturing dendritic cell;
Described biological polyoses microcapsule is to adopt following method preparation: 240mg Span65 is dissolved in the 10ml dichloromethane, adds the 1ml w/v and be 1% sodium alginate soln, and adding petroleum ether 9ml again, to regulate water identical with oil phase proportion; Ultrasonication formed emulsion in 8 minutes; Ultrasonication slowly dripped glacial acetic acid 600 μ l in the time of 5 minutes; Add dichloromethane 5ml, mixed back 1600rpm centrifugal 15 minutes, syringe is taken out the sub-cloud oil phase, adds dichloromethane 10ml again, fully mixes the back recentrifuge and discards oil phase; Add volume fraction 0.5% acetic acid solution 15ml, ultrasonication 8 minutes drips 20mM CaCl in the time of 5 minutes
2Solution 4ml; Change liquid over to the 100ml beaker, with NaOH regulator solution pH value to 7.4, stirring at room 30 minutes; Change solution over to centrifuge tube, add dehydrated alcohol 10ml, mixed back 2000rpm centrifugal 15 minutes; Abandon supernatant, ultrasonication is 3 minutes behind the adding dehydrated alcohol 15ml; Washed with dichloromethane was 1 time after identical washing process repeated 3 times, absolute ethanol washing 2 times; Solution adds the 6ml buffer, ultrasonic 3 minutes after abandoning supernatant; Being formulated as follows of described sodium alginate soln: take by weighing the 1g sodium alginate and be added in the 100ml ultra-pure water, stir 6 hours to dissolving, adding HEPES is 10mM to final concentration, and using 1.5M NaOH regulator solution pH is 7.4.
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| CN1736484A (en) * | 2004-08-19 | 2006-02-22 | 张阳德 | Method for preparing nanometer particle of galactose sodium alginate-polylysine |
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| CN1736484A (en) * | 2004-08-19 | 2006-02-22 | 张阳德 | Method for preparing nanometer particle of galactose sodium alginate-polylysine |
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