CN100398641C - Dendritic cell culturing method and kit - Google Patents
Dendritic cell culturing method and kit Download PDFInfo
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- CN100398641C CN100398641C CNB2003101079949A CN200310107994A CN100398641C CN 100398641 C CN100398641 C CN 100398641C CN B2003101079949 A CNB2003101079949 A CN B2003101079949A CN 200310107994 A CN200310107994 A CN 200310107994A CN 100398641 C CN100398641 C CN 100398641C
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Abstract
The present invention provides a human dendritic cell culture method, a culture medium and a kit. The method of the present invention is used for culturing dendritic cells and has the characteristics of convenient operation, high proportion for the induction of mononuclear cells into dendritic cells, long time culture without easy pollution, shortened time for the inducing maturation of dendritic cells, etc. The present invention can be used in the basic research and the clinical application research of dendritic cells.
Description
Technical field
The present invention relates to field of biology, relate more specifically to a kind of cultural method, substratum and test kit of human dendritic cell.
Background technology
Dendritic cell (Dendritc Cell, DC) be a kind of special cell of a class that exists in the normal human with powerful angtigen presentation function, be called " the natural adjuvant " of body, can directly absorb, processing and antigen-presenting, stimulating intravital primary tape T cell activation, is " the startup person " of immune response.DC can also promote the propagation and the activation of B cell by direct or indirect mode in addition, the regulation and control humoral immunoresponse(HI); Thereby stimulate the memory T cell activation to induce secondary immune response; , the natural immunity nonspecific with NK interaction influence replied.Therefore, DC is an immune response " perpetrator " in the body.DC is in the key link of immunity, can absorb and processing treatment antigen and transfer the body active specific immunotherapy and react.
There are three big immunologic functions, i.e. immune defense, immunological homeostasis and immunosurveillance in the immunity of human body (immune) system.By immune defense, human body is removed pathogenic micro-organism and other antigenicity foreign matters; By immunological homeostasis, human body is removed damage or old and feeble cell; By immunosurveillance, human body is removed the cell of undergoing mutation or cancerating.Immune these three kinds of functions are for the physiological equilibrium of keeping human body and healthy very important.Under certain condition, immune dysfunction can make human body enter morbid state, as malignant tumour takes place.
Healthy human body is an orderly society by cellularity, and the cell distribution of carrying out specific function is in specific zone.Under the normal circumstances, in this society, when proliferation of cells occurs over just the body needs, renewal as hematopoietic cell, fetal development, injury repairing, immunne response process medium size lymphocyte clone's expansion etc., proliferation of cells is subjected to strict and meticulous regulation and control stop after satisfying the needs of human body.Malignant cell is the illegal cell that is evolved into by normal cell in orderly cell society, and it possesses the feature of two meetings to normal cellularity great bodily injury: 1, unrestricted hyperplasia; 2, outgrowth cell is occupied the manor that belongs to other cell.The hyperplasia of malignant cell occurs in body not to be needed to make normal cell can't carry out normal physiological function cellulous the time, finally destroyed whole cell society.
Studies show that of modern molecular biology, human malignant tumor be the gene structure of human body cell or the result that functional status changes.Proto-oncogene (Proto-oncogene) is the normal gene in the normal cell, and the normal differentiation of its coded product pair cell and hyperplasia are very important.Proto-oncogene (Proto-oncogene) can be converted into oncogene (Oncogene) under certain condition.Textural anomaly of oncogene and overactivity can make normal cell be converted into the tumour cell that cancerates.But the human immune system that the tumour cell that has taken place to cancerate can occur identification and the novel substance of attacking, these novel substances are called as tumour antigen.
Tumour antigen can be divided into two classes: tumour specific antigen and tumor associated antigen.Tumour specific antigen is meant the antigen that only is present in certain tumour cell and is not present in normal cell or other tumor cell surface.This type of antigen can be used the evidence of animal tumor transplant rejection, so claim tumor-specific transplantation antigen again.This antigen can be used as the target spot of immune system recognition and attack, and the immune response that it excites (immune surveillance function) can make whole tumour cell be subjected to the attack of immunocyte and be killed.Tumor associated antigen is meant non-a certain tumour cell antigen peculiar, that also exist on other tumour cell or normal cell.This type of antigenic expression amount obviously increases when tumour takes place.As alpha-fetoprotein (AFP), carcinomebryonic antigen (CEA).This antigen also can be used as the target spot of immune system recognition and attack, and the immune response that it excites (immune surveillance function) can make highly to be expressed the whole tumour cell of this antigen and be subjected to the attack of immunocyte and be killed.
The immune surveillance function of human body depends on the immunocyte with anti-tumor activity of human body, and in these immunocytes, the T lymphocyte is most important.Human T cell can be divided into two classes according to surface marker and function.The first kind is the CD4+T cell, helper cell-1 (Th1) and helper cell-2 (Th2).Second class is the CD8+T cell, i.e. cytotoxic T cell (Tc or CTL).
If will excite the anti tumor immune response of T cell mediated, tumour antigen must (antigen presenting cell be processed to little peptide section (tumor antigen peptide) in APC) at antigen presenting cell.Antigen presenting cell is meant and can absorbs, processing treatment antigen, and a para-immunity cell of T, bone-marrow-derived lymphocyte is given in the antigen presentation that will handle.APC mainly comprises the target cell of mononuclear macrophage, dendritic cell, B cell and endotheliocyte, tumour cell and virus infection etc.
APC is different to the approach of offering of exogenous antigen and endogenous antigen.APC to exogenous antigenic processing treatment and the process of offering is: (1) exogenous antigen is formed phagosome through engulfing or pinocytosis in the APC absorption born of the same parents, and the latter and lysosome merge and form phagolysosome; (2) antigen is degraded to micromolecule polypeptide by proteolytic ferment in phagolysosome inner acidic environment, wherein has the immunogenic antigen peptide that is called; (3) after synthetic MHC II quasi-molecule enters golgi body in the endoplasmic reticulum, carry,, antigen peptide is combined with MHC II quasi-molecule in the vesicle form antigen peptide-MHC II quasi-molecule mixture by merging with phagolysosome by secretory vesicle; (4) this mixture is expressed in the APC surface, can be by corresponding CD4+ helper T cell identification combination.
(endogenous antigen is phalangeal cell self a synthetic antigen to APC to endogenous antigen, as tumour antigen and viral protein antigen etc.) the processing treatment and the process of offering be: endogenous antigen is phalangeal cell self a synthetic antigen, as tumour antigen and viral protein antigen etc.The endogenous antigen processing treatment and the process of presenting are as follows: (1) endogenous antigen can be present in the proteasome in the kytoplasm after generating in cell, i.e. small molecules polymerization polypeptide body (LMP) is degraded into micromolecule polypeptide; (2) micromolecule polypeptide is with after heat shock protein 70/90 combine in kytoplasm, and (TAP) is transported in the endoplasmic reticulum through transporter of antigen peptide, modifies to become by processing to have immunogenic antigen peptide; (3) antigen peptide combines with synthetic MHC I quasi-molecule in the endoplasmic reticulum, forms antigen peptide-MHC I quasi-molecule mixture; (4) latter changes golgi body over to, by secretory vesicle it is transported to the APC surface again, for the combination of corresponding CD8+T cell recognition.
The T cell-mediated immune responses is divided into two classes, and a class is cell-mediated by CD4+Th1, and is another kind of cell-mediated by CD8+Tc.
In process by the CD4+Th1 cell-mediated immune responses, the CD4+Th cell by surperficial TCR-CD3 mixture acceptor molecule and APC surface corresponding antigens peptide-MHC II quasi-molecule mixture specific recognition, combine and produces first activation signal, the adhesion molecule on CD4+Th cell and APC surface generation second activation signal that interacts.Under the effect of above-mentioned two signals, the CD4+Th cell-stimulating is also expressed the acceptor of IL-2, IL-4, IL-12.Meanwhile and the cytokines such as APC synthesis secretion IL-1, IL-12 of CD4+Th cytosis.Under the effect of cytokines based on IL-12, the CD4+Th cell proliferation and differentiation is the CD4+Th1 cell.
The CD4+Th1 cell discharges cytokines such as IL-2, TNF, IFN-γ.IL-2 stimulates the CD4+Th cell proliferation and differentiation, activation NK cell and CD8+Tc cell.The NK cell is the effector cell that a class works in early days in the tumour generation, does not need just energy killing tumor cell of presensitization.TNF vasoactive endotheliocyte makes it expression of adhesion molecules (ICAM-1 etc.), secretion IL-8 and MCP-1.Under the effect of adhesion molecule and chemokine, neutrophil leucocyte in the blood, lymphocyte and monocyte are divided a word with a hyphen at the end of a line, adhere to, are exosmosed, answer in the process in tumour, the synthetic polypeptide antigen is processed the surface that back and self MHC I quasi-molecule combined and be transported to target cell in target cell in target cell (tumour cell).The Tc cell produces first activation signal of Tc cell by the identification of surperficial TCR-CD3 coreceptor and in conjunction with the antigen peptide-MHC I quasi-molecule mixture on target cell surface.At the collaborative stimulation molecule of the normal shortage of tumor cell surface, can not stimulate the CD8+Tc cell to produce second activation signals.In this case, first activation signals can make CD8+Tc cell expressing IL-2 and γ-IFN acceptor.Be activated the acceptor of cytokines such as expression IL-12 after the IL-2 of this kind CD8+Tc cell acceptance activation CD4+T emiocytosis and the γ-IFN effect.If target cell (as APC) surface expression work in coordination with stimulation molecule (B7, ICAM-1, LFA-3), then can corresponding acceptor (CD28 with the CD8+Tc cell surface, LFA-1 LFA-2) in conjunction with two second signals that produce the CD8+Tc cell-stimulatings, makes it express the acceptor of cytokine such as IL-12.Above-mentioned activatory CD8+Tc cell is after accepting effect of cytokines such as cell scavenger cell excretory IL-12 and γ-IFN, and further proliferation and differentiation is a responsiveness Tc cell.Responsiveness Tc cell recognition, combination are expressed the target cell of specific antigens material with its cracking.A Tc cell can kill and wound a plurality of target cells continuously.The pore-forming protein albumen of activated Tc cell expressing, granzyme and Fas part are Tc cell killing target cell important medium, and the former makes target cell that the perviousness cracking take place, and both start the programmed death of target cell the back.Cytotoxic T lymphocyte by the cell immune response CD8+ of T cell mediated is important tumor-killing cell, its kill mechanism has two: one, by the tumour antigen on its antigen receptor tumor cell, and at the auxiliary activation down back of the helper T cell of CD4+ direct killing tumour cell; The 2nd, the cytotoxic T lymphocyte secretion gamma-interferon (IFN-γ) of activatory CD8+, the tumour necrosis factor indirect killing tumor cells of cytokine such as (TNF).
In the world, used the tumour that refusion treats and non-hodgkin lymphoma has been arranged, melanoma, prostate cancer and multiple myeloma.
The U.S. is that research dendritic cell treatment malignant tumour is leading country at most, also.The research group of University of Washington at Seattle has carried out the clinical II phase of the treatment of dendritic cell and has tested to 33 patients with prostate cancer that shifted, the result is that 2 tumour kitchen range all disappears, and 6 former and metastatic tumour kitchen range obviously dwindle.Leading work is being made respectively by two scientific research groups of Stanford Univ USA aspect the treatment of multiple myeloma and B cell lymphoma cell patient's dendritic cell.The dendritic cell that the scientific research group of U.S. Miami University reprints with the specific polypeptide of cervical cancer is the external clone who has induced out the cytotoxic T cell with potent anti-tumor activity.
Germany and other national present Research: the associating group from Germany and Sweden has carried out the treatment of dendritic cell to 16 example melanoma patients in late period, there are 5 examples to obtain certain result of treatment, wherein the 2 routine patients melanoma metastasis that shows as a plurality of organs disappears fully, and 3 examples show as part and disappear in addition.The scientific research group of the Zurich medical university of Switzerland is obtaining progress aspect melanoma patient's the dendritic cell treatment.The research group of Austrian Innsbruck university, the dendritic cell of loading with the renal cell carcinoma cell lysate that shifts has induced the immune response of intensive antitumor cell the renal cell carcinoma patient.
Dendritic cell is applied to anti-infectious immunity and has also obtained certain curative effect, adopts the dendritic cell of antigen sensibilization to feed back therapy for treating hepatitis B, autoimmune disorder etc. all under study for action.
China recent years is used also day by day deeply the research and development of dendritic cell.Adopt the Dendritic Cell-based Vaccine Therapy tumour to obtain certain achievement, National Drug Administration's also official approval dendritic cell treatment tumour at present enters the clinical I phase and studies, can predict, dendritic cell treatment tumour and infectious diseases will form huge market, have the significant social economic benefit.
The cultivation of dendritic cell is one of committed step of dendritic cell basis and clinical study, although formed comparatively sophisticated Dendritic Cells Induced and cultural method at present, has for example had several different methods can obtain human dendritic cell at present.
A kind of method of acquisition dendritic cell commonly used is induced from the CD34+ stem cell in DC differentiation marrow, the Cord blood and is contained more relatively CD34+ cell, after in the microenvironment of GM-CSF and TNF-α, inducing for 2 weeks, can be divided into the ripe DC of CD1a+CD83+HLA-DR+, and has amplification effect, about 10-30 initiator cell amount doubly.Adding IL-4 can increase the purity of DC cell, but the output that can not increase, but Flt-3, SCF or IL-3 can improve the DC cell concentration of results greatly.In the culturing process in early days, there is the DC that is in the propagation phase of sufficient amount to be used for gene transfection; But this method needs a large amount of cytokines, spends higher.
Another kind method is to separate DC in the peripheral blood.Because DC expression specificity antibody not, can be by respectively being that the cell-specific negative antibody is selected DC (removing T, B, NK and monocyte in the peripheral blood mononuclear cell); Perhaps express the characteristic separation activatory DC of CD83, CMRF-44 molecule by ripe DC.Because the density of DC is lower, also can use methyl urografic acid methylglucamine salt gradient centrifugation enrichment DC in addition.From peripheral blood, directly separate DC, fast simple, can directly obtain without the process of external long-term cultivation, and have the activity of stronger homologous stimulus T cell proliferation.
Also there is the people from peripheral blood lymphocytes, to induce DC.It is more clearly to induce understand at present each period of DC differentiation and maturation from monocyte, control easily.GM-CSF and IL-4 can induce the monocyte of CD14+ to break up to DC, cultivate the immature DC that can obtain CD1a+CD83-after 5-7 days, have stronger phagocytic activity.Can promote the DC maturation after adding TNF α, CD40L.Although quantitatively monocyte does not have amplification effect to DC differentiation, to contain a large amount of monocytes in the peripheral blood and can separate by its adherent characteristic, institute thinks that most clinical trials adopt.Monocytic ratio is higher in the peripheral blood after this external application GM-CSF, G-CSF mobilize, and contains the precursor cell of some amount, can obtain propagation.But it is low that shortcoming is a yield, and ripe required time is long.
In sum, the cultural method of present dendritic cell is comparatively complicated, and the reagent of employing is more, and each is inconsistent for standard, and incubation time is longer, is difficult to form easy cultural method.Therefore, many in addition investigators are difficult to turn out at short notice the dendritic cell of quality homogeneous.
Therefore, this area presses for the new method for preparing dendritic cell efficiently, fast of exploitation.
Summary of the invention
Purpose of the present invention just provides a kind of method of dendritic cell and relevant substratum and test kit thereof of preparing efficiently, fast.
In a first aspect of the present invention, a kind of Dendritic Cells Induced factor composition is provided, it contains human interleukin 4 by using, human stem cell stimulating factor and human granulocyte-macrophage colony stimulating factor, and human interleukin 4 by using: human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (1 ± 0.2): (1 ± 0.2): (3 ± 0.5), and human interleukin 4 by using, human stem cell stimulating factor and human granulocyte-macrophage colony stimulating factor account for the 0.1-90% of composition total weight.
In another preference, described human interleukin 4 by using: human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (1 ± 0.1): (1 ± 0.1): (3 ± 0.2).
The present invention also provides a kind of maturing dendritic cell inducible factor composition, it contains human tumor necrosis factor-alpha, human gama-interferon and HUMAN HEAT SHOCK PROTEINS 70, and human tumor necrosis factor-alpha: human gama-interferon: the weight ratio of HUMAN HEAT SHOCK PROTEINS 70 is (1 ± 0.2): (1 ± 0.2): (8 ± 2), and human tumor necrosis factor-alpha, human gama-interferon and HUMAN HEAT SHOCK PROTEINS 70 account for the 0.1-90% of composition total weight.
In another preference, described human tumor necrosis factor-alpha: human gama-interferon: the weight ratio of HUMAN HEAT SHOCK PROTEINS 70 is (1 ± 0.1): (1 ± 0.1): (8 ± 1).
In a second aspect of the present invention, provide a kind of dendritic cell liquid substratum, it contains essential carbon source, nitrogenous source, VITAMIN, mineral substance and the water of mammalian cell growth, and the above-mentioned Dendritic Cells Induced factor composition of the present invention, and human interleukin 4 by using, human stem cell stimulating factor and the total concn of human granulocyte-macrophage colony stimulating factor in substratum are 40-90ng/ml.
A kind of liquid substratum of dendritic cell that is used to induce maturing dendritic cell also is provided, it contains essential carbon source, nitrogenous source, VITAMIN, mineral substance and the water of mammalian cell growth, and the above-mentioned maturing dendritic cell inducible factor composition of the present invention, and human tumor necrosis factor-alpha, human gama-interferon and the total concn of HUMAN HEAT SHOCK PROTEINS 70 in substratum are 40-90ng/ml.
In a third aspect of the present invention, provide a kind of dendritic cell to cultivate test kit, it contains container and is loaded on the above-mentioned Dendritic Cells Induced factor composition of the present invention in the container or contains the liquid substratum of dendritic cell of said composition.
Also provide a kind of dendritic cell to cultivate test kit, it contains container and is loaded on the above-mentioned Dendritic Cells Induced factor composition of the present invention in the container or contains the liquid substratum of dendritic cell of said composition.
In another preference, contain two kinds of inducible factor compositions of the present invention or corresponding substratum in the described test kit simultaneously.
In another preference, contained substratum comprises two kinds of inducible factor compositions and serum-free dendritic cell substratum in the described test kit.
In a fourth aspect of the present invention, a kind of method for preparing dendritic cell is provided, may further comprise the steps:
(a) under 37 ± 1 ℃, the culture condition of 5% carbonic acid gas, cultivated peripheral blood lymphocytes 2-4 days, wherein human interleukin 4 by using, human stem cell stimulating factor and the total concn of human granulocyte-macrophage colony stimulating factor in substratum are 40-90ng/ml in the substratum, and human interleukin 4 by using: human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (1 ± 0.2): (1 ± 0.2): (3 ± 0.5) obtain dendritic cell;
(b) under 37 ± 1 ℃, the culture condition of 5% carbonic acid gas, obtain in the culturing step (a) dendritic cell 2-5 days, wherein human tumor necrosis factor-alpha, human gama-interferon and the total concn of HUMAN HEAT SHOCK PROTEINS 70 in substratum are 40-90ng/ml in the substratum, and human tumor necrosis factor-alpha: human gama-interferon: the weight ratio of HUMAN HEAT SHOCK PROTEINS 70 is (1 ± 0.2): (1 ± 0.2): (8 ± 2), thus make maturing dendritic cell
(c) separate sophisticated dendritic cell.
In another preference, described human interleukin 4 by using: human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (1 ± 0.1): (1 ± 0.1): (3 ± 0.2); And
Described human tumor necrosis factor-alpha: human gama-interferon: the weight ratio of HUMAN HEAT SHOCK PROTEINS 70 is (1 ± 0.1): (1 ± 0.1): (8 ± 1).
Description of drawings
Fig. 1 is the purifying color atlas of people IL-4.
Fig. 2 is HSP 70 SDS-PAGE figure.
Embodiment
The present invention is the extensive studies discovery through going deep into, and cultivates peripheral blood lymphocytes under the Dendritic Cells Induced combinations of factors of specified proportion, can significantly improve the yield of dendritic cell.In addition, under the maturing dendritic cell inducible factor combination of specified proportion, cultivate dendritic cell, can significantly shorten the required time of maturing dendritic cell.Finished the present invention on this basis.
Prepare sophisticated dendritic cell and divide two stages: promptly form stage and stage of maturity.
The specificity factor combination in the stage that is used to form is human interleukin 4 by using, human stem cell stimulating factor and human granulocyte-macrophage colony stimulating factor.Preferably, human interleukin 4 by using: human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (1 ± 0.2): (1 ± 0.2): (3 ± 0.5),, more preferably be (1 ± 0.1): (1 ± 0.1): (3 ± 0.2).
In the present invention, i.e. (1 ± 0.2): (1 ± 0.2): the ratio of (3 ± 0.5) is represented 0.8-1.2: 0.8-1.2: 2.5-3.5, the rest may be inferred for other ratios.
In a preference, in the mixture that human interleukin 4 by using, human stem cell stimulating factor and human granulocyte-macrophage colony stimulating factor constitute, contain the hGM-CSF600 microgram in every milligram of inducible factor, hIL-4 is 200 micrograms, hSCF200 microgram.
The specificity factor combination that is used for the stage of maturity is human tumor necrosis factor-alpha, human gama-interferon and HUMAN HEAT SHOCK PROTEINS 70.Preferably, human tumor necrosis factor-alpha: human gama-interferon: the weight ratio of HUMAN HEAT SHOCK PROTEINS 70 is (1 ± 0.2): (1 ± 0.2): (8 ± 2) more preferably are (1 ± 0.1): (1 ± 0.1): (8 ± 1).
In a preference, in the mixture that human tumor necrosis factor-alpha, human gama-interferon and HUMAN HEAT SHOCK PROTEINS 70 constitute, contain hTNF α 100 micrograms in every milligram of inducible factor, gamma-interferon 100 micrograms and hHSP70800 microgram
Being used for above-mentioned six kinds of factors of the present invention can buy, and also can prepare with the ordinary method of this area.
Except above-mentioned six kinds of factors, also can contain other materials in above-mentioned two kinds of inducible factor mixtures of the present invention, as additives such as water, sanitas, antioxidants.
The present invention also provides the substratum that contains above-mentioned Dendritic Cells Induced factor composition and/or maturing dendritic cell inducible factor composition.
The present invention also provides and has contained above-mentioned Dendritic Cells Induced factor composition and/or maturing dendritic cell inducible factor composition, or the test kit of corresponding substratum.
A kind of preferred dendritic cell is cultivated test kit and is comprised container, and the human dendritic cell substratum, the Dendritic Cells Induced factor, maturing dendritic cell inducible factor, dendritic cell culture bag, the dendritic cell that lay respectively in each container are preserved pipe, cell transport case.
A kind of preferred human dendritic cell special culture media is a kind of serum free medium that does not contain animal proteinum, and its prescription is as shown in table 3.Certainly, other dulbecco minimum essential medium Dulbeccos that are usually used in dendritic cell also can be used for the present invention.
Major advantage of the present invention is,
(a) significantly improve the yield of dendritic cell.The yield of dendritic cell improves 2-5 doubly than traditional cultural method;
(b) significantly shorten sophisticated time of dendritic cell, maturation time shortens to 3-5 days.
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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The preparation of embodiment 1, human dendritic cell inducible factor
Comprise three kinds as the human dendritic cell inducible factor: rhIL-4, hSCF, hGM-CSF, they can also can prepare according to a conventional method from buying such as companies such as Simga.In the present embodiment, in order to obtain purer IL-4, the escherichia coli fermented broth that contains IL-4 has been carried out following purifying:
(1) recombinant human interleukin--4's purifying
After expressing the colibacillus engineering fermentation culture of IL-4, collect thalline, the back of weighing adds 50mM Tris/HCl, pH=8.0,1mM edta buffer liquid, carrying out ultrasonic bacteria breaking in adding 1: 20 ratio.Centrifugal back collecting precipitation washs twice with the precipitation inclusion body of collecting with the damping fluid that contains 2%Triton-X100, is dissolved in the 50mM Tris/HCl that contains the 5M Guanidinium hydrochloride, and pH=8.0 is in the 1mM edta buffer liquid.Solubilization of inclusion bodies is centrifugal removal precipitation after 2 hours, and supernatant liquor is slowly joined 50mM Tris/HCl, pH=8.0 by 1: 10 (vol/vol), 1mM EDTA, 1mM PMSF, 0.2M Guanidinium hydrochloride, 0.5M arginine, 1mM GSH/0.5mMGSSG renaturation is with in the damping fluid, and at room temperature renaturation is 24 hours.Remove the supernatant liquor of post precipitation, adopt Pellicon ultra-filtration membrane born of the same parents (molecular weight 5000Dal), concentrate and be converted to damping fluid 50mM Tris/HCl, PH=8.0+0.10M NaCl.After HitrapQ-Sepharose anion column chromatography and Sour15 S cation seperation column chromatography purification, the purity of the recombinant human IL-4 that obtains behind the purifying is greater than 98%, endotoxin content＜1EU/mg.
Q-Sepharose anion column chromatography
Chromatography column: HitrapQ-Sepharose FF post 5ml
Chromatography instrument: Akta Explore100 chromatographic system
Buffer A: 50mM Tris/HCl, PH=8.0+0.10M NaCl
Sample preparation: all pre-temperature of all samples and damping fluid is to 4 ℃, and chromatography carries out at 4 ℃
Wash-out and component are collected: after adopting the damping fluid column equilibration, directly go up sample, collect directly and collect not in conjunction with component.
Source15S cation seperation column chromatography
Chromatography column: ReSourceS 1ml post
Chromatography instrument: Akta Explore100 chromatographic system
Buffer A: 50mM PH=4.5 sodium-acetate buffer
Buffer A: 50mM PH=4.5 sodium-acetate buffer+1M NaCl
Sample preparation: sample is gone up in the 50mM PH=4.5 sodium-acetate buffer dilution back that will go up 4 times of volumes of sample adding of step collection
Wash-out: adopt the 50mM PH=4.5 sodium-acetate buffer that contains 1M NaCl to carry out linear gradient elution after going up sample, collect required component.
The IL-4 purifying chromatogram result of purifying as shown in Figure 1.Show the pure product of IL-4 (purity is greater than 99%) that obtained.
(2) preparation of human dendritic cell inducible factor
The IL-4 of as above preparation is mixed by following weight ratio with hSCF, the hGM-CSF of purchase, add 4% recombinant human albumin again, standby after the freeze-drying.
The prescription of table 1 human dendritic cell inducible factor
| Prescription | rhIL-4 | hSCF | hGM-CSF |
| Prescription 1a | 1 | 1 | 3 |
| Prescription 2a | 0.8 | 1.2 | 3 |
| Prescription 3a | 1.2 | 0.8 | 3 |
| Prescription 4a | 1 | 0.9 | 3.5 |
| Prescription 5a | 1.1 | 1.1 | 2.5 |
The preparation of embodiment 2, the ripe inducible factor of human dendritic cell
Comprise three kinds as the ripe inducible factor of human dendritic cell: gamma-interferon, humanTNF-and HSP 70, they can also can prepare according to a conventional method from buying such as companies such as Simga.In the present embodiment, in order to obtain purer Hsp70 and reducing cost, the escherichia coli fermented broth that contains Hsp70 has been carried out following purifying:
(1) recombinant human heat shock protein(HSP)-70 purifying
Extract the total RNA of human dendritic cell with Trizol (Gibco company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA is formed cDNA as template through reverse transcription.According to disclosed Hsp70 sequence synthesized primer thing, encoding sequence by PCR acquisition Hsp70 is inserted into expression vector pQE-9 (Qiagen Inc.Chatsworth, CA) multiple clone site on then, transform available from Qiagen the E.coli bacterial strain of commodity M15/rep4 by name then.According to kalamycin resistance (Kan
r) the screening transformant.
Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD
600) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM, to induce the expression of Hsp70.
HSP70 is the stronger acidic protein of a kind of wetting ability, how to exist with soluble form behind expression in escherichia coli.Behind the broken bacterium of high-pressure homogenization method, the high speed frozen centrifugation is collected supernatant.Behind low-concentration ethanol precipitation, DEAE anionresin, Phenyl-Sepharose hydrophobic chromatography and S200 gel filtration chromatography, the purity of the rhHSP70 of final preparation is reached more than 95%.
The DEAE-Sepharose column chromatography
Chromatography column: DEAE-Sepharose FF (Pharmacia), 2.6 * 10cm
Chromatography instrument: Akta Explore100 chromatographic system
Sample title and processing:
The all pre-temperature of all samples and damping fluid is to 4 ℃, and chromatography carries out at 4 ℃
Buffer A: 50mM Tris/HCl, pH=8.01mM EDTA
Buffer B: 50mM Tris/HCl, pH=8.01mM EDTA+1M NaCl
Elution protocol
Flow velocity 4ml/min
10 column volumes of column equilibration contain the HSP70 component with 20%B liquid wash-out behind the last sample, use 100%B liquid wash-out impurity again after, wash 5 column volumes of chromatography column with 0.5N NaOH solution.
Phenyl-Sepharose HP hydrophobic chromatography
Chromatography column: Phenyl-Sepharose HP (Pharmacia), 2.6 * 10cm
Chromatography instrument: Akta Explore100 chromatographic system
Sample title and processing:
Adding solid ammonium sulfate to final concentration in the component of anion column chromatography gained is 1.5M, leaves standstill centrifugal removal precipitation behind the 30min, and all pre-temperature of sample and damping fluid is to 4 ℃, and chromatography carries out at 4 ℃
Buffer A: 50mM Tris/HCl, pH=7.51mM EDTA+1.5M (NH4) 2SO4
Buffer B: 50mM Tris/HCl, pH=7.51mM EDTA
Elution protocol
Flow velocity 4ml/min
10 column volumes of column equilibration, be washed till ultraviolet absorption value behind baseline values with A liquid behind the last sample, in 20 column volumes, make linear gradient elution, collect the component that contains HSP70 with 0-100%B liquid, use 5 column volumes of 100%B wash-out again, 0.5M sodium hydroxide cleans chromatography column.
Sephacryl S-200 HR column chromatography
Chromatography column: Sephacryl S-200 HR (Pharmacia), 2.6 * 100cm
Chromatography instrument: Akta Explore100 chromatographic system
Elution protocol: flow velocity is 1ml/min, collects required component
Applied sample amount: the component of collecting behind the 25ml drainage column
Collect required component.
The SDS-PAGE result of the Hsp70 of purifying as shown in Figure 2.Detected result shows the pure product of HSP70 (purity is greater than 95%) that obtained.
(2) preparation of the ripe inducible factor of human dendritic cell
The Hsp70 of as above preparation is mixed by following weight ratio with gamma-interferon, the humanTNF-of purchase, add 4% recombinant human albumin again, standby after the freeze-drying.
The prescription of the ripe inducible factor of table 2 human dendritic cell
| Prescription | Gamma-interferon | The humanTNF- | People Hsp70 |
| Prescription 1b | 1 | 1 | 8 |
| Prescription 2b | 0.8 | 1.2 | 8 |
| Prescription 3b | 1.2 | 0.8 | 8 |
| Prescription 4b | 1 | 0.9 | 10 |
| Prescription 5b | 1.1 | 1.1 | 6 |
Inducing of the dendritic cell of embodiment 3, OVA antigen sensibilization
The about 20ml of separation of human peripheral blood mononuclear cell, slowly adding adds the 50ml Falcon centrifuge tube (cell suspension volume equates with the parting liquid volume) of 20ml lymphocyte separation medium in advance, centrifugal 30 minutes of back 400 * g room temperature, draw the interface cell with flat mouthful of Pasteur's dropper, join in the 30ml RPMI1640 substratum, centrifugal 10 minutes of 200 * g washs 3 times, collects cell.
To be made into 4 * 10 with containing the dendritic cell substratum through the mononuclearcell of centrifuge washing
6The cell suspension of/ml plants dendron shape cell culture bags, and the culturing bottle that fills cell is put 37 ℃, 5% carbonic acid gas, cultivates adherent 2 hours in the incubator of saturated humidity.Culture bag is taken out, jiggle ten for several times, non-adherent cell is hanged, discard suspension cell, slowly in bag, add 10ml RPMI1640 substratum again, rock the residual non-adherent cell of flush away gently.
(promptly the total concn of three kinds of Dendritic Cells Induced factors is the 60ng/ml nutrient solution to add the dendritic cell substratum of the 30ml 60ng/ml Dendritic Cells Induced factor in the culture bag that keeps attached cell, the prescription of three kinds of factors sees Table 1), put 5% carbonic acid gas, 37 ℃, continue in the incubator of saturated humidity to cultivate.
Be cultured to the 3rd day, replenishing 20ml in the culture bag contains the dendritic cell fresh culture of the 60ng/ml Dendritic Cells Induced factor (promptly the total concn of three kinds of Dendritic Cells Induced factors is the 60ng/ml nutrient solution, the prescription of three kinds of factors sees Table 2), continue to cultivate 1 day.At the 4th day that cultivates, visible cell became the burr shape, and assembles agglomerating, add OVA antigen 50 μ g/ml, cultivated 6 hours, and replenished maturing dendritic cell inducible factor 15 μ g/ml, continue to cultivate 1 day with the 50ml syringe, blow and beat the culture bag bottom with the 50ml syringe gently in culture bag, part half adherent dendritic cell is hanged, nutrient solution is sucked the 50ml centrifuge tube, centrifugal 10 minutes of 200 * g, collect cell, this cell is the dendritic cell of OVA antigen sensibilization.
The composition of serum-free dendritic cell substratum that is used for present embodiment is as shown in table 3.
The composition (g/L) of table 3 serum-free dendritic cell substratum
| Calcium chloride (CaCl 2) | 116.60 |
| Copper sulfate (CuSO 4·5H 2O) | 0.0013 |
| Iron nitrate (Fe (NO 3) 3·9H 2O) | 0.05 |
| Ferrous sulfate (FeSO 4·7H 2O) | 0.417 |
| Repone K (KCl) | 311.80 |
| Magnesium chloride (MgCl 2) | 28.64 |
| Sal epsom (MgSO 4) | 48.84 |
| Sodium-chlor (NaCl) | 6995.50 |
| Sodium bicarbonate (NaHCO 3) | 2438.00 |
| SODIUM PHOSPHATE, MONOBASIC. (NaH 2PO 4·H 2O) | 62.50 |
| Sodium phosphate dibasic (Na 2HPO 4) | 71.02 |
| Zinc sulfate (ZnSO 4·7H 2O) | 0.432 |
| Glucose | 3151.00 |
| Xanthoglobulin sodium salt (Hypoxanthine-Na) | 2.39 |
| Linolic acid (Linoleic Acid) | 0.042 |
| Thioctic Acid (Lipoic Acid) | 0.105 |
| Phenol red (Phenol red) | 8.10 |
| Carnitine (Putrescine-2HCl) | 0.081 |
| Sodium.alpha.-ketopropionate (Sodium Pyruvate) | 55.00 |
| Thymidine (Thymidine) | 0.365 |
| The L-L-Ala | 4.45 |
| L-arginine hydrochloride (L-Arginine hydrochloride) | 147.50 |
| L-aspartic acid (L-Asparagine-H 2O) | 7.50 |
| L-aspartic acid (L-Aspartic acid) | 6.65 |
| L-halfcystine (L-Cysteine-HCl-H 2O) | 17.56 |
| L-Gelucystine (L-Cystine-2HCl) | 31.29 |
| L-L-glutamic acid (L-Glutamic acid) | 7.35 |
| L-glutaminate (L-Glutamine) | 365.00 |
| G glycine (Glycine) | 18.75 |
| L-Histidine (L-Histidine-HCl-H 2O) | 31.48 |
| L-Isoleucine (L-Isoleucine) | 54.47 |
| L-leucine (L-Leucine) | 59.05 |
| L-lysine hydrochloride (L-Lysine hydrochloride) | 91.25 |
| L-methionine(Met) (L-Methionine) | 17.24 |
| L-phenylamino acid (L-Phenylalanine) | 35.48 |
| L-proline(Pro) (L-Proline) | 17.25 |
| L-Serine (L-Serine) | 26.25 |
| L-Threonine (L-Threonine) | 53.45 |
| L-tryptophane (L-Tryptophan) | 9.02 |
| L-Sodium L-tyrosinate (L-Tyrosine-2Na-2H 2O) | 55.79 |
| L-Xie Ansuan (L-Valine) | 52.85 |
| VITAMIN (VITAMINS) | |
| Vitamin H (Biotin) | 0.0035 |
| D-calcium pantothenate (D-Calcium pantothenate) | 2.24 |
| Choline chloride 60 (Choline chloride) | 8.98 |
| Folic acid (Folic acid) | 2.65 |
| I-inositol (i-Inositol) | 12.60 |
| Niacinamide (Niacinamide) | 2.02 |
| Vitamin B6 hydrochloride (Pyridoxine hydrochloride) | 2.00 |
| Riboflavin (Riboflavin) 0.219 | |
| Thiamine hydrochloride (Thiamine hydrochloride) | 2.17 |
| Vitamin B12 (Vitamin B12) | 0.68 |
| The insulin human | 10 |
| Human transferrin | 5.5 |
| The recombinant human albumin | 0.1 |
| Cholesterol | 0.5 |
| Pluronic F-68 | 1 |
| Dextran | 1M |
| Selenium | 0.005 |
The result:
(a) under the various human dendritic cell inducible factor prescriptions of table 1, the inventive method all can significantly improve the yield of dendritic cell.The yield of final dendritic cell improves 2-5 doubly than traditional cultural method;
(b) under the prescription of the ripe inducible factors of various human dendritic cells of table 2, the inventive method all can significantly shorten the sophisticated time of dendritic cell, the differentiation and maturation of general dendritic cell needs about 7 days, adopts the inventive method and can shorten to 3-5 days (average 4 days).Its important mechanism is to adopt gamma interferon and HSP70 acting in conjunction, can reduce the generation of the dendritic cell SC factor, thereby shortens the culture cycle of dendritic cell.
Inducing of the dendritic cell of embodiment 4, OVA antigen sensibilization
Repeat the step of embodiment 3, difference only is that Dendritic Cells Induced factor total concn becomes 40ng/ml or 90ng/ml (prescription 1a) by 60ng/ml in the culturing process; Perhaps maturing dendritic cell inducible factor total concn becomes 40ng/ml or 90ng/ml (prescription 1b) by 60ng/ml.
The result: under the Dendritic Cells Induced factor total concn of 40ng/ml or 90ng/ml, significantly improve the yield of dendritic cell equally, the yield of final dendritic cell improves 2 times and 6 times than traditional cultural method;
Under maturing dendritic cell inducible factor total concn at 40ng/ml or 90ng/ml, equally all can significantly shorten the sophisticated time of dendritic cell, be 5 days or 4 days.
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 (6)
1. Dendritic Cells Induced factor composition, it is characterized in that, it contains human interleukin 4 by using, human stem cell stimulating factor and human granulocyte-macrophage colony stimulating factor, and human interleukin 4 by using: human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (0.8～1.2): (0.8～1.2): (2.5～3.5), and human interleukin 4 by using, human stem cell stimulating factor and human granulocyte-macrophage colony stimulating factor account for the 0.1-90% of composition total weight.
2. composition as claimed in claim 1 is characterized in that, described human interleukin 4 by using: the human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (0.9～1.1): (0.9～1.1): (2.8～3.2).
3. the liquid substratum of a dendritic cell, it contains essential carbon source, nitrogenous source, VITAMIN, mineral substance and the water of mammalian cell growth, it is characterized in that, it also contains the described Dendritic Cells Induced factor composition of claim 1, and human interleukin 4 by using, human stem cell stimulating factor and the total concn of human granulocyte-macrophage colony stimulating factor in substratum are 40-90ng/ml.
4. a dendritic cell is cultivated test kit, it is characterized in that, it contains container and the described Dendritic Cells Induced factor composition of claim 1 or the liquid substratum of the described dendritic cell of claim 3 that are loaded in the container.
5. a method for preparing dendritic cell is characterized in that, may further comprise the steps:
(a) under 36～38 ℃, the culture condition of 5% carbonic acid gas, cultivated peripheral blood lymphocytes 2-4 days, wherein human interleukin 4 by using, human stem cell stimulating factor and the total concn of human granulocyte-macrophage colony stimulating factor in substratum are 40-90ng/ml in the substratum, and human interleukin 4 by using: human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (0.8～1.2): (0.8～1.2): (2.5～3.5) obtain dendritic cell;
(b) under 36～38 ℃, the culture condition of 5% carbonic acid gas, obtain in the culturing step (a) dendritic cell 2-5 days, wherein human tumor necrosis factor-alpha, human gama-interferon and the total concn of HUMAN HEAT SHOCK PROTEINS 70 in substratum are 40-90ng/ml in the substratum, and human tumor necrosis factor-alpha: human gama-interferon: the weight ratio of HUMAN HEAT SHOCK PROTEINS 70 is (0.8～1.2): (0.8～1.2): (6～10), thus make maturing dendritic cell
(c) separate sophisticated dendritic cell.
6. method as claimed in claim 5 is characterized in that, described human interleukin 4 by using: the human stem cell stimulating factor: the weight ratio of human granulocyte-macrophage colony stimulating factor is (0.9～1.1): (0.9～1.1): (2.8～3.2); And
Described human tumor necrosis factor-alpha: human gama-interferon: the weight ratio of HUMAN HEAT SHOCK PROTEINS 70 is (0.9～1.1): (0.9～1.1): (7～9).
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| CN105670994A (en) * | 2016-02-28 | 2016-06-15 | 深圳爱生再生医学科技有限公司 | DC (dendritic cell) inducer and application thereof |
| CN106282111A (en) * | 2016-08-30 | 2017-01-04 | 孟玲 | A kind of Dendritic Cells Induced factor composition |
| CN113249322A (en) * | 2021-05-18 | 2021-08-13 | 蓝莲(杭州)生物科技有限公司 | Culture solution and culture method for DC cells |
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| CN1408850A (en) * | 2001-09-18 | 2003-04-09 | 王虹 | Immortal technology for human blood dentritic cell |
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| CN1408850A (en) * | 2001-09-18 | 2003-04-09 | 王虹 | Immortal technology for human blood dentritic cell |
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