CN113403274B - Cell therapeutic composition, preparation method thereof and application of cell therapeutic composition as allergic reaction and autoimmune disease therapeutic drug - Google Patents
Cell therapeutic composition, preparation method thereof and application of cell therapeutic composition as allergic reaction and autoimmune disease therapeutic drug Download PDFInfo
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Abstract
The present invention provides a cell therapeutic composition comprising IgE (CH 3 ) I gE (CH) obtained by co-culturing DC and CI K cells 3 ) DC-CI K cells are co-cultured with allogeneic MSC cells to prepare cellsThe invention also provides an application of the cell therapeutic composition in preparing a cell medicament for treating allergic reaction and autoimmune diseases. The invention uses IgE (CH) 3 ) DCCI k+mscs co-cultured cells treat allergic reactions and autoimmune diseases, which have been shown to produce more favorable results without any side effects.
Description
Technical Field
The invention relates to the field of cellular immunity, in particular to a cell therapeutic composition, a preparation method thereof and application thereof in treating allergic reaction and autoimmune diseases.
Background
Currently, the treatment of allergic reactions, such as allergic asthma, uses mainly a broad spectrum immunosuppressive glucocorticoid. Although the medicines have a certain effect on controlling allergic reaction, the medicines have obvious side effects due to the inhibition effect on most immune cells, such as low immune function of organisms and easy induction of diseases such as infection, tumor and the like after long-term use.
Therefore, how to develop a therapeutic agent for allergic reaction without side effects or with less side effects has been one of the directions that those skilled in the art have focused on developing.
Disclosure of Invention
The present invention has been made in order to solve the above problems, and an object of the present invention is to provide a cell therapy composition and a method for preparing the same, which can effectively treat allergic diseases caused by allergens and acute transfer plant anti-host diseases and even epidemic new crown diseases at present, and has no side effects.
The purpose of the invention is realized in the following way:
the invention provides a cell therapeutic composition comprising therapeutically effective amounts of DC cells, CIK cells and MSC cells.
The above cell therapeutic composition, wherein the DC cells and CIK cells are autologous cells, the MSC cells are allogeneic cells, and the DC cells carry CH of IgE 3 A protein fragment.
The invention also provides a preparation method of the cell therapeutic composition, which comprises the following steps:
(1) Preparing autologous peripheral blood mononuclear cells;
(2) Isolation and culture of DC cells;
(3) Induction culture of CIK cells;
(4) IgE-added CH in DC cells 3 Protein fragments, preparation of IgE (CH) 3 ) A DC cell;
(5)IgE(CH 3 ) Co-culture of DC cells with CIK cells to prepare IgE (CH 3 ) DC-CIK cells;
(6)IgE(CH 3 ) The DC-CIK cells were co-cultured with allogeneic MSC cells to prepare cell therapeutic compositions.
The method for preparing a cytotherapeutic composition as described above, wherein in step (4), the CH 3 The concentration of the protein fragment is 50ug-500ug/1×10 7 DC cells.
The method for preparing a cytotherapeutic composition as described above, wherein in step (4), the CH 3 The protein fragment is obtained by eukaryotic cell gene expression.
The method for preparing a cell therapeutic composition, wherein in step (5), igE (CH 3 ) DC cells and CIK cells were mixed and co-cultured in a cell number ratio of 1:3-5.
The method for preparing a cell therapeutic composition, wherein in step (6), MSC cells and IgE (CH) 3 ) DC-CIK cells were co-cultured in a cell number ratio of 1:10-50.
IgE (CH) 3 ) Cell therapeutic composition obtained by co-culturing DC-CIK cells and MSC cells and used for treatingCell medicines for treating anaphylaxis and autoimmune diseases.
IgE(CH 3 ) Glycosylation after gene expression binds to mannose receptors on DC cells, co-cultures with CIK, DC is a professional presenting cell, DC and IgE (CH 3 ) Identification of presentation followed by co-culture with CIK promotes proliferation of CIK, while CIK cells also promote maturation of DCs by secretion of complementary cytokines from DCs and CIK. In IgE (CH) 3 ) The cell activity of CIK is stimulated in the co-culture process of DC and CIK, so that T killer cells such as CIK are endowed with IgE (CH) 3 ) The memory of the cell line is used for eliminating pathogenic somatic cells in the body and reducing the occurrence of allergic diseases, namely cell-mediated cellular immune effect.
DC internalize IgE (CH) carrying during culture 3 ) The MHC II molecules of the DC cells are activated to form a complex, and then presented to the Th cells, which recognize the B cells through the signal molecules in vivo, causing B cell proliferation, antibody production and antibody class switching, which is a B cell mediated humoral immune effect.
The invention selects MSC cells and IgE (CH) 3 ) DC-CIK cells were then co-cultured in IgE (CH 3 ) MSC can secrete multicellular factors through contact and internalization among co-cultured cells in a specific microenvironment of a culture system of DC-CIK, particularly secrete a T effector cell under the condition that the tilting factor can be trained, so that the MSC can be induced to differentiate into cells with different phenotypic characteristics under the specific culture condition, and can be injected into a body to more effectively remove secreted IgE or bound IgE (CH) 3 ) Is a disease-causing somatic cell.
MSC (human umbilical cord mesenchymal stem cells) is an early-stage gene-free MHC and has good regulation effect on an immune system, and many experiments prove that MSC has good results for treating allergic diseases caused by allergic sources and acute transfer plant anti-host diseases and even epidemic new crown diseases at present.
The present invention relates to the treatment of allergic diseases: (1) t cell immune effects; (2) DC rendering CH 3 Cellular immune effects of antigen production; (3) MSC (Mobile switching center) S Single immunoregulatory effect and MSC and upper part thereofAnd the DCCIK cells cooperate and link functional effects. The present invention uses IgE (CH) 3 ) The dccik+mscs co-cultured cells treat allergic reactions and autoimmune diseases, and experiments prove that the dccik+mscs co-cultured cells produce more favorable results without any side effects.
Detailed Description
The invention is further illustrated, but not limited, by the following examples. The reagent materials described in the examples below are commercially available common materials, except where sources are noted, and the reagents are formulated in a conventional manner. Methods not described in detail in the examples are all routine in the art.
In the examples of the invention, SD rats were used as the experimental animals.
Example 1: acquisition of IgE by genome-wide PCR 3 Gene fragment and construction of recombinant plasmid
Two pairs of primers P1 and P2 were synthesized by PCR. The cleavage sites at the 5 'and 3' ends of the primers P1 and P2 were introduced into EcoRV and EcoRI cleavage sites, respectively, to construct pVAC-CH 3 Recombinant plasmids.
P1:5'-GGGATATCATGCCGACAGATCATGAGCCACG-3' (EcoRV cleavage site underlined)
P2:5'-GGGAATTCTCACTGGGGAAGGGTGATGGAAC-3' (EcoRI cleavage site underlined)
Using SD rat whole genome DNA as a template, a 100. Mu.L reaction system was established, comprising 10. Mu.L of 10 XPCR reaction buffer, 25mM MgCl 26. Mu. L, DMSO 5. Mu.L, dNTP 8. Mu.L, 4. Mu.L of each of the upstream and downstream primers, 4. Mu.L of the rat whole genome DNA, and 60. Mu.L of water. Then, PCR was performed, and 3.2. Mu.L of TaqDNA polymerase was added thereto after 5min of pre-denaturation at 95 ℃. The reaction conditions are as follows: denaturation at 94℃for 1min, annealing at 56℃for 1min and extension at 72℃for 2min was performed for 39 cycles. The last cycle was extended at 72℃for 10min and stored at 4 ℃.
Construction of recombinant plasmid pVAC-CH3
Using whole genome DNA as template, using primers P1, P2 to amplify out CH of IgE 3 The fragment was ligated into PMD18-Tvector, and the recovered small fragment was ligated with the large fragment recovered by double digestion of the pVAC empty vector by double digestion of T-CH3 with EcoRI and EcoRV. ConnectionThe product transformation competent cell DH5 alpha, zeocin anti-double-element screening, selecting positive clone, culturing in LB culture medium overnight, extracting plasmid DNA, enzyme cutting identification and DNA sequence analysis, and naming the correct sequencing as pVAC-CH 3 。
Amplification of recombinant plasmids
The recombinant plasmid pVAC-CH3 is used for transforming competent cells DH5 alpha, culturing overnight, picking a monoclonal in the next morning, inoculating in 10mL of LB culture medium containing zeocin antibiotics for culturing for 8 hours, inoculating in 300mL of LB culture medium containing zeocin antibiotics according to the ratio of 1:100, shaking at 37 ℃ for 2.5 hours, and culturing for 16 hours under vigorous shaking at 37 ℃.
Large-scale extraction and purification of recombinant plasmids
The bacterial pellet was resuspended in 50mL of STE pre-chilled with ice, washed once, and the supernatant discarded by centrifugation at 4100rpm for 20min at 4℃with an appropriate rotor. The bacterial precipitate is resuspended in 10mL of solution I, 1mL of newly prepared lysozyme solution is added, 20mL of newly prepared solution II is added, the bottle cap is closed, the centrifuge bottle is slowly reversed for several times, the contents are fully mixed, and the mixture is placed at room temperature for 5-10min. 15mL of ice-chilled solution III are added. The bottle mouth was sealed and the centrifuge bottle was shaken several times to mix the contents. Placing on ice for 10min to form a white flocculent precipitate.
The rotor was stopped naturally by centrifugation at 11000rpm for 30min at 4℃with a suitable rotor. The supernatant was carefully transferred in its entirety to another bottle and the pellet in the centrifuge tube was discarded. The volume of the supernatant was measured, transferred into a clean centrifuge tube together with 0.6 times of the volume of isopropyl alcohol, and left to stand at room temperature for 10min. The nucleic acid precipitate was recovered by centrifugation at 8000rmp for 15min at room temperature. Carefully pour the supernatant, open the centrifuge tube, and invert onto paper towels to remove residual supernatant. The bottom and walls of the deposited tube were washed with 70% ethanol at room temperature. The ethanol was decanted, all droplets attached to the walls of the flask were aspirated with a Pasteur pipette connected to a vacuum apparatus, the centrifuge tube was opened and placed onto a paper towel, the residual traces of ethanol were evaporated to dryness, and the nucleic acid pellet was solubilized with 2mLTE (pH 8.0). Phenol, phenol: the plasmids were purified 1 time in chloroform and chloroform. OD was measured after dilution of nucleic acid pellet 1:100 with TE (pH 8.0) 260 /OD 280 Meter (D)Calculation of plasmid DNA concentration (1 OD 260 =50 μg plasmid DNA/mL), the extracted plasmid was singly digested and subjected to agarose electrophoresis to examine the effect, and then the DNA was stored at-20 ℃.
Example 2: CH (CH) 3 Eukaryotic expression, extraction and purification of protein fragments
CHO cells with good growth status were fine-bubbled at 1×10 per well 5 The cells were inoculated in 6-well plates and washed 2 times with serum-free antibiotic-free RPMI 1640 medium when the cells were grown to 80% by total anchorage. Mu.g of DNA was dissolved in 100. Mu.L of serum-free antibiotic-free RPMI 1640 medium, designated as solution A. 10 mu.L of LLiopofectAMINETM 2000 was dissolved in 90. Mu.L of serum-free antibiotic-free RPMI 1640 medium and left for 10min, designated as solution B. A, B the two solutions were mixed and incubated at room temperature for 30min to allow the DNA-lipome complex to form. Adding 800. Mu.L of serum-free and antibiotic-free DMEM culture solution into the complex, gently mixing, slowly dripping into the washed cells, and adding CO at 37deg.C 2 Culturing in incubator for 6 hr. The transfection liquid is sucked and removed, 1mL of antibiotic-free RPMI 1640 culture liquid containing 20% calf serum is added for continuous culture, cells are collected for detection of transient expression of target proteins after 48h of transfection, and CHO cells which are transfected and successfully expressed are obtained through screening.
Through eukaryotic expression of CHO cells, CH is finally obtained by extraction and purification 3 The protein fragment was ready for use.
Example 3: preparation of Chinese parasol pollen extract
The phoenix tree pollen is a common allergen, allergic diseases such as bronchial asthma, allergic rhinitis and the like are often induced, specific IgE exists in serum of a pollen patient, and fluctuation of the IgE content in the blood is large, and stability is poor, so that an experimental animal model with stable IgE secretion is established for the correctness of the experiment, and the allergic reaction of the phoenix tree pollen is similar to that of a human being by using a domestic common SD rat, and the relationship between the mechanism and treatment of the allergic diseases of the human being is also researched by the mediation of IgE.
The phoenix tree pollen is naturally dried to constant weight, degreased with diethyl ether for several times until the diethyl ether layer is colorless, and 10mLCOCA's (5.0 g sodium chloride, 2.75g sodium bicarbonate, 4mL phenol, distilled water to 1000 mL) solution (1:10, w/v) is added per gram of pollen after thorough volatilization of diethyl ether. Leaching at 4deg.C for 48 hr with magnetic force under stirring for 20min, centrifuging to obtain supernatant with protein content of about 1mg/mL, dialyzing the leachate in dialysis bag with PBS until the external solution is colorless, aseptically filtering, packaging, and freeze-preserving.
Example 4: preparation of SD rat MSC cells
Healthy 6-10 SD rats (200 g/rat) are not limited in male and female, and are sacrificed in vacuum, and 75% ethanol is soaked for 5min in sterile ultra-clean bench rats with femur and tibia on both sides. The muscle is peeled off by aseptic shearing, the femur and tibia which are separated cleanly are put into PBS liquid, the femur and tibia are sheared off by large-size shearing, PBS liquid is sucked by a 1mL aseptic syringe to repeatedly flush the bone marrow cavity of the femur and tibia, then the femur and tibia are sucked out and put into a 10mL centrifuge tube to be centrifuged for 5min at 400 Xg, the supernatant is discarded, and a 24-hole plate and an MSC 3X 10 are used for counting 4 1 mM SC culture solution (YOUKANG) was placed in the well at 37deg.C with 5% CO 2 Culturing in a fine bubble incubator with saturated humidity.
Example 5: igE (CH) 3 ) Preparation of DC-CIK+MSC coculture cells
The tail vein of the SD rat (experimental group) is adopted to draw blood (or open chest heart blood sampling), and an SD rat allergy model (comprising the preparation of negative control serum sampling and preservation) is prepared after blood sampling. The experiment requires allogeneic MSC, autologous DCCIK.
After 400g of freshly separated blood components were centrifuged for 10 minutes, the upper plasma components were removed.
Adding 0.9% physiological saline injection into the residual blood in equal proportion, and gently mixing.
5ml of lymphocyte separation solution was added to a 15ml centrifuge tube, and the mixture of blood and physiological saline was slowly dropped onto the top layer, and centrifuged at 400g for 20 minutes.
The layered leukocyte layer liquid was gently aspirated, placed in a new centrifuge tube, added with a certain amount of physiological saline, gently mixed, and centrifuged at 400g for 10 minutes.
The supernatant was removed, fresh physiological saline was again added, and after gentle mixing, 400g was centrifuged for 10 minutes.
The supernatant was removed and fresh physiological saline was replenished.
Calculated yield and survival rate: 100ul of cell suspension was aspirated, and an equal volume of 0.2% trypan blue was added and mixed well. The total cell density was calculated by counting the total cells with a hemocytometer, and the viable cell density was calculated by counting the viable cells. This procedure was repeated three times, and the average densities were taken separately, and the amount of cells harvested was converted from the volume of the inoculated suspension, and the survival rate was obtained as the number of living cells compared with the total number of cells.
After centrifugation of 400g of peripheral blood mononuclear cell suspension for 10 minutes, the supernatant was removed.
Cells were resuspended in DCCIK cell basal medium, i.e., serum-free medium, according to 1X 10 5 Cell culture at 37℃and 5% CO 2 The culture was allowed to stand in a saturated humidity incubator for 3 hours.
The cell culture flask was gently shaken, the suspension cells were removed, transferred to a new flask, and IFN-. Gamma.1000U/ml was added, and the flask was placed at 37℃with 5% CO 2 And (5) a saturated humidity incubator, and performing induced culture on CIK cells.
Supplementing DCCIK basal medium into original cell culture flask, adding 1000IU/ml GM-CSF and 500IU/ml IL-4, placing at 37deg.C and 5% CO 2 The saturated humidity incubator, the induced culture of DC cells.
The next day, CD was added to CIK cell culture flasks 3 Monoclonal antibody 100ng/ml and IL-2 1000U/ml, placed in a well at 37℃and 5% CO 2 And (5) standing and culturing in a saturated humidity incubator. Fresh medium serum-free medium + IL-2 500u/ml was added every 2-3 days and passaged.
Addition of CH to the next day DC cells 3 A protein fragment expressed by the gene. CH (CH) 3 The concentration of the protein fragments is 50ug-500ug/1×10 7 DC cells.
On day 3, two cytokines such as GM-CSF and IL-4 are supplemented to DC cells according to the amount of the culture medium and the concentration, and the DC cells are continuously induced and cultured to obtain IgE (CH) 3 ) DC cells.
On day 5, igE (CH 3 ) TNF-alpha 200IU/ml was added to DC cells in the amount of medium, and the mixture was placed in a 5% CO2 saturated humidity incubator at 37℃for 48 hours to induce IgE (CH) 3 ) DC cells mature.
On day 7, igE (CH) 3 ) DC cells and CIK cells were counted and purified according to IgE (CH 3 ) DC cells, CIK cells are mixed according to the cell number ratio of 1:3-5, fresh serum-free culture medium and IL-2 500U/ml are supplemented, and the cell density is regulated to about 3-5 multiplied by 10 6 Individual cells/ml, transferred to a fresh flask, placed at 37℃in 5% CO 2 Saturated humidity incubator, co-culture IgE (CH 3 ) The DC cells and the CIK cells,
fresh serum-free medium + IL-2 500u/ml was added every 3 days and passaged according to the proliferation of cells.
The IgE (CH) was prepared on days 10-12 3 ) DC-CIK cells were transferred to flasks where SD rat stem cells MSC (P3) were cultured. MSC cells and IgE (CH) 3 ) DC-CIK cells are mixed according to the cell number ratio of 1:10-50, and co-cultured at 37C 5% saturation temperature for static culture.
After 72 hours of co-cultivation, 200g of the co-cultivated cells were centrifuged for 5 minutes, the supernatant was removed, and washed with physiological saline 2 times (200 g. Times.5 minutes), and by cell counting, they were purified according to IgE (CH) 3 ) DC-CIK+MSC coculture cells 1X 10 7 Is used for intravenous injection in allergy model mice.
Example 6: SD rat allergy model experiment design
5 normal SD rats (without anaphylactic stimulation) are sampled and a little blood is taken as a negative serum sample to be stored, 2ml of the extract (allergen) of the phoenix tree pollen is taken for subcutaneous multipoint injection immunization of the SD rats in the third day, the positive serum sample is sampled and stored after three weeks of immunization and after a period of days after the injection of the freeze-dried powder of the atomized extract, and thus the SD rat animal experimental model for establishing allergic diseases is obtained.
IgE (CH) prepared in example 5 3 ) Cells co-cultured with DC-CIK+MSC cells were injected once daily by tail vein injection for five allergy model SD rats, 1×10 each time 7 The tail vein blood was collected three consecutive days after four days to prepare serum as the serum of the experimental group (preservation).
EXAMPLE 7 IgE ELISA
Determination of SD rat IgE concentration in samples using double antigen sandwich method: coating a micropore plate with purified antigen to prepare solid-phase antigen, sequentially adding immunoglobulin E (IgE) into the coated micropore, combining with HRP-marked antigen to form antigen-antibody-enzyme-labeled antigen complex, and thoroughly washing and adding a substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and to final yellow under the action of acid. The shade of color and immunoglobulin E (IgE) in the sample are positively correlated. 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 a standard curve.
| 1 | 30 times concentrated washing liquid | 20ml x 1 bottle | 7 | Stop solution | 6ml x 1 bottle |
| 2 | Enzyme-labeled reagent | 6ml x 1 bottle | 8 | Standard solution (8 mug/ml) | 0.5ml×1 bottle |
| 3 | Enzyme-labeled coated board | 12 holes x 8 strips | 9 | Standard substance diluent | 1.5ml×1 bottle |
| 4 | Sample diluent | 6ml x 1 bottle | 10 | Description | 1 part of |
| 5 | Color reagent A liquid | 6ml x 1 bottle | 11 | Sealing plate film | 2 sheets of |
| 6 | Color reagent B liquid | 6ml x 1 bottle | 12 | Sealed bag | 1 number of |
The operation steps are as follows:
A. dilution of standard: one stock of the original standard was provided and diluted in small tubes according to the following chart.
| IgE standard content | Dilution of | OD 450nn | |
| 3μg/ml | Standard No. 5 | 150 μl of the original multiple standard was added to 150 μl of the standard diluent | 2.42 |
| 1.5μg/ml | No. 4 standard substance | 150 μl of standard No. 5 is added to 150 μl of standard diluent | 2.16 |
| 0.5μg/ml | Standard No. 3 | 150 μl of standard No. 4 is added to 150 μl of standard diluent | 1.85 |
| 0.2μg/ml | Standard No. 2 | 150 μl of standard No. 3 was added to 150 μl of standard diluent | 1.55 |
| 0.1μg/ml | Standard No. 1 | 150 μl of No. 2-7-standard was added to 150 μl of standard diluent | 0.90 |
Quantitative standard curve for preparing content of IgE standard substance
The samples of the stored serum in examples 6 and 7 were sampled and the three stored serum samples were each synchronously tested.
B. SD rat tail vein blood collection: respectively 5 parts of negative samples (non-sensitized), 5 parts of positive serum (sensitized) and 5 parts of experimental group serum (MSC-FC DCCIK), and respectively arranging blank holes (blank control holes are not added with samples and enzyme-labeled reagents, and the rest steps are the same), standard holes and holes of samples to be tested. And (3) accurately adding 50 μl of standard substance on the enzyme-labeled coated plate, and adding 25 μl of sample diluent into the sample hole to be detected to obtain the actual concentration of the sample.
Elisa detects IgE content in serum of SD rats (negative, positive and experimental groups):
| experimental grouping | No. 1 | No. 2 | No. 3 | No. 4 | No. 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 |
| Laboratory group serum | 0.202 | 0.353 | 0.26 | 0.30 | 0.25 |
Comparison with standard sample curve review reveals that: the present invention uses IgE (CH) 3 ) The co-culture of cells with DC-CIK+MSC for treating allergic reactions and autoimmune diseases produces more favorable results without any side effects.
IgE is a related molecule of various allergic diseases in humans, type i allergic diseases including bronchial asthma allergic rhinitis, atopic dermatitis, food allergy, environmental allergy, etc., and the allergic incidence has been gradually increased in the last 10 years, about 2 million people worldwide have allergic diseases, and it is estimated that the global economic costs for preventing allergic diseases have far exceeded the sum for tuberculosis and aids. At present, host T cell inflammatory factors and B cell proliferation and differentiation and related factors caused by new crown disease infection are popular worldwide, and the IgE of patients is suddenly and greatly increased to form a cytokine storm syndrome (Cytokin Storm Syndrome, CSS).
MSC S A large amount of immune regulation factors are mainly generated in allergic and inflammatory environments, cell tilting factors and growth factors regulate immune allergic reactions, and MSC can promote in-situ tissue stem cell proliferation and tissue repair. MSC (Mobile switching center) S Treatment of acute graft versus host disease (aGVHD), inhibition of excessive T cell proliferation down regulates pro-inflammatory cytokines and suppresses excessive IgE elevation causing severe rash reactions.
LeBlane discussion: human umbilical cord mesenchymal stem cells (human umbilical cord, MSC, hUCMSC) S ) Is a group of multipotent cells which support the recovery of hematopoietic remodeling and differentiation into various tissue cells, and has the functions of low immunogenicity and immunoregulation, thereby effectively alleviating the symptoms of GVHD. MSC (Mobile switching center) S Synergistic effects with DC-CIK in combination with hematopoietic stem cell transplantation, SAya-lia et al: detection of hmscs by Flow Cytometry (FCM) S Correlation of Th1, th2 cytokine levels with the immunoregulatory mechanism of MSC in culture supernatant of 4d co-cultured with DC-CIK cells at 1:10. Nishimura et al, "discuss the effect of hMSC on allogeneic DC-CIK cell molecules", observed that mice receiving allogeneic bone marrow cell transplantation in combination with CIK cell therapy survived all but only slightly chronic subclinical Graft Versus Host Disease (GVHD), but that CIK cells infused with IFN-r knockdown showed fatal GVHD, indicating that CIK alleviating GVHD symptoms was associated with IFN-r, whereas DC-CIK cell populations were CIK-based cells, and were seen to have a significant effect on GVHD.
IgE can severely cause autoimmune diseases due to chronic inflammatory diseases resulting from the loss or limitation of tissue function caused by the disruption of autoimmune tolerance and the reaction of T cells and antibodies with their own cell and tissue antigens, but the mechanism by which autoimmune tolerance is disrupted is currently unknown. Type I diabetes, rheumatoid arthritis, lupus erythematosus multiple sclerosis, and myasthenia gravis. Current treatments for autoimmune diseases suppress the systemic immune system, thereby increasing the production of infection and neoplastic disease in patients using MSC-IgE (CH 3 ) DCCIK may have very good results.
The present invention uses IgE (CH) 3 ) Dccik+mscs co-cultured cells for the treatment of allergic reactions and autoimmune diseases are expected to produce more favorable results, DC presenting IgE (CH 3 ) MSC is added in the co-culture with CIK to co-culture, the contact action between cells causes the direct contact of cytoplasmic membranes and the generation of extracellular matrix (ECM), stem cell differentiation is influenced by the extracellular matrix, a few stem cells are positioned in the extracellular matrix, the latter often is the products of the stem cells and the sub-cells thereof have different structural characteristics in different in-vitro culture environments or in-vivo tissues, proteoglycan, mucin and the like are generated to cause the interaction between cells, wherein the chemotactic factors secreted by MSCs, the training functions, such as the training of CIK cells and CD 8 + T cells, and the like. Related literature reports related theories concerning cell biology and molecular biology concerning co-culture of MSCs with DC-CIK.
The mechanism of action of MSCs immunomodulation is not completely understood by the scholars, and experiments have shown that cell contact or indirect contact is through soluble cytokines secreted by MSCs such as: TGF-beta, PGE2, etc., meisel et al believe that MSC is involved in activating indoleamine 2,3 dioxygenase (IDO) after IFN-r stimulation, aggarwal reports that MSC makes Th 1 Cell secretion IFN-r is reduced, while Th 2 The secretion of IL-4 by cells is increased. Luan Xiying MSCs are reported to inhibit IFN-r, IL-2 secretion by PHA activated lymphocytes. There are also reports that MSCs promote secretion of IL-2 and IL-10 from freshly isolated lymphocytes, that MSCs co-culture with DCCIK found that Th1 cytokines and Th2 cytokines and the interconversion of Th1 and Th2 factors-MSCs co-culture with sensitized DCCIK produced mainly cellular immunity, and that MSC present some research results in that some cells were induced to differentiate into functional cells and even to repair tissue organ functions, and that they were in the direction of cytogenesis in the microenvironment throughout the whole process, where stem cell action was fully manifested, in IgE (CH 3 ) In the co-culture of DC+CIK, MSC promotes T cell clearance, pathogenic cells of IgE and blocks IgE sensitization pathway through the conduction of various cytokines.
The DC cells are antigen presenting cells (Antigen Presenting Cell, APC) in the immune system, and the DC endocytosesAntigen uptake or antigen uptake mediated by DC receptor, then antigen processing, degradation to polypeptide fragments, and binding to MHC molecules to form MHC molecule complexes, transfer to DC surface and binding to T cell surface TCR to present antigen peptides to CD 4 + T cells are processed by binding to MHC-1 molecules or by presenting them in the MHC class II pathway, depending on the source of the antigen presented. In vitro induction amplification of DCCIK cell immune effect, CIK is a novel immunocompetent cell, and co-culture of DC cells and CIK is two important parts of cellular immunotherapy. The former recognizes antigens, activates the acquired immune system, and the latter exerts its own cytotoxic effects through DC signaling and secretes large amounts of cytokines to clear pathogenic cells (including Cancer cells).
When the DCs and the CIK cells are co-cultured, the DCs and the CIK cells are mutually regulated, the CIK cells can promote the maturation of the DCs, and meanwhile, the DC cells can enhance the proliferation capacity and the killing activity of the CIK cells. Examples: igE (CH) 3 ) Co-culturing DC and CIK, and marking CD80 on the surface of the DC; CD86; CD40 human leukocyte antigen expression was increased. DC presentation IgE (CH) 3 ) Antigen process, secretion cytokines IL-1 alpha, IL-8, TNF-alpha, INF-alpha and GM-CSF play a regulatory role, and DC secreted chemotactic factors mediate and activate CIK cells. DCs can also activate T cells of different subclasses, differentiating Th cells in different directions, inducing a B cell immune response.
The above embodiments are provided for illustrating the present invention and not for limiting the present invention, and various changes and modifications may be made by one skilled in the relevant art without departing from the spirit and scope of the present invention, and thus all equivalent technical solutions should be defined by the claims.
Claims (7)
1. A cell therapeutic composition comprising a therapeutically effective amount of a DC cell, a CIK cell and a MSC cell, wherein the DC cell and the CIK cell are autologous cells, wherein the MSC cell is allogeneic cells, and wherein the DC cell carries IgE in CH 3 Protein fragments carried by CH with IgE added to DC cells 3 A protein fragment.
2. 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) IgE-added CH in DC cells 3 Protein fragments, preparation of IgE (CH) 3 ) A DC cell;
（5）IgE(CH 3 ) Co-culture of DC cells with CIK cells to prepare IgE (CH 3 ) DC-CIK cells;
（6）IgE(CH 3 ) The DC-CIK cells were co-cultured with allogeneic MSC cells to prepare cell therapeutic compositions.
3. The method of claim 2, wherein in step (4), the CH is used as a carrier for the cell therapy composition 3 The concentration of the protein fragment is 50ug-500ug/1×10 7 DC cells.
4. The method of claim 2, wherein in step (4), the CH is used as a carrier for the cell therapy composition 3 The protein fragment is obtained by eukaryotic cell gene expression.
5. A method of preparing a cytotherapeutic composition as claimed in claim 2 wherein in step (5) IgE (CH 3 ) DC cells and CIK cells were mixed and co-cultured in a cell number ratio of 1:3-5.
6. The method of claim 2, wherein in step (6), the MSC cells and IgE (CH 3 ) DC-CIK cells were co-cultured in a cell number ratio of 1:10-50.
7. IgE (CH) as claimed in claim 2 3 ) Co-culture of DC-CIK cells and MSC cellsThe application of the obtained cell therapeutic composition in preparing cell medicines for treating anaphylactic reaction is provided.
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