CN113403274A - Cell therapy composition, preparation method thereof and application of cell therapy composition as medicine for treating anaphylactic reaction and autoimmune disease - Google Patents
Cell therapy composition, preparation method thereof and application of cell therapy composition as medicine for treating anaphylactic reaction and autoimmune disease Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention provides a cell therapy composition comprising I gE (CH)3) IgE (CH) from Co-culture of DC and CI K cells3) DC-CI K cells, and then co-culturing with allogeneic MSC cellsThe invention also provides an application of the cell therapy composition in preparing cell medicines for treating anaphylactic reaction and autoimmune diseases. The invention uses IGE (CH)3) DCCI K + MSCs co-cultured cells for treating anaphylaxis and autoimmune diseases are proved to produce more favorable results through experiments and have no side effect.
Description
Technical Field
The invention relates to the field of cellular immunity, in particular to a cell therapy composition, a preparation method thereof and application of the cell therapy composition in treating anaphylactic reaction and autoimmune diseases.
Background
At present, the broad-spectrum immunosuppressant of glucocorticoid class is mainly used for treating anaphylactic reaction, such as allergic asthma. Although the medicine has a certain effect on controlling anaphylactic reaction, the medicine has an obvious side effect because of inhibiting most immune cells, and if the medicine is used for a long time, the immune function of the organism is low, and diseases such as infection, tumor and the like are easily induced.
Therefore, it is one of the directions in which those skilled in the art have been working to develop a therapeutic agent for anaphylaxis with no or less side effects.
Disclosure of Invention
The object of the present invention is to solve the above problems and to provide a cell therapy composition which can effectively treat allergic diseases caused by allergens and acute metastatic plant anti-host diseases and current epidemic new crown diseases without any side effects, and a preparation method thereof.
The purpose of the invention is realized as follows:
the invention provides a cell therapy composition comprising therapeutically effective amounts of DC cells, CIK cells and MSC cells.
The above cell therapy composition, wherein,the DC cells and the CIK cells are autologous cells, the MSC cells are allogeneic cells, and the DC cells carry CH of IgE3A protein fragment.
The invention also provides a method for preparing a cell therapy composition, comprising the following steps:
(1) preparing autologous peripheral blood mononuclear cells;
(2) isolation and culture of DC cells;
(3) induction culture of CIK cells;
(4) addition of CH to IgE to DC cells3Preparation of IgE (CH) protein fragment3) A DC cell;
(5)IgE(CH3) Co-culture of DC cells and CIK cells to produce IgE (CH)3) DC-CIK cells;
(6)IgE(CH3) The DC-CIK cells and allogeneic MSC cells are co-cultured to prepare the cell therapeutic composition.
The method for preparing a cytotherapeutic composition as described above, wherein, in the step (4), said CH3The concentration of the protein fragment is 50ug-500 ug/1X 107DC cells.
The method for preparing a cytotherapeutic composition as described above, wherein, in the step (4), said CH3The protein fragment is obtained by adopting eukaryotic cell gene expression.
The process for preparing a cell therapy composition as described above, wherein, in the step (5), IgE (CH)3) The DC cells and the CIK cells are mixed and cultured according to the cell number ratio of 1: 3-5.
The method for preparing a cell therapy composition as described above, wherein, in the step (6), the MSC cells are contacted with IgE (CH)3) The DC-CIK cells are mixed and co-cultured according to the cell number ratio of 1: 10-50.
IgE (CH) described above3) The cell therapeutic composition obtained by co-culturing DC-CIK cells and MSC cells can be used as cell medicines for treating anaphylaxis and autoimmune diseases.
IgE(CH3) Glycosylation after gene expression binds to mannose receptors on DC cells, which are professional presenting cells, and are co-cultured with CIK (cytokine induced killer), DC and IgE (CH)3) IdentificationThe proliferation of CIK can be promoted by co-culturing with CIK after the CIK is presented, and the maturation of DC is promoted by CIK cells, and the functions are completed by secreting and complementing cytokines by DC and CIK. In IgE (CH)3) The cell activity of CIK is excited in the co-culture process of DC and CIK, so that T killer cells such as CIK and the like are endowed with IgE (CH)3) The memory of the human body can eliminate pathogenic somatic cells in the human body, reduce the occurrence of allergic diseases, namely cell-mediated cellular immune effect.
DCs internalize to carry IgE (CH) during culture3) The activated DC cell recognizes B cell through signal molecule, and causes B cell proliferation, antibody production and antibody class conversion, which is B cell mediated humoral immunity effect.
The present invention selects MSC cells and IgE (CH)3) Co-culture of DC-CIK cells in IgE (CH)3) In a specific microenvironment of a culture system of the DC-CIK, the MSC can secrete a plurality of cytokines, particularly secrete a tipping factor to train T effector cells under a co-culture condition through contact and internalization among co-cultured cells, so that the T effector cells can be induced to differentiate into cells with different phenotypic characteristics under the specific culture condition, and can effectively clear secreted IgE or combined IgE (CH) when injected into a body3) The disease-causing somatic cell of (1).
MSC (human umbilical cord mesenchymal stem cells) is not expressing MHC genes in the early stage and has good regulation effect on an immune system, and a plurality of experiments prove that MSC has good results for treating allergic diseases caused by allergens, acute transfer plant anti-host diseases and current epidemic new crown diseases.
The present invention relates to the treatment of allergic diseases: t cell immune effect; ② DC presents CH3Cellular immune effects produced by the antigen; ③ MSCSA single immunoregulatory effect and the synergistic and linked functional effects of the MSC and the DCCIK cells. The present invention utilizes IgE (CH)3) The DCCIK + MSCs co-cultured cells are used for treating anaphylactic reaction and autoimmune diseases, and experiments prove that the DCCIK + MSCs co-cultured cells can produce more favorable results and do not have any side effect.
Detailed Description
The present invention will be further described with reference to the following examples, but is not limited thereto. The raw materials of the reagents described in the following examples are commercially available raw materials except for the source, and the reagents are prepared by a conventional method. The methods not detailed in the examples are all conventional in the art.
In the embodiments of the invention, SD rats are used as verification experimental animals.
Example 1: whole genome PCR for obtaining CH of IgE3Gene fragment and construction of recombinant plasmid
Two pairs of primers P1 and P2 were synthesized for PCR. The 5 'and 3' cleavage sites of primers P1 and P2 were introduced into EcoRV and EcoRI cleavage sites, respectively, to construct pVAC-CH3A recombinant plasmid.
P1: 5'-GGGATATCATGCCGACAGATCATGAGCCACG-3' (EcoRV cleavage site underlined)
P2: 5'-GGGAATTCTCACTGGGGAAGGGTGATGGAAC-3' (EcoRI cleavage site underlined)
With SD rat whole genome DNA as a template, a 100 mu L reaction system is established, which comprises 10 mu L of 10 XPCR reaction buffer, 25mM MgCl26 mu L, DMSO5 mu L, dNTP8 mu L, 4 mu L of each of upstream and downstream primers, 4 mu L of rat whole genome DNA and 60 mu L of water. Then, PCR reaction was carried out, and 3.2. mu.L of TaqDNA polymerase was added after pre-denaturation at 95 ℃ for 5 min. The reaction conditions are as follows: denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 1min, and extension at 72 ℃ for 2min for a total of 39 cycles. The final cycle of extension at 72 ℃ for 10min and storage at 4 ℃.
Construction of recombinant plasmid pVAC-CH3
Amplifying CH of IgE by using whole genome DNA as template and primers P1 and P23And (3) connecting the fragment into a PMD18-Tvector, performing double digestion on T-CH3 by using EcoRI and EcoRV, and connecting the recovered small fragment with the recovered large fragment by double digestion of the pVAC empty vector. Transforming the ligation product into competent cells DH5 alpha, screening zeocin anti-biotin, selecting positive clones, culturing in LB culture medium overnight, extracting plasmid DNA, enzyme digestion identification and DNA sequence analysis, and naming the ones with correct sequencing as pVAC-CH3。
Amplification of recombinant plasmids
Recombinant plasmid pVAC-CH3, transformed competent cell DH5 alpha, cultured overnight, the next morning picked single clone, inoculated in 10mL of LB medium containing zeocin antibiotic and cultured for 8h, then inoculated in 300mL of LB medium containing zeocin antibiotic according to the proportion of 1:100, shaken for 2.5h at 37 ℃, and cultured for 16h under vigorous shaking at 37 ℃.
Mass extraction and purification of recombinant plasmids
Centrifuge at 4100rpm for 20min at 4 ℃ with a suitable rotor, discard the supernatant and harvest the bacteria, resuspend the bacterial pellet in 50mL STE chilled with ice, wash once and discard the supernatant. Resuspending the bacterial pellet in 10mL of solution I, adding 1mL of newly formulated lysozyme solution, adding 20mL of newly formulated solution II, capping the bottle, slowly inverting the flask several times, mixing the contents well, and standing at room temperature for 5-10 min. 15mL of solution III pre-cooled with ice are added. The mouth of the bottle was sealed and the flask shaken several times to mix the contents. Placing on ice for 10min to form a white flocculent precipitate.
The rotor was centrifuged for 30min at 11000rpm at 4 ℃ with a suitable rotor, causing the rotor to stop spinning naturally. Carefully transfer the whole supernatant to another bottle and discard the pellet in the centrifuge tube. The volume of the supernatant was measured, and the supernatant was transferred into a clean centrifuge tube together with 0.6 times the volume of isopropyl alcohol and mixed well, and left at room temperature for 10 min. The nucleic acid pellet was recovered by centrifugation at 8000rmp for 15min at room temperature. The supernatant was carefully decanted, the centrifuge tube was opened with the lid open, and decanted onto a paper towel to remove the residual supernatant. The bottom and wall of the deposition tube were washed with 70% ethanol at room temperature. The ethanol was decanted, any droplets adhering to the wall of the vial were aspirated by a pasteur pipette connected to a vacuum, the centrifuge tube was inverted at the opening onto a paper towel to evaporate any remaining traces of ethanol, and the nucleic acid pellet was dissolved using 2ml of LTE (pH 8.0). Using phenol, phenol: the plasmid was purified by extracting chloroform 1 time each. OD measurement after 1:100 dilution of nucleic acid precipitate with TE (pH8.0)260/OD280The concentration of plasmid DNA (1 OD) was calculated26050ug plasmid DNA/mL), the extracted plasmid was single digested and subjected to agarose electrophoresis to detect the effect, and the DNA was stored at-20 ℃.
Example 2: CH (CH)3Eukaryotic expression of protein fragmentsExtraction and purification of
The CHO fine bubbles in good growth state were injected at a rate of 1X 10 per well5One was seeded in 6-well plates and when the cells grew to 80% total anchorage, the cells were washed 2 times with serum-free and antibiotic-free RPMI 1640 medium. Mu.g of DNA was dissolved in 100. mu.L of serum-free and antibiotic-free RPMI 1640 medium, designated solution A. 10 μ L of LLiopfectAMINETM2000 was dissolved in 90 μ L of serum-free and antibiotic-free RPMI 1640 medium and left for 10min, called liquid B. A, B the two solutions were mixed and incubated at room temperature for 30min to allow the formation of DNA-liposome complexes. Adding 800 μ L serum-free antibiotic-free DMEM culture solution into the complex, gently mixing, slowly adding dropwise into the washed cells, and adding CO at 37 deg.C2Culturing in an incubator for 6 h. And (3) removing the transfection solution, adding 1mL of antibiotic-free RPMI 1640 culture solution containing 20% calf serum for continuous culture, collecting cells after transfection for 48h, detecting transient expression of target protein, and screening to obtain transfected and successfully expressed CHO cells.
Obtaining CH by CHO cell eukaryotic expression, final extraction and purification3The protein fragment is ready for use.
Example 3: preparation method of phoenix tree pollen leaching solution
Phoenix tree pollen is a common allergen and can often induce allergic diseases such as bronchial asthma, allergic rhinitis and the like, specific IgE exists in serum of a patient with pollinosis, the fluctuation of the content of the IgE in blood is large, and the stability is poor, so that an experimental animal model with stable IgE secretion is established for the correctness of the experiment, a domestic common SD rat is used, the allergic reaction of the SD rat is similar to that of a human, and the relationship between the mechanism and treatment of the allergic diseases of the human is researched through the mediation of the IgE.
Phoenix tree pollen is naturally dried to constant weight, and is degreased for several times by ether until the ether layer is colorless, and after the ether is completely volatilized, the ratio (1:10, w/v) of 10 mLCOCa's (5.0g sodium chloride, 2.75g sodium bicarbonate, 4mL phenol, distilled water to 1000mL) solution is added into each gram of pollen. Leaching at 4 deg.C for 48 hr while magnetically stirring for 20min, centrifuging to obtain supernatant with protein content of about 1mg/mL, dialyzing with PBS in dialysis bag until the external liquid is colorless, aseptically filtering, packaging, and freezing.
Example 4: preparation of SD rat MSC cells
Healthy 6-10 SD rats (200 g/rat) are subjected to vacuum sacrifice and soaked in 75% ethanol for 5min to obtain sterile super clean bench rats with bilateral femurs and shines. Aseptic scissors are used for muscle stripping, the femur and the tibia which are completely separated are placed into PBS (phosphate buffer solution), the femur and the tibia are cut by a large-size scissors, 1mL aseptic syringe is used for absorbing the PBS to repeatedly wash marrow cavities of the femur and the tibia, then the femur and the tibia are sucked out and placed into a 10mL centrifugal tube to be centrifuged at 400 Xg for 5min, supernatant is discarded, 24-pore plates are counted and assisted, and MSC 3X 10 is placed in a 10mL centrifugal tube for 5min4Per well 1 mM MSC culture solution (Youkang) at 37 deg.C and 5% CO2Culturing in a fine bubble incubator with saturated humidity.
Example 5: IgE (CH)3) Preparation of DC-CIK + MSC cocultured cells
Respectively drawing blood (or drawing blood from a chest and a heart) from the tail vein of an SD rat (experimental group), and preparing an SD rat allergy model (including preparing negative control serum for sampling and storing) after blood drawing. The experiment requires allogeneic MSC, autologous DCCIK.
After centrifugation of 400g of freshly separated blood components for 10 minutes, the upper plasma component was removed.
Adding 0.9% physiological saline injection into the residual blood in an equal time, and gently mixing.
5ml of the lymphocyte separation medium was added to a 15ml centrifuge tube, and about 5ml of a mixture of blood and physiological saline was slowly dropped on the upper layer, and 400g of the mixture was centrifuged for 20 minutes.
Gently sucking out the stratified leukocyte layer liquid, placing the leukocyte layer liquid in a new centrifuge tube, adding a certain amount of normal saline, gently mixing uniformly, and centrifuging at 400g for 10 minutes.
The supernatant was removed, fresh physiological saline was added, and after gently mixing, 400g was centrifuged for 10 minutes.
The supernatant was removed and fresh physiological saline was added.
Calculating yield and survival rate: 100ul of cell suspension was aspirated, an equal volume of 0.2% trypan blue was added, and mixed well. The total cell density was calculated by counting the total number of cells using a blood count plate, and the viable cell density was calculated by counting the number of viable cells. The method is repeated for three times, the average density is respectively obtained, the harvested cell quantity is converted according to the volume of the inoculated suspension, and the survival rate is obtained by comparing the number of the living cells with the total number of the cells.
After centrifuging 400g of the peripheral blood mononuclear cell suspension for 10 minutes, the supernatant was removed.
The cells were resuspended in DCCIK cell basal medium, serum-free medium, at 1X 105Cell/ml seeding Density cells were seeded in cell 6 plates at 37 ℃ in 5% CO2And (5) carrying out static culture for 3 hours in a saturated humidity incubator.
Gently shaking the cell culture flask, taking out suspended cells, transferring to a new culture flask, adding IFN-gamma 1000U/ml, standing at 37 deg.C and 5% CO2And (5) a saturated humidity incubator for induced culture of CIK cells.
Supplementing DCCIK basic culture medium in original cell culture bottle, adding GM-CSF 1000IU/ml and IL-4500 IU/ml, placing at 37 deg.C and 5% CO2And (5) a saturated humidity incubator for inducing and culturing the DC cells.
The next day, CD was added to CIK cell culture flasks3Monoclonal antibody 100ng/ml and IL-21000U/ml, placing in a well at 37 deg.C and 5% CO2And (5) carrying out static culture in a saturated humidity incubator. Fresh medium serum-free medium + IL-2500U/ml was added every 2-3 days and passaged.
Addition of CH to the following day DC cells3Protein fragments of gene expression. CH (CH)3The concentration of the protein fragment is 50ug-500ug/1 × 107DC cells.
On day 3, two cytokines such as GM-CSF and IL-4 were added to the DC cells according to the amount of the culture medium and the above concentrations, and the DC cells were further induced and cultured to obtain IgE (CH)3) DC cells.
Day 5, IgE (CH)3) Adding TNF-alpha 200IU/ml into DC cell according to the amount of culture medium, standing at 37 deg.C in 5% CO2 saturated humidity incubator for 48 hr to induce IgE (CH)3) The DC cells were matured.
On day 7, IgE (CH) was collected separately3) DC cells and CIK cells were counted according to IgE (CH)3) DC cell CIK cell is 1:3-5 cell number ratio, and the two cells are mixed and supplemented with fresh bloodless bloodThe clear culture medium and IL-2500U/ml are added, and the cell density is adjusted to about 3-5 multiplied by 106The cells/ml were transferred to a new flask and placed at 37 ℃ in 5% CO2Saturated humidity incubator for co-cultivation of IgE (CH)3) The DC cells and the CIK cells are selected,
fresh serum-free medium + IL-2500U/ml was added every 3 days and passaged according to cell proliferation.
The IgE (CH) is administered on days 10-123) The DC-CIK cells were transferred to a culture flask in which the SD rat stem cell MSC (P3) was cultured. MSC cells and IgE (CH)3) Mixing the DC-CIK cells according to the cell number ratio of 1:10-50, and co-culturing at 37C 5% saturation temperature for static culture.
After 72 hours of co-culture, 200g of co-cultured cells were centrifuged for 5 minutes, the supernatant was removed, washed 2 times with physiological saline (200 g. times.5 minutes), counted by cells, and IgE (CH) was counted3) Co-cultured cells of DC-CIK + MSC (dendritic cell-cytokine induced killer) 1X 107The amount of (a) is ready for intravenous injection into allergic model mice.
Example 6: SD rat allergy model experimental design
A little blood is drawn from 5 normal SD rats (without allergic stimulation) and stored as a negative serum sample, 2ml of the blood is taken from 1mg/ml of phoenix tree pollen leachate (allergen) on the third day and used for subcutaneous multipoint injection immunization of the SD rats, the immunization is carried out once a week, and a positive serum sample is taken from the tail vein after every other day and is stored after being stimulated by atomized leachate freeze-dried powder after three weeks of immunization, so that the SD rat animal experimental model for allergic diseases is obtained.
IgE (CH) prepared in example 53) The cells co-cultured by DC-CIK + MSC cells are injected into five allergy model SD rats by tail vein injection once a day, and the injection is used for 1 multiplied by 10 each time7Individual cells (cell count), three consecutive days, and four days later, tail vein blood was collected to prepare serum as test group serum (preservation).
Example 7 IgE enzyme-linked immunoassay
The concentration of SD rat IgE in the sample was determined using a double antigen sandwich method: coating a microporous plate with purified antigen to prepare solid phase antigen, sequentially adding immunoglobulin E (IgE) into the coated microporous plate, combining with HRP-labeled antigen to form an antigen-antibody-enzyme-labeled antigen complex, thoroughly washing, and adding substrate TMB for color development. TMB is converted to blue by the catalysis of HRP enzyme and to the final yellow by the action of acid. The shade of the color is positively correlated with the amount of immunoglobulin E (IgE) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and the concentration of human immunoglobulin E (IgE) in the sample was calculated from the standard curve.
| 1 | 30 times concentrated washing liquid | 20ml X1 bottle | 7 | Stopping liquid | 6ml X1 bottle |
| 2 | Enzyme labeling reagent | 6ml X1 bottle | 8 | Standard solution (8 mug/ml) | 0.5ml X1 bottle |
| 3 | Enzyme-labeled coated plate | 12 holes by 8 strips | 9 | Standard substance diluent | 1.5ml × 1 bottle |
| 4 | Sample diluent | 6ml X1 bottle | 10 | Description | 1 part of |
| 5 | Color developing agent A liquid | 6ml X1 bottle | 11 | Sealing plate film | 2 pieces of paper |
| 6 | Developer B liquid | 6ml X1 bottle | 12 | Sealing bag | 1 is provided with |
The method comprises the following operation steps:
A. and (3) diluting the standard: one stock standard was provided and diluted in a small tube according to the following chart.
| Content of IgE standard | Dilution of | OD450nn | |
| 3μg/ml | Standard substance No. 5 | Adding 150 μ l of original standard substance into 150 μ l of standard substance diluent | 2.42 |
| 1.5μg/ml | Standard substance No. 4 | 150 μ l of No. 5 standard substance was added to 150 μ l of the standard substance dilution | 2.16 |
| 0.5μg/ml | No. 3 standard product | 150 μ l of No. 4 standard substance was added to 150 μ l of the standard substance dilution | 1.85 |
| 0.2μg/ml | Standard article No. 2 | 150 μ l of No. 3 standard substance is added into 150 μ l of standard substance diluent | 1.55 |
| 0.1μg/ml | Standard article No. 1 | 150 μ l of No. 2-No. 7-standard substance was added to 150 μ l of the standard substance dilution | 0.90 |
Quantitative standard curve for preparing IgE standard substance content
The above examples 6 and 7 were sampled and stored with serum, and the three kinds of stored serum samples were simultaneously tested.
B. Blood was taken from the tail vein of SD rats: respectively comprises 5 parts of negative sample (non-sensitized), 5 parts of positive serum (sensitized) and 5 parts of experimental group serum (MSC-FC DCCIK), and is provided with a blank hole (the blank control hole is not added with a sample and an enzyme-labeled reagent, and other steps are the same), a standard hole and a sample hole to be detected. And accurately adding 50 mul of standard sample on the enzyme-labeled coating plate, and adding 25 mul of sample diluent in a sample hole to be detected to obtain the actual concentration of the sample.
Elisa tests the content of IgE in serum of SD rats (negative, positive and experimental groups):
| experiment grouping | Number 1 | Number 2 | No. 3 | Number 4 | Number 5 |
| Blank space | 0.07 | 0.069 | 0.090 | 0.059 | 0.089 |
| Negative serum | 0.092 | 0.102 | 0.089 | 0.141 | 0.150 |
| Positive serum | 1.83 | 1.79 | 2.01 | 2.10 | 1.99 |
| Serum of experimental group | 0.202 | 0.353 | 0.26 | 0.30 | 0.25 |
And comparing with the standard sample curve to find that: the present invention utilizes IgE (CH)3) The DC-CIK + MSC co-cultured cells can produce more favorable results for treating anaphylaxis and autoimmune diseases without any side effect.
IgE is related to various allergic diseases of human beings, type I allergic diseases comprise bronchial asthma allergic rhinitis, atopic dermatitis, food allergy, environmental allergy and the like, the incidence rate of allergy gradually rises in the last 10 years, about 2 hundred million people all over the world suffer from allergic diseases, and the global prevention and treatment of allergic diseases are estimated to be far more than the sum of the allergic diseases for tuberculosis and AIDS from economic cost. At present, new host T cell inflammatory factors and B cell proliferation and differentiation and related factors caused by new crown disease infection are epidemic worldwide, and are accompanied with the sudden and large rise of IgE of patients to form a Syndrome of cytokine Storm (Cytokin Storm Syndrome, CSS).
MSCSMainly produces a large amount of immune regulatory factors, cell tilting factors and growth factors to regulate immune allergic reaction under the allergic and inflammatory environments, and simultaneouslyMSC can also promote in situ tissue stem cell proliferation and tissue repair. MSCSTreating acute graft versus host disease (aGVHD), inhibiting excessive T cell proliferation down-regulates proinflammatory cytokines and levels excessive IgE elevation causing severe rash response.
Leblank discusses: human umbilical cord mesenchymal stem cells (human mesenchymal cord, MSC, hUCMSC)S) Is a group of multipotential cells which can support the restoration of hematopoietic reconstruction and differentiate into various tissue cells, and has low immunogenicity and immunoregulation function, thereby effectively relieving the symptoms of GVHD. MSCSHas synergistic effect with DC-CIK and combined transplantation of hematopoietic stem cells respectively, and is disclosed in the paper of SAya-lian et al: detection of hMSCs by Flow Cytometry (FCM)SAnd (3) the correlation between the content of Th1 and Th2 cytokines and the immune regulation mechanism of MSC in the culture supernatant of the DC-CIK cells cultured at a ratio of 1:10 for 4 days. Nishimura et al, "discussing the effect of hMSC on allogeneic DC-CIK cell molecules", observed that mice receiving allogeneic bone marrow cell transplantation combined with CIK cell therapy all survived, only slight chronic subclinical transplantation anti-host disease (GVHD) appeared, but IFN-r gene knock-out CIK cells appeared fatal GVHD, indicating that CIK alleviates GVHD symptoms and is related to IFN-r, while DC-CIK cell population is CIK-dominant cells, which was found to have significant effect on GVHD.
IgE can cause severe autoimmune diseases, and is a chronic inflammatory disease caused by the fact that autoimmune tolerance is broken, T cells and antibodies react with self cell and tissue antigens to cause the loss or limitation of tissue functions, but the mechanism of breaking autoimmune tolerance is not clear at present. The autoimmune diseases include type I diabetes, rheumatoid arthritis, lupus erythematosus, multiple sclerosis, and myasthenia gravis. The treatment of autoimmune diseases now suppresses the systemic immune system, thereby increasing the incidence of infection and neoplastic disease in patients, using MSC-IgE (CH)3) DCCIK may have good results.
The present invention utilizes IgE (CH)3) The DCCIK + MSCs co-cultured cells for treating anaphylaxis and autoimmune diseases are expected to produce more favorable results, and the DCs present IgE (CH)3) Adding MSC into the CIK for co-culture, and performing co-culture between cellsThe stem cell differentiation is affected by the extracellular matrix, and more or less stem cells are located in the extracellular matrix, while the latter is usually the stem cells and the products of their daughter cells, which have different structural characteristics in different in vitro culture environments or in vivo tissues, and the production of proteoglycan and mucin, etc. causes the interaction between cells, in which the MSCs secrete chemokines, and the training functions, such as training CIK cells and CD, are involved8 +T cells, and the like. The literature reports that the co-culture of MSCs and DC-CIK relates to the theory of cell biology and molecular biology.
The mechanism of action of immune modulation of MSCs is not fully understood by the authors, and experiments have shown that cell contact or indirect contact is via soluble cytokines secreted by MSCs such as: TGF-. beta.s, PGE2, etc., Meisel et al, thought to be associated with MSC activation of indoleamine 2,3 dioxygenase (IDO) following IFN-r stimulation, Aggarwal reported that MSC caused Th1The secretion of IFN-r by cells is reduced, while Th2IL-4 secretion by cells is increased. Koelreuterin reports that MSCs inhibit secretion of IFN-r and IL-2 by PHA-activated lymphocytes. The MSCs are reported to promote freshly separated lymphocytes to secrete IL-2 and IL-10, the MSCs and DCCIK are reported to be co-cultured to find that the interconversion of Th1 cytokines and Th2 cytokines and Th1 and Th2 factors, namely the co-culture of the MSCs and sensitized DCCIK mainly generates cellular immunity, and the MSC has the results of some current researches that certain cells are induced to differentiate into functional cells and even repair the functions of tissues and organs, and can be compliant with the generation direction of the cells in the microenvironment in the whole generation process, wherein the function of stem cells is fully expressed and the IgE (CH)3) In co-culture of DC + CIK, MSC contributes to T cell clearance, IgE-pathogenic cells and block IgE sensitization pathways by conduction of various cytokines.
DC cells are Antigen Presenting Cells (APC) in the immune system, and the DC cells can swallow Antigen or take DC receptor mediated Antigen, then the Antigen is processed, degraded into polypeptide fragments, combined with MHC molecules to form MHC molecule complexes, transferred to DC surface and combined with T Cell surface TCR to present Antigen peptide to CD4 +T cells and processes thereofMHC class 1 molecules bind or are presented in the MHC class II pathway, depending on the source of the antigen. DCCIK cells are induced and amplified in vitro to belong to the cellular immune effect, CIK is a novel immunocompetent cell, and the co-culture of DC cells and CIK is two important parts of cellular immunotherapy. The former recognizes antigens, activates the adaptive immune system, and the latter exerts its own cytotoxic effects through DC signaling and secretes a large amount of cytokines to eliminate pathogenic cells (including Cancer cells).
The DC and the CIK cells are mutually regulated during co-culture, the CIK cells can promote the maturation of the DC, and the DC cells can enhance the proliferation capacity and the killing activity of the CIK cells. Examples are: IgE (CH)3) Co-culturing the DC and CIK, and marking the surface of the DC with CD 80; CD 86; increased expression of CD40 human leukocyte antigen. DC presentation of IgE (CH)3) The antigen process, secretion cytokines IL-1 alpha, IL-8, TNF-alpha, INF-alpha and GM-CSF, etc. play a regulating role, and chemotactic factors secreted by DC mediate and activate CIK cells. DCs can also activate different subsets of T cells, causing Th cells to differentiate in different directions, thereby inducing B cell immune responses.
The above embodiments are provided only for illustrating the present invention and not for limiting the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and therefore all equivalent technical solutions should also fall within the scope of the present invention, and should be defined by the claims.
Claims (8)
1. A cell therapy composition comprising a therapeutically effective amount of DC cells, CIK cells and MSC cells.
2. The cell therapy composition according to claim 1, wherein the DC cells and CIK cells are autologous cells, the MSC cells are allogeneic cells, and the DC cells carry CH of IgE3A protein fragment.
3. A method of preparing a cell therapy composition comprising the steps of:
(1) preparing autologous peripheral blood mononuclear cells;
(2) isolation and culture of DC cells;
(3) induction culture of CIK cells;
(4) addition of CH to IgE to DC cells3Preparation of IgE (CH) protein fragment3) A DC cell;
(5)IgE(CH3) Co-culture of DC cells and CIK cells to produce IgE (CH)3) DC-CIK cells;
(6)IgE(CH3) The DC-CIK cells and allogeneic MSC cells are co-cultured to prepare the cell therapeutic composition.
4. The method of claim 3, wherein in step (4), the CH is present in the composition3The concentration of the protein fragment is 50ug-500 ug/1X 107DC cells.
5. The method of claim 3, wherein in step (4), the CH is present in the composition3The protein fragment is obtained by adopting eukaryotic cell gene expression.
6. The method of claim 3, wherein in step (5), IgE (CH) is3) The DC cells and the CIK cells are mixed and cultured according to the cell number ratio of 1: 3-5.
7. The method of claim 3, wherein in step (6), the MSC cells are contacted with IgE (CH)3) The DC-CIK cells are mixed and co-cultured according to the cell number ratio of 1: 10-50.
8. The IgE (CH) according to claim 33) Application of cell therapeutic composition obtained by co-culturing DC-CIK cells and MSC cells in preparation of cell medicines for treating anaphylaxis and autoimmune diseases.
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