CN108642013A - From being detached in Cord blood after CD34 candidate stem cells expand culture, induction prepares Dendritic Cells method to one kind on a large scale - Google Patents

From being detached in Cord blood after CD34 candidate stem cells expand culture, induction prepares Dendritic Cells method to one kind on a large scale Download PDF

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CN108642013A
CN108642013A CN201810411706.5A CN201810411706A CN108642013A CN 108642013 A CN108642013 A CN 108642013A CN 201810411706 A CN201810411706 A CN 201810411706A CN 108642013 A CN108642013 A CN 108642013A
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dendritic cells
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王毅
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Avic (ningxia) Biological Ltd By Share Ltd
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Abstract

The invention belongs to cell therapy fields, it is related to the isolating hematopoietic stem cells from discarded Cord blood to induce into DC Dendritic Cells, for a kind of new application of cell therapy, more particularly to from being detached in Cord blood after CD34 candidate stem cells expand culture, induction prepares Dendritic Cells method on a large scale.Pass through the research of the induction preparation Dendritic Cells after detaching the expansion culture of CD34 candidate stem cells in Cord blood.Karyocyte is mainly detached from Cord blood using Wealthlin cell processing kits, then magnetic bead separation kit isolated CD34+ candidate stem cells from karyocyte are utilized, amplification culture medium is added to the continuous amplification cultivation of CD34+ candidate stem cells 31 days, CD34 cells are harvested in amplification procedure, inducing culture is added to induce precursor, to prepare DC Dendritic Cells on a large scale.This method can largely prepare DC Dendritic Cells, solve from Cord blood detach monocyte induction DC Dendritic Cells quantity it is few, the low problem of purity.

Description

One kind induces on a large scale after detaching the expansion culture of CD34 candidate stem cells in Cord blood Prepare Dendritic Cells method
Technical field
The invention belongs to cell therapy field, it is related to the isolating hematopoietic stem cells from discarded Cord blood and induces into DC dendrons Shape cell is used for a kind of new application of cell therapy, more particularly to detaches CD34 candidate stem cells from Cord blood and expand culture Extensive induction prepares Dendritic Cells method afterwards.
Background technology
Dendritic Cells was found in 1973 by Canadian scholar STEINMAN, was that function known today is strongest Antigen presenting cell stretches out many dendron samples or pseudo- Microfilament when its maturation due to gains the name.Dendritic Cells (DENDRITICCELL, DC) plays an important role in primary link-antigen of immune response is offered, and is generally acknowledged at present body The most powerful professional antigen presenting cells (ANTIGEN-PRESENTINGCELL, APC) of interior function.It can activate tranquillization type T Cell is primarily involved in the humoral immune reaction that cellular immunity and T cell rely on, plays a crucial role in anti-tumor immune response. Under inverted phase contrast mirror, Dendritic Cells suspension growth differs in size, form irregular, stretches out the thick of different number around Carefully differ, the cytoplasmic processes that form is totally different, what is had is short and thick, and end is in the pseudopodium sample of blunt circle；Have thin and grow, form many ginsengs The dendroid of poor uneven multiple-limb, surface are in burr sample；Also the two having has concurrently.Cell process is ceaselessly put in culture solution It moves or quickly and continuously stretches, its direction and form is caused to be constantly changing.Core is big and irregular, is often in distorted shape, there is refractive power With apparent beating.There is dense granule in cytoplasm, is mitochondria；Occasionally with the presence of the strong fat drips of refractivity.
The generation of DC is in two stages：It is divided into immature DC stage and immature DC by environmental stimuli (such as from progenitor cells Bacterial product, tumor cell lysate and and various cell factors) the differentiation and maturation stage.
Internal DC originates from pluripotential hemopoietic stem cell.By sources its differentiation pathway is divided into two：1. myeloid stem cell is in GM- It is divided into DC, referred to as marrow sample DC, also referred to as DCL under the stimulation of the CSF factors, has common precursor thin with monocyte and granulocyte Born of the same parents；Including Langerhans cell, the DCS etc. of mesothelium (or corium) DCS and monocyte derived.2. dry from lymph sample Cell, referred to as lymph sample DC or Plasmacytoid DC, i.e. DC2 have common precursor with T cell and NK cells.Dendron shape is thin Born of the same parents (DC) although lazy weight peripheral blood mononuclear cells 1%, surface have abundant antigen presenting molecule (I Hes of MHC- MHC- II), costimulating factor (CD80/B7-1, CD86/B7-2, CD40, CD40L etc.) and adhesion factor (ICAM-1, ICAM- 2, ICAM-3, LFA-1, LFA-3 etc.), it is powerful professional antigen presenting cell (APC).At present to DC subgroups and differentiation The research method that is mainly derived from vitro culture, the classification of natural DC subgroups is still up for further studying in vivo.
The development of DC is divided into ripe and mezzanine level, and there are the two different biological characteristics to seek peace cell phenotype.Normally In the case of, internal most of DC cells are in mezzanine level, are distributed widely in each peripheral tissues of whole body, immature DC can Express I/class Ⅱmolecules of MHC, IGGFC receptors (FC Γ R), C3B receptors (C3BR) and certain toll-like receptors such as TLR2, TLR4 And TLR9, height expression phagocytosis associated receptor (FC receptors, complement receptors, mannose receptor), without expression or low expression costimulation Molecule and adhesion molecule (CD14, CD54, CD40, CD80).Immature DC intake, working process antigenic capacity are strong, and offer to resist Original excitation immune response ability is weak.By a series of processes such as antigen uptake, inflammatory factor activation, the immature state transformations of DC For mature cell, ripe DC then high expression major histocompatibility complexs The costimulations such as (MAJORHISTOCOMPATIBILITYCOMPLEX, MHC) class Ⅱmolecule and CD54, CD40, CD80, CD86 point Son and adhesion molecule, CD83, CD25 are the characteristic mark of maturation DC.It is absorbed, working process antigenic capacity is weak, and is offered Antigen, startup immune response ability are strong.The maturation of DC with migrating, after absorbing antigen in peripheral tissues, passes through simultaneously Extend dendron shape protrusion, changes the modes such as chemokine receptor expression and enter maturation in lymph node and lymphatic vessel, and activated t cell Reaction.
DC is that the important professional antigen of a major class is in delivery cell APC, though negligible amounts in vivo, its antigen offers energy Power is much stronger than other APC.Immature DC has extremely strong endocytosis ability, by film shrinkage and forms big vesica, is formed in liquid phase Effect is gulped down, the antigen of extremely low concentration can be made to be presented, also endocytosis can be carried out to mannosylated antigen by receptor-mediated.It is taken the photograph by DC The antigen molecule taken handles by II classpaths of MHC, is processed into antigen polypeptide, then compound with MHC class Ⅱmolecules-Antigenic Peptide Object form presents on the surfaces DC, and can maintain the immune stimulating function of long period (24~48H).Immature DC, which has, to be gulped down It is fast to drink speed, gulps down the big feature of drink amount, is that the main of internal antigen takes the photograph taker.
After absorbing antigen, immature DC gradually changes to ripe DC, and shows very strong immune activation ability.DC Dendritic Cells is in addition to the first signal for providing MHC class Ⅱmolecules-antigenic peptide complexes, DC cells also being total to by its height expression Stimulation molecule (CD80/B7-1, CD86/B7-2, CD40 etc.) provides sufficient second signal for T cell, to promote swashing for T cell It is living.And DC cells can be promoted the activation of NK cells with secrete cytokines and enhance NK cytotoxicity, and DC also participates in B The growth of cell and differentiation and antibody tormation.
DC is the cell that can uniquely activate tranquillization type T cell to generate primary immune response in vivo, and can be put by dotted Big stimulation, activation T cell proliferation.Therefore, in inducing T cell activation or tolerance course, DC plays highly important effect. DC plays an important role to selection course of the T cell in thymus gland as important thymic stroma cell.The height expression of the surfaces DC MHC-II class molecules, the own MHC molecules on double positive thymocytes surfaces identification DC after TCR rearrangements, by the positive selects Survival；In Solid phase, the self peptide-MHC compounds on the surfaces T cell identification DC pass through Apoptosis mechanism by Solid phase And it is eliminated.DC in thymus gland is interacted by ICAM-1, CD40L, CD30 and the FAS expressed with T cell, participates in T cell To the central tolerance of self peptide.
DC Dendritic Cells antineoplastic immune flows：1. the intake and presentation of tumor associated antigen；2. activating tumour related T cells with antigenic specificity；3. guiding tumor associated antigen specific T-cells to tumor locus, tumour cell is killed.Body is anti-swollen The immune immune response for relying primarily on cytotoxic T lymphocyte (CYTOTOXICTLYMPHOCYTE, CTL) of tumor is swollen to kill Oncocyte, and CTL itself does not identify complete tumour antigen molecule, can only be by the tumour antigen that is presented by MHC molecule Polypeptide carrys out tumor cell and kills.In most of malignant tumour, the MHC Antigenic Peptides of tumor cell surface, costimulation point Son and expression of adhesion molecule are relatively low, cannot effectively inducing T cell activation, therefore need the synergistic effect of antigen presenting cell.
Major part DC is in non-maturity state in human body, expresses low-level costimulating factor and adhesion factor, external to swash The ability for sending out mixed lymphocytes breeder reaction of the same race is relatively low, but immature DC has extremely strong antigen phagocytic activity, is absorbing Antigen (including processing in vitro) or while being stimulated by certain factors, are divided into ripe DC, and ripe DC expression is high-caliber total Stimulating factor and adhesion factor.DC migrates into secondary lymphatic organ during maturation, by the peripheral tissues of contact antigen, It is contacted with T cell and excites immune response.In addition to inducing cellular immune, DC can also enhance humoral immunity:：On the one hand, DC is logical The generation for promoting antigentic specificity CD4+TH is crossed, antibody tormation is promoted；On the other hand, DC high expresses FCR, CR, can make DC film tables Face can adhere to a certain amount of antigen for a long time, stimulate memory B cell by long-time, B cell is made to maintain immunological memory, promote static B cell expresses B7 molecules, makes it have antigen presentation function, directly adjusts B cell growth by discharging soluble factor and divides Change.
The antitumor mechanism of DC Dendritic Cells is as follows：1. DC Dendritic Cells can high expression I classes of MHC- and MHC- II Class molecule, MHC molecule capture the tumour antigen of processing with it and are combined, and form peptide-MHC molecule compound, and submission is to T cell, To start the reaction of MHC-I class Restricted CTLs and the restrictive CD4+THL reactions of II classes of MHC-.Meanwhile DC also passes through its high table The costimulatory molecules (CD80/B7-1, CD86/B7-2, CD40 etc.) reached provide second signal necessary to T cell activation, start Immune response.2. DC is combined with T cell can largely secrete IL-12, IL-18 activation T cell Proliferations, induction CTL is generated, main TH1 type immune responses are led, tumor clearance is conducive to；And it being capable of activated NK, activation perforin P granzyme Bs and FASL/FAS Jie The approach enhancing NK cytotoxicity led；3. DC secretes chemotactic factor (CF) (CHEMOTACTICCYTOKINES, CCK) single-minded chemotactic Primary tape T cell promotes T cell aggregation, enhances the excitation of T cell.Keep effect T cells in tumor locus long-term existence, Tumor vessel may be influenced by discharging certain anti-angiogenesis substances (such as IL-12, IFN- Γ) and preceding angiogenesis factor Formation.Above-mentioned CCK further activates DC in a manner of positive feedback paracrine, raises the expression of IL-12 and CD80, CD86；Together When DC also directly to CD8+T presented by cells Antigenic Peptides, make CD8+T cell activations under the CD4+T cells auxiliary of activation, CD4+ and CD8+T cells can also enhance antitumor immunity of organism response further by secrete cytokines or direct killing.
DC Dendritic Cells is distributed widely in each internal organs of the whole body other than brain, only accounts for the 1% of peripheral blood mononuclear cells, by In internal negligible amounts, scientific research and clinical needs cannot be met.So external extensive Fiber differentiation DC Dendritic Cells skills The progress of art makes it possible DC vaccine antineoplastons.
DC Dendritic Cells currently used for treatment is mostly from marrow or peripheral blood CD34+ hematopoietic progenitor cells and peripheral blood Monocyte, wherein the DC with cells of monocytic origin is most widely used.The method for obtaining DC Dendritic Cells has；1. will derive from Marrow or peripheral blood CD34+ hematopoietic progenitor cells are common by the SCF factors and the GM-CSF factors and tumor necrosis factor Α in vitro Culture, obtains a large amount of DC.2. by being induced under the GM-CSF factors and IL-4 collective effects from the monocyte detached in peripheral blood It is divided into immature DC, and tumour antigen is added and tumor necrosis factor Α Co stituations are divided into ripe DC.In addition, grinding Study carefully and prepare DC using the mononuclearcell of derived from cord blood, with cord blood mononuclear cells, in the GM-CSF factors, IL-4 and bleeding of the umbilicus It is divided into immature DC under Cytokine, promotes bleeding of the umbilicus list within a short period of time with umbilical cord blood plasma substitution tumor necrosis factor Α A monocyte differentiation makes full use of the cell factor being rich in bleeding of the umbilicus at DC, reduces the dosage of recombinant cytokine, provides One relatively easy, efficient approach that DC is prepared by Cord blood.
Using specific polypeptide substance known to tumour, as antigen load object, targeting is preferable, and it is anti-to can avoid autoimmunity The generation answered, it is safer.The prostate-specific antigen such as Zhuan Zhixiang, prostate-specific membrane antigen and prostatic acid phosphatase Enzyme polypeptide combines the self DC of sensitization and treats hormone-refractory prostate cancer, as a result significantly improves patient immune function, effectively swashs Specific T cell immunity response is sent out, the state of an illness is under control and mitigation.
Use intact tumor cells as antigen load DC Dendritic Cells：The tumour cell of inactivation is irradiated through radioactive ray, is swollen Oncocyte multigelation supernatant or tumour cell freeze thawing caudacoria fragment or ultrasonication method prepare the protein extraction of tumour cell Extremely weak antineoplastic immune effect is all only generated after the direct vivo immunization of object, is seldom individually used for clinic as tumor vaccine. But if impacting primed dendritic shape cell in vitro with them, these have loaded the dendritic cell-based tumor vaccines of cellularity tumour antigen It feeds back in vivo, then can generate stronger antitumor curative effect.Due to its not yet clear specific tumour of current most of tumor types Antigen, and full Cell tumor antigen is easily obtained and prepares, and in the case that without clear tumour antigen, full cellular antigens are loaded In Dendritic Cells, Dendritic Cells is allowed to go to extract kinds of tumors antigen, process and present, to induce for difference The ctl clone of antigenic determinant, the method that can yet be regarded as easily and effectively are suitble to clinical application.
Antigen gene imports DC Dendritic Cells：The gene of tumour specific antigen is imported into dendron shape by viral vectors In cell, specific tumor antigen is allowed to be expressed in DC Dendritic Cells, and then stimulates activation T cell.The method of importing is main It is by retrovirus, adenovirus etc..There is research to confirm, is transfected using the recombinant virus for carrying tumour specific antigen gene The vaccine-induced CTL abilities of DC higher than the DC that Antigenic Peptide or holoantigen load.Its reason may be that the expression of antigen after transfecting exists Inside DC Dendritic Cells, endogenous antigen is more advantageous to processing of the DC Dendritic Cells to antigen.Although virus coat protein can As immune primary stimuli DC activated dendritic cells, virus specific immunity response is caused to generate, but virus has with host cell The danger of integration, this method are yet to be completed.It replaces tumour antigen to stimulate Dendritic Cells with tumour cell MRNA, core can be passed through Acid amplification, amplification is to sufficient amount of MRNA from the limited tumor tissues in source, and can be obtained by the methods of differential screening The tumour MRNA of tumor cell specific expression impacts Dendritic Cells with this tumour MRNA through differential screening, can avoid Autoantigen stimulates Dendritic Cells to induce the danger of autoimmune disease, there is its unique application value.
Cytokine gene imports DC：Load IL-2, the cells such as IL-12, TNF- Α that tumour antigen stimulation DC secretes because Son can enhance the ctl response of DC Dendritic Cells Induceds.The growth of DC Dendritic Cells is also required to cytokine profiles joint thorn Swash, the gene of cell factor or chemotactic factor (CF) is imported into DC Dendritic Cells, its anti-tumor effect can be enhanced.Cao great Yong etc. makes The DC Dendritic Cells that IL-2 genes have been imported with the liver cancer cells of multigelation impact sensitization, induces compared with liver cancer cells sensitization The higher levels of CTL of DC.
With deepening continuously for cell therapy research, people produce height to how DCS cells start NK cell activations Pay attention to.More and more research shows that DCS mainly passes through cell factor and cell-cell contacts two ways induced NK cell Activation.Initially it is believed that IL-12 plays a significant role in NK cell activations, and IL-12 can be gathered in immunological synapse with High concentration mode acts on NK cells, to promote the activation of NK cells.There is scientist's research in the activation of NK cells It was found that IL-12 does not play a major role, MAILLIARD et al. researchs find DCS secretions IFN-A/IFN-B, IL-18 and IL-15 can also promote NK cell differentiations proliferation, migration and generate IFN-R and enhance the cell-mediated cellulotoxic effects of NK.Into one Step research finds that IFN-A/IFN-B, IL-18 and IL-15 can adjust the ligand expression of NK cell NKG2D receptors, makes NK cells Activation, IL-18 can enhance NK cells and NK promoted CCL21 sensibility to be migrated to peripheral lymphoid organs, promote NK cells It is contacted with DCS and makes its activation.The researchs such as nUCCI find that the IL2 of DC secretions also assists in NK cell activation processes.In addition, DCS swashs Another essential condition of NK cells living is contacted between cell-cell.DCS activates NK cell processes by cell contact In, other than cytoskeletal components participate in, it is also necessary to the participation of some adhesion molecules.Immature DC can express CD48 and CD70 Both molecule can be combined respectively with NK cell surface receptors 2B4, CD27, induced NK cell activation.Ripe DC cells can raise With lower own face some developed by molecule, for the combination of NK cell receptors.JINUSHI etc. the study found that DC in I types Under the action of IFN, the expression (MICB is the ligand of NK cell activation receptors NKG2D) of its surface MICB can be raised, and is lowered Its surface C type Lectin-related molecule expression (c-type Lectin-related molecule be the cell inhibiting receptor NKR2-P1B of NK and The ligand of NKR2P1D), its activation for inhibiting signal is reduced to enhance NK cell activation signals, to make NK cell activations.
With Dendritic Cells (DC) separation, amplification in vitro, culture technique maturation it is perfect, it infectious diseases, from The research in the fields such as body immunity disease, prevention and treatment of malignant tumors and graft-rejection regulation and control is luxuriantly risen.Especially because it is increasing The important function of strong body specificity antineoplastic immunity ability and the research emphasis as tumor biotherapy.
The research group headed by LEVY of medical college of Stanford Univ USA is anti-in first Application antiidiotype in 1996 The self DC Dendritic Cells of body extracorporeal shock sensitization is adopted the method for feedback, treats non-Hodgkin's B Lymphomas, as a result Induction patient produces significant cell and humoral immune reaction, has 1 knurl to completely disappear in the patient of 4 through treatment, and another 1 Example knurl is obviously reduced, no side effects.
In recent years, foreign countries had carried out the research of numerous application Dendritic Cells (DC) treating cancers.IADHAMS etc. is with certainly Body tumour cell loaded dendritic cell (DC) treats unresectable HCC liver cancer cells and obtains positive effect.LEE etc. uses broken HCC liver cancer cells product loaded dendritic cells (DC) the treatment late period HCC liver cancer patient split, I clinical trial phase work well. CHI etc. has found that conventional radiotheraphy combination intratumor injection adoptive immunity Dendritic Cells (DC) treats intractable HCC liver cancer, shows good Good application prospect.KUANG etc. is used for the fixed self HCC liver cancer cells loaded dendritic cell (DC) of formaldehyde to prevent art afterwards The II phase randomized clinical trial recurred afterwards, effect are significant compared with control group.The dendron that NAKAMOTO etc. obtains autologous peripheral blood Row conduit hepatic artery embolism art is fed back after shape cell (DC) amplification in vitro, as a result, it has been found that DC Dendritic Cells is around knurl and tumor Internal portion existed for 17 days, and with the infiltration of monocyte and lymphocyte, it is clinical and have no any serology and from The foundation of adverse reaction is immunized in body, fully confirms that Dendritic Cells (DC) feeds back the safe efficient of scheme, shows good answer Use foreground.
The self DC Dendritic Cells of the external sensitization of German scholar application idiotype protein polypeptide, adoptive therapy is more than 7 Hair property patients with malignant myeloma.Its practice is that idiotype protein is isolated from multiple myeloma patients serum, after enzymic digestion degradation It is purified into polypeptide with protein purification system, then the self DC dendrons shape again with the external sensitization CD34+ cell origins of this polypeptide is thin Such DC Dendritic Cells is subcutaneously injected to multiple myeloma patients, and primary uniqueness was then subcutaneously injected every 2 weeks by born of the same parents Type polypeptide continuous 3 times, as a result, it has been found that in 4 multiple myeloma patients checked, there is antiidiotype in 2 blood The level of IGM and IGG antibody increases about 8 times and 12 times respectively, and the Specific T cell immunity of idiotype protein is also observed Reaction, shows the validity and potential applicability in clinical practice of the therapy.
Report in recent years in relation to DC Dendritic Cells for leukemia treating research is more, in view of immature DC dendron shape Cell has certain phagocytic activity, the reports such as FUJII, in order to induce autogenous cell poison T in vitro with DC Dendritic Cells Cell is subsequently used for the immunization therapy of leukaemia, by peripheral blood CD34+ cells granulocyte/macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF- Α) in vitro culture goes out DC Dendritic Cells, this mixing DC groups (including it is typical Ripe DC Dendritic Cells and immature DC Dendritic Cells) extrinsic protein antigen-keyhole limpet hemocyanin can be absorbed (KEYHOLDLIMPETHEMOCYANIN, KLH) and it is offered to give CD4+ and CD8+T cells, induces keyhole limpet hemocyanin (KLH) specificity cell toxicity T lymphocyte.And they select 3 acute myeloid leukemia (AML) patients, will put After leukaemia cell and DC Dendritic Cells, the T cell of penetrating property inactivation are incubated jointly, as a result wherein 2 induce and can kill in vain The autogenous cell toxic T lymphocyte of blood disease cell, to lay the foundation for cytotoxic T lymphocyte adoptive immunotherapy.
MARTEN etc. prepares the I of DC Cell vaccines, II using autologous tumor cell lysates antigen pulse DC Dendritic Cells 15 advanced renal cell cancer patients have been treated in phase clinical trial, and as a result 1 part is alleviated, and 7 lesions are stablized, 7 lesion growths, 3 Delayed allergy (DTH) is positive, has no adverse reaction.
The domestic basic research for surrounding Dendritic Cells (DC) development in recent years is also very much.Cao Xue great waves academicians preside over country " 863 " 95 major project " therapy of tumor of the antigen presenting cell System-mediated of genetic modification is studied ".Cao Xue great waves institute The clinical research that scholar treats late stage colorectal cancer patients in the DC for carrying out antigen sensibilization, as a result shows that the effective percentage for the treatment of group reaches 46.2%, the effective percentage of chemotherapy group is 22.5%.
Ye Shenglong etc. is with bases such as Melanoma antigen gene -3 (MAGE-1), interleukin 12 (IL-12), interleukin 18s (IL-18) Because modification Dendritic Cells (DC) induces the stronger CTL killed to HCC liver cancer cell specificities, this achievement in vitro It is expected to apply in the prevention of HCC liver cancer as a kind of novel tumor seedling.Xie Bin etc. confirms receptor tyrosine kinases (C- MET), the effect of E- calcium mucin, Β-catenin in the invasion transfer of HCC liver cancer cells, for by above-mentioned protein load dendron Good thinking is provided to the control of liver cancer cells invasion transfer after shape cell (DC).Numerous studies show：HCC liver cancer is exempted from Epidemic disease treatment using Dendritic Cells (DC) submission tumour antigen can apparent inducing antitumor cytotoxic T lymphocyte (CTL), Clinical effectiveness is good and non-evident effect, the tumor vaccine based on Dendritic Cells (DC) structure have become ideal treatment The immune complementary therapy of liver cancer.
The prostate-specific antigen (prostate-specific membrane antigen) and prostatic acid phosphatase polypeptide such as Zhuan Zhixiang The self DC dendritic cells in treatment hormone-refractory prostate cancer of joint sensitization, as a result significantly improves patient immune function, has Effect excites Specific T cell immunity response, and the state of an illness is under control and mitigation.
Lu Daopei etc. has treated 111 acute white blood patients with self DC Combined with Dendritic CIK cell, wherein 94 Complete incidence graph, 5 years disease-free survival probability (DFS) be 66%, hence it is evident that be higher than chemotherapy or autologous hematopoietic stem cell transplantation for treatment Person；14/16 remaining leukaemia chromosome and (or) genetic marker are turned out cloudy；The liver and spleen and marrow of 3/6 hepatosplenomegaly patient The outer lump of the marrow of outer leukaemia person, which is obviously reduced, even to disappear, and disease-free survival is so far.
Zhongxin Hospital, Qingdao's biological therapy center uses the side of self DC Dendritic Cells and CIK cell combined chemotherapy Method has successfully treated 1 advanced pulmonary adenocarcinoma, and by the treatment of a course for the treatment of, patient's cervical lymph node is by walnut size reduction To shelled peanut size, treatment results are determined as that part is alleviated.It is swollen to late period that this therapy compensates for single biological therapy The efficient lower defect of tumor, while also improving the immunity of organisms reduced due to chemotherapy.
The research and development of DC cell clinical application industrialization, by the immunocyte of Cord blood or derived from peripheral blood through external After large-scale culture amplification in input cancer patient's body, it is made to play antitumor action in patient's body.Through clinical research for many years Comparison show that the generally applicable various oncotherapy of clinic of DC cell therapies is notable to most tumor efficiency, especially to maligna Plain tumor, prostate cancer, kidney, carcinoma of urinary bladder, oophoroma, colon and rectum carcinoma, breast cancer, cervical carcinoma, various lung cancer, laryngocarcinoma, nose The therapeutic effects such as pharynx cancer, cancer of pancreas, liver cancer, gastric cancer are preferable.Compared with other antitumor drugs, adoptive immunotherapy can be direct Kill cancer cell, and adjust and enhancing body itself immune function, it has also become tumor operation, radiotherapy, chemotherapy it is important auxiliary Help therapy.
Although the distribution of internal Dendritic Cells is extensive, quantity is atomic.Such as in human peripheral blood single nucleus cell, tree Prominent shape cell only accounts for 1% hereinafter, so micro Dendritic Cells, causes Dendritic Cells after the end of the seventies is identified, though It is attracted great attention with its strong antigen presentation function, but eventually because cell quantity is few, isolated culture method is immature and gives Research brings numerous difficulties, cause once to get into a difficult position in addition some scholars for Dendritic Cells examine whether can be deep The attitude for entering down to produce suspection is being transferred to low ebb to the research of Dendritic Cells in the mid-80.Until 1992, The laboratories STEINMAN, which have been initially set up, uses recombination granulocyte-macrophage colony stimutaing factor (GM-CSF) from mouse bone marrow cells The method that middle large-scale culture prepares Dendritic Cells, hereafter successfully adds interleukin-4 (IL-4) using GM-CSF quickly Tumor necrosis factor Α (TNF- Α) is added to expand from CD34+ candidate stem cells from human peripheral blood single nucleus cell, or with GM-CSF Increase to a large amount of Dendritic Cells, so that the research of Dendritic Cells is reentered climax, and obtain the progress advanced by leaps and bounds.At present The main method for clinically obtaining DC Dendritic Cells is the autologous peripheral blood mononuclear cell using tumour patient, but there are numbers Amount less, antigen submission energy force difference the problems such as.
Had in recent years document report by certain methods by candidate stem cell in vitro after expanding on a large scale, add The DC Dendritic Cells that quantity is big, function is strong can be induced by entering specific cell factor, make large-scale production, the storage of cell It is possibly realized with clinical application.And be taken as containing a large amount of candidate stem cell in the fresh Cord blood of postpartum waste, compared with Stem cell in peripheral blood and marrow has stronger proliferation potential and differentiation potential.
September exists within Xu Li, Kang Zizhen, Wang Bin, Cai Haibo, Tan Wensong, 2002 years《Journal of Immunology》The 5th phase of volume 18 delivers One《Two-step method is from CD34+ Dendritic Cells Induced from Peripheral Blood Stem Cells》, it is disclosed that research purpose：How research is from having The candidate stem cell of limit obtains a large amount of Dendritic Cells (DC), for the further biochemical characteristic of research Dendritic Cells and faces Bed application provides technical support.Using immunomagnetic beads method, ((MACS) is enriched with ten cells of CD34 to method, is first added in cultivating system The cell factors such as FLT3 ligand (FL), thrombopoietin (TPO) and stem cell factor (SCF), then to the cell of enrichment into Row amplification, the cell after amplification induce 7D under the action of GM-CSF and IL-4, the cell flow cytometry analysis after induction Dendritic Cells characteristic surface markers CDIA.As a result two-step method can obtain a large amount of DC, use TPO/FI/ in the first step SCF/IL3/IL6 can obtain most DC to expand CD34+ cells.((3 weeks), two-step method can expand under identical incubation time Increase and 274 times of DC, has been more than greatly the amplification times (24 times) of direct induction.Also, with the growth of incubation time, The amplification times of DC constantly rise.Its used technical solution is：1, sterile immediately after cell origin health parturient childbirth Newborn (adding heparin sodium 20U/ML) Cord blood is taken, average blood sampling volume is 40-80ML, and sample sorts in acquisition 8H, bleeding of the umbilicus It is diluted with PBS buffer solution, FICOLL (collect boundary layer mononuclearcell (MNC), wash by 1.077 density gradient centrifugation of relative density It washs spare.2, isolating and purifying for CD34+ cells is detached using absorption monoclonal antibody Beads enrichment system (CMACS), institute It is QBEND/10 with AntiCD3 McAb 4+ antibody, magnetic bead is the anti-human IGG1 immunomagnetic beads of mouse (MILTENYIBIOTECH companies).Label is thin Born of the same parents elute removal CD34- cells, splitter are removed magnetic field by the MINIMACS splitters being fixed in high-intensity magnetic field, With MACS wash buffer splitters, CD34+ cells are collected.3, the amplification of CD34+ cells:CD34+ cells with The density of 5X104CELLS/ML is inoculated in 24 orifice plates, and basal medium is IMDM (GIBCO companies), adds 10%FBS (GIBCO companies), 2MMOL/L paddy ammonia phthaleins ammonia, bis- coloured glaze base of 100U/ML penicillin, 10 microgram G/ML streptomysins and 50 micromoles/L Ethyl alcohol is cultivated 1-3 weeks in 37 DEG C, the incubator of 5%CO2 saturated humidities.Cell is by SCF (20NG/ML), IL-3 (5NG/ ML), IL-6 (20NG/ML), FLT3-L (25NG/ML), TPO (10U/ML) constitute different combination of cytokines and are expanded. The induction of Dendritic Cells:The CD34+ cells for expanding 1-3 weeks and not expanding are connect with the initial density of 2X105CELLS/ML For kind in 24 orifice plates, cell induces 6-7D by the combination of cytokines that GM-CSF (50NG/ML) and IL-4 (20NG/ML) are constituted.
Invention content
It is by being lured after detaching the expansion culture of CD34 candidate stem cells in Cord blood the invention belongs to cell therapy field Lead preparation Dendritic Cells.Karyocyte is mainly detached from Cord blood using Wealthlin cell processing kits, it is then sharp With magnetic bead separation kit, isolated CD34+ candidate stem cells, addition amplification culture medium make CD34+ from karyocyte The continuous amplification cultivation of hemocytoblast 31 days harvests CD34- cells in amplification procedure, inducing culture is added and is carried out to precursor Induction, to prepare DC Dendritic Cells on a large scale.This method can largely prepare DC Dendritic Cells, solve from Cord blood Middle separation monocyte induction DC Dendritic Cells quantity is few, the low problem of purity.
The technical scheme is that from being detached in Cord blood after CD34 candidate stem cells expand culture, induction is made on a large scale Standby Dendritic Cells method, includes the following steps：
(1) Cord blood is detached by WEALTHLIN cell processing kits, obtains the higher monocyte of enough purity；
(2) obtained monocyte is detached by magnetic bead separation kit, obtains the higher CD34+ hematopoiesis of purity Stem cell；
(3) IL-3, SCF, FLT3, the CD34+ candidate stem cells that TPO amplification culture mediums obtain purifying is used to expand Increase, fresh amplification culture medium was replaced per 2-3 days, after cell quantity, which reaches passage, to be required, suspension cell is transferred to 6CM Plate, T75 bottles, continues to cultivate in 1L culture bags by T25 bottles；
(4) cell suspension is taken out from 1L culture bags, cell is collected by centrifugation, six orifice plates are added after cell is resuspended with culture medium In；After 4-6 hours, suspension cell is sucked, GM-CSF, IL-4 is added in adherent cell collecting, and FLT3 inducing cultures are lured It leads, changes liquid every 2-3 days half amounts, CEA carcinomebryonic antigens are added within 5 days, GM-CSF, IL-4, TNF-A, FLT3, IL6, IL-1 are added within 6 days Β, PGE2 inducing culture are induced, and are changed liquid every 2-3 days half amounts, are collected ripe Dendritic Cells within 8-12 days.
Further, in the step (1), the Cord blood 100ML of anticoagulant heparin is centrifuged into 10MIN with 2094G, is drawn Supernatant liquid, 60 DEG C of inactivation 30MIN, 4 DEG C (2851G) centrifuges 30MIN, and it is spare that absorption supernatant obtains blood plasma；It will be in above-mentioned steps It centrifuges remaining haemocyte to be resuspended with the isometric physiological saline of the blood plasma of absorption, Cord blood is slowly added in A liquid, then B liquid is poured into again, is slowly rotated mixing, is unscrewed lid after mixing, mixed liquor is stored at room temperature 30MIN, the supernatant that will be obtained It is transferred in 8-9 clean 50ML centrifuge tubes, 1210G centrifuges 5MIN, removes supernatant, retains the cell of tube bottom, use physiology The suspension of tube bottom cell is incorporated into a 50ML centrifuge tube by brine, and C liquid is separately added into 2 new 50ML centrifuge tubes, and often pipe adds Enter 25ML, and cell suspension is slowly added in C liquid, 720G centrifuges 20MIN, and cell is made to be layered, and tunica albuginea is drawn with aseptic straw Confluent monolayer cells, are used in combination physiological saline to be settled to 50ML, and 1210G centrifuges 5MIN, washing cell 3 times；
The A liquid is cell maintenance liquid, can be phosphate buffer (PBS)；
The B liquid is erythroprecipitin agent, and the aqueous solution containing 0.4% carboxymethyl starch and 0.4% calcium gluconate should Solution ph 7.4；
The C liquid is Sodium Amidotrizoate-ficoll 400#, contain 0.3% Sodium Amidotrizoate and 8% ficoll 400#It is water-soluble Liquid, PH 7.
Further, in the step (2), karyocyte is resuspended with buffer solution, the suspension for drawing 1X108 cell exists Centrifugal force 300G is centrifuged 10 minutes, and supernatant is sucked out completely, 300UL buffer solutions is added, cell is resuspended, and adds the FCR examinations of 100UL Agent, the CD34+ microballons of 100UL, is wrapped up after being sufficiently mixed with masking foil, and 30MIN is incubated in 4 DEG C of refrigerators, and the slow of 10ML is added Fliud flushing is washed, and centrifuges 10MIN in centrifugal force 300G, and 500UL buffer solutions are added with pipettor in complete Aspirate supernatant Cell is resuspended；The MS pillars prepared with 500UL wash buffers, the cell liquid used is added in pillar, and column is flowed through in collection The unlabeled cells of son are repeated three times with 500UL wash buffer pillars, pillar are removed from separator, and place it in suitable It on the collecting pipe of conjunction, draws on 1ML buffer solutions to pillar, by promoting plunger in pillar, gets express developed out and use magnetic labels The CD34 candidate stem cells of label.
Further, in the step (3), amplification culture medium LONZAX-VIVOTM15 serum-free cell culture mediums, 10% autologous plasma, the use concentration of the use a concentration of 50NG/ML, FLT3 of the use a concentration of 20NG/ML, SCF of IL-3 For 50NG/ML, a concentration of 80NG/ML of use of TPO；
The LONZAX-VIVOTM15 serum-free cell culture mediums are 1L, contain L-Glutamine, phenol red.
Further, in the step (4), inducing culture LONZAX-VIVOTM15 serum-free cell culture mediums, 10% autologous plasma, the use a concentration of 100NG/ML, IL-4 of the use a concentration of 2UG/ML, GM-CSF of CEA carcinomebryonic antigens Use a concentration of 50NG/ML, TNF-A use a concentration of 50NG/ML, FLT3 a concentration of 50NG/ML of use, IL6's makes With a concentration of 20NG/ML, a concentration of 1UG/ML of use of the use a concentration of 10NG/ML, PGE2 of IL-1 Β；
The LONZAX-VIVOTM15 serum-free cell culture mediums are 1L, contain L-Glutamine, phenol red.
Compared with prior art, this method can largely prepare DC Dendritic Cells, solve and detach list from Cord blood Nucleus induces DC Dendritic Cells quantity few, the low problem of purity.
Description of the drawings
Fig. 1 is DC Dendritic Cells morphological feature figures；
Fig. 2 is to detach monocyte from Cord blood within the 0th day to carry out CD14 flow cytometer detections；
The 0th day monocyte isolated from Cord blood of Fig. 3 carries out CD34 flow cytometer detections；
Fig. 4 isolates and purifies to obtain the progress of CD34+ cells from Cord blood using U.S.'s day Ni magnetic beads for purifying kits on the 0th day CD34 flow cytometer detections；
The 0th day monocyte isolated from Cord blood of Fig. 5 detects DC cell surface markers；
Cytokine induction is added 10 days in Fig. 6 after isolated CD34+ cells amplification cultivation in Cord blood；
Cytokine induction is added 10 days in Fig. 7 monocytes；
Cytokine induction is added 12 days in Fig. 8 after isolated CD34+ cells amplification cultivation in Cord blood；
Cytokine induction is added 12 days in Fig. 9 monocytes；
The DC Dendritic Cells of Figure 10 CD34 derived from hematopoietic precursor cells and the DC surface of dendritic cells of cells of monocytic origin Marker expression situation statistical comparison；
The DC Dendritic Cells of Figure 11 CD34 derived from hematopoietic precursor cells and the DC Dendritic Cells cells of cells of monocytic origin Number comparison diagram.
Specific implementation mode
Embodiment one
The separation of monocyte
One cord blood is detached using WEALTHLIN cell processing kits, obtains the higher monocyte of enough purity, And count cell number and cell survival rate.
The separation of CD34 candidate stem cells
Obtained monocyte is purified using U.S.'s day Ni magnetic bead sortings kit, the higher CD34 of purity is obtained and makes Hemocytoblast, and count cell and cell survival rate.
The amplification of CD34+ candidate stem cells
Using IL-3, SCF, FLT3, TPO, autologous plasma amplification culture medium to the obtained CD34 candidate stem cells of purifying into Row amplification, fresh IL-3, SCF, FLT3, TPO, autologous plasma amplification culture medium, when cell quantity reaches biography were replaced per 2-3 days After generation requires, suspension cell is transferred to 6CM plates, T25 bottles, T75 bottles, continues to cultivate in 1L culture bags.
The harvest of CD34- cells and induction
Cell suspension is taken out from 1L culture bags, cell is collected by centrifugation after cell count and cell survival rate, use culture medium It is added in six orifice plates after cell is resuspended.After 4-6 hours, suspension cell is sucked, GM-CSF, IL-4 is added in adherent cell collecting, FLT3 inducing cultures are induced, and are measured every 2-3 days half and are changed liquid, 5 days addition CEA carcinomebryonic antigens, 6 days addition GM-CSF, IL-4, TNF-A, FLT3, IL6, IL-1 Β, PGE2, autologous plasma inducing culture are induced, and liquid, 8-12 are changed every 2-3 days half amounts It collects ripe Dendritic Cells.
Concrete operation step
The DC Dendritic Cells Induced cultures of one .CD34 derived from hematopoietic precursor cells
1. the separation of karyocyte
(1) the Cord blood 100-135ML of anticoagulant heparin is centrifuged into 10MIN with 2094G, draws supernatant liquid, 60 DEG C of inactivations 30MIN, 4 DEG C (2851G) centrifuge 30MIN, and it is spare that absorption supernatant obtains blood plasma；
(2) physiological saline weight of the remaining haemocyte with isometric (Plasma volumes of absorption) will be centrifuged in above-mentioned steps It is outstanding；
(3) Cord blood is slowly added in A liquid；
(4) B liquid is slowly added in (3) step mixed liquor, slowly rotates mixing, unscrew lid after mixing, will mixes Liquid is stored at room temperature 30MIN；
(5) obtained supernatant is transferred in 8-9 clean 50ML centrifuge tubes, 1210G centrifuges 5MIN；
(6) remove supernatant, retain the cell of tube bottom, with physiological saline by tube bottom cell suspension be incorporated into a 50ML from Heart pipe；
(7) C liquid is separately added into 2 new 50ML centrifuge tubes, often 25ML is added in pipe, and liquid obtained by (6) step is slow Slow to be added in C liquid, 720G centrifuges 20MIN, and cell is made to be layered；
(8) tunica albuginea confluent monolayer cells are drawn with aseptic straw, physiological saline is used in combination to be settled to 50ML, 1210G centrifuges 5MIN, washes Wash cell；
(9) (8) step, washing cell 2-3 times are repeated；
(10) the karyocyte sum obtained is calculated, trypan blu e dyeing is used in combination to calculate cell survival rate.
2.CD34 the separation of candidate stem cell
(1) monocyte is resuspended with buffer solution, the suspension for drawing 1X108 cell centrifuges 10 points in centrifugal force 300G Supernatant is sucked out in clock completely；
(2) 300UL buffer solutions are added with pipettor and cell is resuspended；
(3) the FCR reagents of 100UL, the CD34+ microballons of 100UL are added with pipettor；
(4) it is wrapped up with masking foil after being sufficiently mixed, 30MIN is incubated in 2-8 DEG C of refrigerator；
(5) buffer solution that 10ML is added is washed, and centrifuges 10MIN, complete Aspirate supernatant in centrifugal force 300G；
(6) 500UL buffer solutions are added with pipettor and cell is resuspended；
(7) the MS pillars for using 500UL wash buffers to prepare；
(8) cell liquid used is added in pillar, collects the unlabeled cells for flowing through pillar；
(9) 500UL wash buffer pillars are used, are repeated three times；
(10) pillar is removed from separator, and is placed it on suitable collecting pipe；
(11) it draws on 1ML buffer solutions to pillar, by promoting plunger in pillar, gets express developed out with magnetic labels mark The CD34 candidate stem cells of note；
(12) the CD34 candidate stem cell sums obtained are calculated, trypan blu e dyeing is used in combination to calculate cell survival rate.
3.CD34+ the amplification of candidate stem cell
(1) CD34 candidate stem cells (20NG/MLIL-3,50NG/MLSCF, 50NG/MLFLT3, the 80NG/ that will be obtained MLTPO, 10% autologous plasma) amplification culture medium adjusts into 3X105/ML cell inoculation to six orifice plates, per hole in be inoculated with 2.5ML is placed in 37 DEG C, 5%CO2 incubator cultures；, in incubation six orifice plates are gently shaken per 12H；
(2) 3-4 days when the cell density in six orifice plates reaches 80-100%, by six orifice plates suspension cell inhale Go out, carries out cell count and dyed with trypan blu e to calculate cell survival rate, and carry out the detection of streaming CD34 indexs, then use (20NG/MLIL-3,50NG/MLSCF, 50NG/MLFLT3,80NG/MLTPO, 10% autologous plasma) amplification culture medium is resuspended thin By in 1X106/ML cell inoculation to T25 bottles after born of the same parents, it is placed in 37 DEG C, 5%CO2 incubator cultures；
(3) 3-4 days when the cell density in T25 bottles reaches 80-100%, by the suspension cell in T25 bottles It is sucked out, carries out cell count and dyed with trypan blu e to calculate cell survival rate, and carry out the detection of streaming CD34 indexs, then use (20NG/MLIL-3,50NG/MLSCF, 50NG/MLFLT3,80NG/MLTPO, 10% autologous plasma) amplification culture medium is resuspended thin By in 1X106/ML cell inoculation to T75 bottles after born of the same parents, it is placed in 37 DEG C, 5%CO2 incubator cultures；
(4) 3-4 days when the cell density in T75 bottles reaches 80-100%, by the suspension cell in T75 bottles It is sucked out, carries out cell count and dyed with trypan blu e to calculate cell survival rate, and carry out the detection of streaming CD34 indexs, then use (20NG/MLIL-3,50NG/MLSCF, 50NG/MLFLT3,80NG/MLTPO, 10% autologous plasma) amplification culture medium is resuspended thin By in 1X106/ML cell inoculation to GT-T610 (A) cell culture bags of 1L after born of the same parents, observed daily using microscope, Cell count is carried out every sampling in 3 days, is dyed with trypan blu e and calculates cell survival rate, and carry out the detection of streaming CD34 indexs, and It carries out half amount and changes liquid, be added that new (20NG/MLIL-3,50NG/MLSCF, 50NG/MLFLT3,80NG/MLTPO, 10% are self Blood plasma) amplification culture medium cultivated, continuous culture to the 31st day (the cell count statistics in 1 cell expansion process of table).
The harvest of CD34- cells and the induction of DC cells
After amplification the 12nd day, suspension cell is sucked out from GT-T610 (A) cell culture bags, carries out cell count and is used in combination Trypan blu e dyeing calculates cell survival rate, is inoculated into six orifice plates by the inoculum concentration of 3X106/ML, and each hole meets 2.5ML, the Suspension cell was sucked in two days, adherent cell collecting, (100NG/MLGM-CSF, 50NG/MLIL-4 and 50NG/ is added MLFLT3) inducing culture is induced；It is placed in 37 DEG C, 5%CO2 incubator cultures；
In induction the 3rd day, half amount is carried out to the cell in six orifice plates and changes liquid, adds (100NG/MLGM-CSF, 50NG/ MLIL-4,50NG/MLFLT3) inducing culture, it is placed in 37 DEG C, 5%CO2 incubator cultures；
When inducing the 5th day, carcinomebryonic antigen (CEA) is added, is placed in 37 DEG C, 5%CO2 incubator cultures；
When inducing the 6th day, (G100NG/MLGM-CSF, 50NG/MLIL-4,50NG/MLTNF-A, 50NG/ is added MLFLT3,20NG/MLIL6,10NG/MLIL-1 Β, 1UG/MLPGE2,10% autologous plasma) inducing culture induced, Liquid is changed every 2-3 days half amounts, collects ripe Dendritic Cells within 8-12 days, cell count is carried out and trypan blu e dyeing calculates cell Survival rate, and carry out flow cytometer detection (HLA-DR, CD80, CD83, CD86).
The Fiber differentiation of the DC Dendritic Cells of two, cells of monocytic origin
1. the separation of monocyte
(1) the Cord blood 100-135ML of anticoagulant heparin is centrifuged into 10MIN with 2094G, draws supernatant liquid, 60 DEG C of inactivations 30MIN, 4 DEG C (2851G) centrifuge 30MIN, and it is spare that absorption supernatant obtains blood plasma；
(2) physiological saline weight of the remaining haemocyte with isometric (Plasma volumes of absorption) will be centrifuged in above-mentioned steps It is outstanding；
(3) Cord blood is slowly added in A liquid；
(4) B liquid is slowly added in (3) step mixed liquor, slowly rotates mixing, unscrew lid after mixing, will mixes Liquid is stored at room temperature 30MIN；
(5) obtained supernatant is transferred in 8-9 clean 50ML centrifuge tubes, 1210G centrifuges 5MIN；
(6) remove supernatant, retain the cell of tube bottom, with physiological saline by tube bottom cell suspension be incorporated into a 50ML from Heart pipe；
(7) C liquid is separately added into 2 new 50ML centrifuge tubes, often 25ML is added in pipe, and liquid obtained by (6) step is slow Slow to be added in C liquid, 720G centrifuges 20MIN, and cell is made to be layered；
(8) tunica albuginea confluent monolayer cells are drawn with aseptic straw, physiological saline is used in combination to be settled to 50ML, 1210G centrifuges 5MIN, washes Wash cell；
(9) (8) step, washing cell 2-3 times are repeated；
(10) the karyocyte sum obtained is calculated, trypan blu e dyeing is used in combination to calculate cell survival rate.
The induction of 2.DC Dendritic Cells
(1) monocyte obtained above is resuspended with serum free medium, according to:3 × 106/ML density inoculating cells In six orifice plates, and it is put into cell incubator and stands 3-4 hours；
(2) after cell is adherent, upper cell liquid is washed off, with being added to factor 100NG/MLGM-CSF, 50NG/ The serum free medium culture attached cell of MLIL-4 and 50NG/MLFLT3 is placed in 37 DEG C, 5%CO2 incubator cultures；
(3) in induction the 3rd day, half amount is carried out to the cell in six orifice plates and changes liquid, add (100NG/MLGM-CSF, 50NG/MLIL-4,50NG/MLFLT3) inducing culture, it is placed in 37 DEG C, 5%CO2 incubator cultures；
(4) when inducing the 5th day, 2UG/ML carcinomebryonic antigens (CEA) is added, are placed in 37 DEG C, 5%CO2 incubator cultures；
(5) when inducing the 6th day, (100NG/MLGM-CSF, 50NG/MLIL-4,50NG/MLTNF-A, 50NG/ is added MLFLT3,20NG/MLIL6,10NG/MLIL-1 Β, 1UG/MLPGE2,10% autologous plasma) inducing culture induced, Liquid is changed every 2-3 days half amounts, collects ripe Dendritic Cells within 8-12 days, cell count is carried out and trypan blu e dyeing calculates cell Survival rate, and carry out flow cytometer detection (HLA-DR, CD80, CD83, CD86).
The DC Dendritic Cells of three .CD34 derived from hematopoietic precursor cells is compared with the DC Dendritic Cells of cells of monocytic origin
Fig. 1 is DC Dendritic Cells morphological feature figures.A is CD34 derived from hematopoietic precursor cells under (400X) inverted microscope DC Dendritic Cells aspect graphs, B be (400X) inverted microscope resulted in monocyte source DC Dendritic Cells aspect graph (tree Prominent shape cell suspends or half adherent growth, differs in size, form irregular, the thickness for stretching out different number around differs, The totally different cytoplasmic processes of form, what is had is short and thick, and end is in the pseudopodium sample of blunt circle；Have thin and grow, formed many irregular Multiple-limb dendroid, surface be in burr sample；Also the two having has concurrently).
Fig. 2 is to detach monocyte from Cord blood within the 0th day to carry out CD14 flow cytometer detections, wherein figure A.p1 is streaming circle Door figure B. compares figure C.CD14 flow cytometer detections.
The 0th day monocyte isolated from Cord blood of Fig. 3 carries out CD34 flow cytometer detections, wherein figure A.p3 is streaming Circle door figure B. compares figure C.CD34 flow cytometer detections.
Fig. 4 isolates and purifies to obtain the progress of CD34+ cells from Cord blood using U.S.'s day Ni magnetic beads for purifying kits on the 0th day CD34 flow cytometer detections, wherein figure A.p4 is streaming circle door figure B. compares figure C.CD34 flow cytometer detections.
The 0th day monocyte isolated from Cord blood of Fig. 5 detects DC cell surface markers, wherein scheming A.CD80 Flow cytometer detection figure B.CD83 flow cytometer detection figure C.CD86 flow cytometer detection figure D.hLA-DR flow cytometer detections.
Cytokine induction is added 10 days in Fig. 6 after isolated CD34+ cells amplification cultivation in Cord blood
Scheme the DC cell CD80 flow cytometer detections of A.CD34 derived from hematopoietic precursor cells
Scheme the DC cell CD83 flow cytometer detections of B.CD34 derived from hematopoietic precursor cells
Scheme the DC cell CD86 flow cytometer detections of C.CD34 derived from hematopoietic precursor cells
Scheme the DC cell HLA-DR flow cytometer detections of D.CD34 derived from hematopoietic precursor cells.
Cytokine induction is added 10 days in Fig. 7 monocytes
Scheme the DC cell CD80 flow cytometer detections of A. cells of monocytic origin
Scheme the DC cell CD83 flow cytometer detections of B. cells of monocytic origin
Scheme the DC cell CD86 flow cytometer detections of C. cells of monocytic origin
Scheme the DC cell HLA-DR flow cytometer detections of D. cells of monocytic origin.
Cytokine induction is added 12 days in Fig. 8 after isolated CD34+ cells amplification cultivation in Cord blood
Scheme the DC cell CD80 flow cytometer detections of A.CD34 derived from hematopoietic precursor cells
Scheme the DC cell CD83 flow cytometer detections of B.CD34 derived from hematopoietic precursor cells
Scheme the DC cell CD86 flow cytometer detections of C.CD34 derived from hematopoietic precursor cells
Scheme the DC cell HLA-DR flow cytometer detections of D.CD34 derived from hematopoietic precursor cells.
Cytokine induction is added 12 days in Fig. 9 monocytes
The DC cell CD80 flow cytometer detections of Fig. 9 .A cells of monocytic origin
The DC cell CD83 flow cytometer detections of Fig. 9 .B cells of monocytic origin
The DC cell CD86 flow cytometer detections of Fig. 9 .C cells of monocytic origin
The DC cell HLA-DR flow cytometer detections of Fig. 9 .D cells of monocytic origin.
The DC Dendritic Cells of Figure 10 CD34 derived from hematopoietic precursor cells and the DC surface of dendritic cells of cells of monocytic origin Marker expression situation statistical comparison.
The DC Dendritic Cells of Figure 11 CD34 derived from hematopoietic precursor cells and the DC Dendritic Cells cells of cells of monocytic origin Number comparison diagram.
Final conclusion
Sample number into spectrum DC1 DC2 DC3 DC4
The sources CD34+ DC cells 2.11×108It is a 1.57×108It is a 1.03×108It is a 9.1×107It is a
Cells of monocytic origin DC cells 1.65×107It is a 1.32×107It is a 1.11×107It is a 7.9×106It is a

Claims (5)

1. a kind of, induction prepares Dendritic Cells method on a large scale after detaching the expansion culture of CD34 candidate stem cells in Cord blood, It is characterized by comprising the following steps：
(1) Cord blood is detached by WEALTHLIN cell processing kits, obtains the higher monocyte of enough purity；
(2) obtained monocyte is detached by magnetic bead separation kit, it is thin obtains the higher CD34+ Hematopoietic Stems of purity Born of the same parents；
(3) IL-3, SCF, FLT3, the CD34+ candidate stem cells that TPO amplification culture mediums obtain purifying is used to expand, often It replaces fresh amplification culture medium within 2-3 days, after cell quantity, which reaches passage, to be required, suspension cell is transferred to 6CM plates, T25 Bottle, continues to cultivate in 1L culture bags by T75 bottles；
(4) cell suspension is taken out from 1L culture bags, cell is collected by centrifugation, be added in six orifice plates after cell is resuspended with culture medium； After 4-6 hours, suspension cell is sucked, GM-CSF, IL-4 is added in adherent cell collecting, and FLT3 inducing cultures are induced, every 2-3 days half amounts change liquid, and CEA carcinomebryonic antigens are added within 5 days, be added within 6 days GM-CSF, IL-4, TNF-A, FLT3, IL6, IL-1 Β, PGE2 inducing cultures are induced, and are changed liquid every 2-3 days half amounts, are collected ripe Dendritic Cells within 8-12 days.
2. one kind according to claim 1 induces on a large scale after detaching the expansion culture of CD34 candidate stem cells in Cord blood Prepare Dendritic Cells method, which is characterized in that in the step (1), by the Cord blood 100ML of anticoagulant heparin with 2094G from Heart 10MIN draws supernatant liquid, and 60 DEG C of inactivation 30MIN, 4 DEG C (2851G) centrifuges 30MIN, and it is spare that absorption supernatant obtains blood plasma； Remaining haemocyte will be centrifuged in above-mentioned steps to be resuspended with the isometric physiological saline of the blood plasma of absorption, and Cord blood is slowly added Enter in A liquid, then pour into B liquid again, slowly rotate mixing, unscrew lid after mixing, mixed liquor is stored at room temperature 30MIN, it will Obtained supernatant is transferred in 8-9 clean 50ML centrifuge tubes, and 1210G centrifuges 5MIN, removes supernatant, retains tube bottom Cell, with physiological saline by tube bottom cell suspension be incorporated into a 50ML centrifuge tube, by C liquid be separately added into 2 new 50ML from Heart pipe, often 25ML is added in pipe, and cell suspension is slowly added in C liquid, and 720G centrifuges 20MIN, so that cell is layered, with sterile suction Pipe draws tunica albuginea confluent monolayer cells, and physiological saline is used in combination to be settled to 50ML, and 1210G centrifuges 5MIN, washing cell 3 times；
The A liquid is cell maintenance liquid, can be phosphate buffer (PBS)；
The B liquid is erythroprecipitin agent, and the aqueous solution containing 0.4% carboxymethyl starch and 0.4% calcium gluconate, this is water-soluble Liquid PH7.4；
The C liquid is Sodium Amidotrizoate-ficoll 400#, contain 0.3% Sodium Amidotrizoate and 8% ficoll 400#Aqueous solution, PH It is 7.
3. one kind according to claim 1 induces on a large scale after detaching the expansion culture of CD34 candidate stem cells in Cord blood Prepare Dendritic Cells method, which is characterized in that in the step (2), karyocyte is resuspended with buffer solution, draws 1X108 The suspension of cell centrifuges 10 minutes in centrifugal force 300G, and supernatant is sucked out completely, 300UL buffer solutions is added, cell is resuspended, add The FCR reagents of 100UL, the CD34+ microballons of 100UL is added to be wrapped up with masking foil after being sufficiently mixed, be incubated in 4 DEG C of refrigerators 30MIN, the buffer solution that 10ML is added are washed, and centrifuge 10MIN in centrifugal force 300G, and complete Aspirate supernatant uses liquid relief Device is added 500UL buffer solutions and cell is resuspended；The MS pillars prepared with 500UL wash buffers, the cell liquid used is added to In pillar, the unlabeled cells for flowing through pillar are collected, with 500UL wash buffer pillars, repeats three times, is removed from separator Pillar, and place it on suitable collecting pipe, it draws on 1ML buffer solutions to pillar, by promoting plunger in pillar, quickly Flush out the CD34 candidate stem cells marked with magnetic labels.
4. one kind according to claim 1 induces on a large scale after detaching the expansion culture of CD34 candidate stem cells in Cord blood Prepare Dendritic Cells method, which is characterized in that in the step (3), amplification culture medium LONZAX-VIVOTM15 serum-frees Cell culture medium, 10% autologous plasma, the use a concentration of 50NG/ML, FLT3 of the use a concentration of 20NG/ML, SCF of IL-3 Use a concentration of 50NG/ML, TPO a concentration of 80NG/ML of use；
The LONZAX-VIVOTM15 serum-free cell culture mediums are 1L, contain L-Glutamine, phenol red.
5. one kind according to claim 1 induces on a large scale after detaching the expansion culture of CD34 candidate stem cells in Cord blood Prepare Dendritic Cells method, which is characterized in that in the step (4), inducing culture LONZAX-VIVOTM15 serum-frees Cell culture medium, 10% autologous plasma, a concentration of 2UG/ML of use of CEA carcinomebryonic antigens, the use of GM-CSF are a concentration of A concentration of 50NG/ML of use of the use a concentration of 50NG/ML, TNF-A of 100NG/ML, IL-4, the use of FLT3 are a concentration of A concentration of 1UG/ of use of the use a concentration of 10NG/ML, PGE2 of the use a concentration of 20NG/ML, IL-1 Β of 50NG/ML, IL6 ML；
The LONZAX-VIVOTM15 serum-free cell culture mediums are 1L, contain L-Glutamine, phenol red.
CN201810411706.5A 2018-05-02 2018-05-02 From being detached in Cord blood after CD34 candidate stem cells expand culture, induction prepares Dendritic Cells method to one kind on a large scale Pending CN108642013A (en)

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