28 results about "K562 cells" patented technology

The application provides a kind of HLA-A24:02 APC cell preparation method and application, it is related to cell preparation technical field.The present application constructs the APC cell (K562-HLA-A24:02) that can replace HLA-A24:02 subtype DC cell, fills the blank of HLA-A24:02 subtype special engineering APC cell, and the cell can realize the antigen presentation function consistent with natural HLA-A24:02 subtype DC cell, provides special tool cell for the immune research for the HLA subtype;The present application constructs APC cell with K562 cell line as base, K562 cell can be in vitro permanent passage amplification, without repeatedly separating and inducing DC cell from human peripheral blood, reduces the use amount of peripheral blood, reduces the raw material dependence and cost of cell acquisition, while avoiding the influence of individual difference of peripheral blood source on experimental results.

The present invention provides an application of thyroid hormones and its analogues in preparation of drugs for treating alpha-thalassemia, especially in preparation of drugs for regulating expression of zeta-globin gene. During differentiation of K562 cells, thyroid hormone analogue (Triac) can significantly up-regulate the expression of zeta-globin gene (HBZ) by 50 folds or above. The expression of zeta-globin gene (hbae5) can also be up-regulated by 30-70 folds in zebrafish treated with thyroid hormones and thyroid hormones analogues. Therefore, according to the present invention, the expression of zeta-globin gene can be significantly activated by thyroid hormones and its analogues, which may develop new potential therapies for patients with alpha-thalassemia. It provides an economical, safe and effective method for treating alpha-thalassemia and can be widely used.

The present application belongs to the field of cell therapy and immunotherapy, and provides a genetically engineered human trophoblast, a preparation method and application thereof. The human trophoblast takes K562 cells as starting cells, and stably expresses membrane-bound interleukin 21, CD137 ligand and Delta-like ligand 1 after genetic engineering. The constructed K562 three-factor trophoblast can significantly improve the expansion efficiency, activation state and functional stability of NK cells and γδT cells. The synergistic mechanism includes enhancing the proliferation, cytotoxicity and stemness maintenance of NK cells and γδT cells through STAT3, NF-κB and Notch signaling pathways, respectively. The human trophoblast has the advantages of good expression stability, significant functional enhancement, and high activity after freezing and recovery.

The application discloses a kind of trichoderma fermentation processing indigo blue method, the product obtained and application.The method includes: preparation trichoderma seed liquid;After primary indigo blue sterilization, access seed liquid, fermentation 5-9 days under 25-30 DEG C, 120-180 rpm, avoid light, obtain secondary processing indigo blue.The application first utilizes trichoderma to carry out biological transformation to indigo blue, can significantly improve the content of its key anti-tumor component indigo carmine with specificity, and the increase is more than 150%.In vitro anti-tumor experiment shows that the inhibitory activity of secondary processing indigo blue prepared by the application to human leukemia K562 cells and human colon adenocarcinoma cell SW480 cell is significantly better than ordinary indigo blue.The product of the application can be used for preparing medicine for preventing and / or treating leukemia, colon adenocarcinoma, process controllable, suitable for industrialized production, and provides a new way for developing efficient indigo blue anticancer new drug.

The invention discloses an application of a natural product WithangatinA in preparation of erythroleukemia induced differentiation treatment drugs, the natural product WithangatinA promotes HEL and K562 cells to be transformed from malignant proliferative cells to normal megakaryocytes by inducing the cell particle size to become larger and significantly increasing multinuclear megakaryocytes and promoting expression of surface labeled molecules differentiated from megakaryocytes at the same time, and the erythroleukemia induced differentiation treatment drugs are obtained. The effect of resisting erythroleukemia is achieved. After the natural product Withangulin A is adopted for treatment, the size and the weight of the spleen of a leukemia mouse can be remarkably reduced, the hematocrit and the platelet number of the mouse are remarkably increased, in addition, treatment of the natural product Withangulin A has no obvious influence on the weight of the mouse, and the safety is high. In-vivo research shows that the medicine can induce megakaryocyte differentiation of erythroleukemia in a mouse body, the effect of resisting erythroleukemia is achieved, and a research basis is provided for development of differentiation treatment medicines of erythroleukemia.

The application discloses application of aza-3 in resisting erythroleukemia, wherein the aza-3 is a compound obtained by modifying a ring of aza-3 with 3alpha-O-(Boc-proline); the application proves that the aza-3 can obviously inhibit HEL and K562 cell proliferation, reduce splenomegaly, improve anemia indexes (RBC, HGB and HCT) and splenic and hepatic pathological infiltration, and has no obvious toxic side effects on main organs such as heart, lung and kidney through in-vitro MTT experiment and a Friend virus-induced erythroleukemia mouse model.

The present invention relates to the field of immune cell therapy, and specifically relates to a method for expanding NK cells and use thereof. The method comprises using OK-432 in combination with irradiated feeder cells, such as K562 cells, to expand and culture NK cells in vitro.

A method for producing γδ T cells modified by a chimeric antigen receptor, comprising: transfecting K562 cells with shFPPS targeted to FPP synthase by means of a lentiviral vector, such that the expression of FPPS in the K562 cells is lowered and a K562-shFPPS cell line with reduced FPPS expression is constructed; adding the K562-shFPPS cell line into a γδ T cell culture system for co-culturing with the γδ T cells, wherein it is found that the K562-shFPPS cell line can facilitate in vitro differentiation and proliferation of the γδ T cells; and adding a CAR-expressing lentiviral vector to the γδ T cell culture system comprising the cell line for co-culturing, wherein it is found that the K562-shFPPS cell line can further effectively improve the transfection rate of CAR genes. The provided solution effectively overcomes the technical challenge of the large-scale production of CAR-γδ T cells, and has a good application prospect.

The invention discloses a method for culturing and amplifying NK (Natural Killer) cells in vitro, which comprises the following steps: adding mononuclear cells into an activation culture medium, then adding the mononuclear cells into a culture flask which is coated with a coating solution in advance, putting the culture flask into an incubator, culturing for 16-22 days under the conditions of 37 DEG C and 5% CO2, and harvesting the NK cells, wherein the culture medium is supplemented and activated on the fifth day of culture; and from the sixth day of culture, supplementing the amplification culture medium once every two days. The invention further discloses a culture medium for culturing the NK cells. The culture medium comprises an activation culture medium and a supplementary amplification culture medium. According to the method for culturing and amplifying the NK cells in vitro, amplification of more than 3000 times can be achieved, the purity of the NK cells is larger than 90% during harvesting, the cell viability is larger than 90%, and the NK cells have good in-vitro killing activity after being co-incubated with the K562 cells. According to the culture method, large-scale amplification of the NK cells can be realized, and a low-cost and high-safety technical solution is provided for the field of immune cell therapy.

The application discloses application of VK4 in preparation of a drug for treating leukemia, and belongs to the leukemia treatment field. The application research finds that VK4 can inhibit proliferation of leukemia cells through cycle arrest and induction of apoptosis, and VK4 can inhibit proliferation of HL60 cells and K562 cells through blocking of a JAK / STAT signal path. The results of Wright's staining, immunohistochemical staining and H&E staining of a mouse leukemia model show that VK4 also has an anti-leukemia effect in vivo, and has no obvious liver and kidney damage, and can provide a new drug selection for clinical treatment of leukemia.

The invention discloses a high-activity NK cell as well as an in-vitro amplification method and application thereof. The method comprises the following steps: S1, selecting a basic culture medium; s2, other culture medium components are added, wherein the following components are added according to the ratio: 5-10 [mu] g / mL of lactobacillus, 30-50 [mu] M of ergothioneine, 80-100 [mu] g / mL of fucoidan polysaccharide, 2-3 [mu] g / mL of holothurin, 3-5 [mu] M of astaxanthin, 150-200 IU / mL of IL-2150, 20 ng / mL of IL-1515, 10 ng / mL of IL-218, 2 mmol / L of stable L-glutamine, 1 mmol / L of human pyruvic acid and 5% v / v of human serum albumin; s3, carrying out feeder layer cell treatment and co-culture, mixing inactivated K562 cells and purified NK cells according to a ratio of 1: 10-1: 20, inoculating to a culture container, and removing feeder layer K562 cells after 10 days; and S4, regulating and controlling culture conditions, maintaining the constant temperature of 37.0 + / -0.5 DEG C, controlling the pH value to 7.2-7.4, culturing in a 2%-5% low-oxygen environment, and culturing for 14-21 days to obtain the high-activity NK cells. According to the in-vitro amplification method of the high-activity NK cells, disclosed by the invention, the core problems of difficult in-vitro proliferation and low activity of the NK cells in the prior art are effectively solved by optimizing the synergistic matching of natural active substances, key cell factors and nutritional supplements in the basic culture medium.

The invention discloses a targeted protein degradation agent utilizing a ubiquitin-proteasome pathway as well as a preparation method and application of the targeted protein degradation agent, and belongs to the technical field of biological medicines. On the basis of a PROTAC (protein targeted degradation chimera) strategy and on the basis of imatinib, different Linkers are introduced to be connected with an E3 ubiquitin enzyme ligand Nutlin-3 derivative, a degradation tag is labeled on BCR-ABL cancer protein, and the degradation agent with the Bcr-Abl protein targeting capacity is constructed. A Western blot experiment shows that the targeted protein degradation agent shows a good degradation effect on the Bcr-Abl protein in K562 cells, and the compound WP shows a degradation effect on concentration dependence and time dependence of the Bcr-Abl protein. Tumor cell proliferation experiments show that the compound has certain inhibitory activity on K562 cells. Wherein when the Linker is dodecane-1, 12-diketone, the anti-proliferative activity is the best. An apoptosis experiment and a period experiment show that the targeted protein degradation agent can realize concentration-dependent promotion of cell apoptosis and retard cells in S and G1 / G0 periods.

The invention discloses an efficient NK (Natural Killer) cell in-vitro amplification method based on cytokine combination, which comprises the following steps: acquiring peripheral blood mononuclear cells, inoculating the peripheral blood mononuclear cells into a serum-free culture medium containing IL-2, IL-15 and IL-21 at the density of 1 * 10 < 6 > / mL, culturing the peripheral blood mononuclear cells at the concentration of 500-1000IU / mL, the concentration of IL-15 at the concentration of 50-100ng / mL and the concentration of IL-21 at the concentration of 10-20ng / mL under the conditions of 37 DEG C and 5% CO2; adding a fresh serum-free culture medium which is twice of the initial volume on the third day of culture, and supplementing the fresh culture medium every 1-2 days to control the cell density to be 0.8-1 * 10 < 6 > / mL; the method comprises the following steps: adding irradiation inactivated K562 cells with the same quantity as the NK cells on the 7th day of culture, adjusting the concentration of IL-2 to 200-500IU / mL, and continuously culturing until the 14th day, so that the NK cells can be amplified by 3000-5000 times, the cost is low, and the safety is high.

The invention discloses an in-vitro amplification method for improving the purity of natural killer cells. The use combination of the cell factors is IL-2, IL-15, IL-18 and IL-21, the culture of the umbilical cord blood NK cells is optimized mainly from the aspects of the factor combination used for culturing the NK cells, the use concentration and the like, CD3-CD56 + positive indexes all reach 80% or above, and the amplification rate reaches 100 times or above. The method is also suitable for large-scale culture of peripheral blood NK cells, the CD3-CD56 < + > positive rate reaches 99.0% or above, the amplification rate reaches 1000 times or above, and the killing rate of K562 cells is 30% or above when the effect-target ratio is 1: 1.

The invention discloses a system and a method for controlling an'optical handle 'to perform cell excitation regulation based on a scanning Gaussian beam, and belongs to the technical field of optics. The system comprises a lighting source, an objective table, an inverted fluorescence microscope, a dichroscope group, a galvanometer, a spatial light modulator, a 1064nm laser, a fluorescence LED light source and a CMOS camera. The method comprises the following steps: preparing a 3-micron polystyrene microsphere as an optical handle, and culturing K562 cells; a scanning Gaussian beam is controlled by a galvanometer to drive an optical handle to rotate; the method comprises the following steps: optically assembling a target cell and a rotating optical handle in a three-dimensional space, and driving the cell to rotate to apply mechanical stimulation; and the cell calcium ion signal change is monitored in real time by utilizing fluorescence imaging. According to the invention, continuous, stable and controllable rotary stimulation on cells is realized, high-throughput parallel control, high three-dimensional space resolution and real-time dynamic monitoring capability are realized, and a brand new tool is provided for cell mechanics research.

A vector construct is described that is a lentiviral construct including DNA encoding for GM-CSF. A vaccine composition is also described that is includes K562 cells transfected with this vector construct, and also possibly including the U266 and H929. Methods are described for using the vaccine composition in methods of immunizing against plasma cell disorders, including multiple myeloma and related disorders.

The present disclosure provides a fully human therapeutic monoclonal antibody or fragment thereof, which is a fully human antibody specifically binding to the RBD region of SARS-CoV-2 S protein obtained from isolating a single B cell clone from a convalescent blood sample of a Covid-19 patient, which does not produce ADE effect on SARS-CoV-2 infection of THP-1 cells and K562 cells. The monoclonal antibody has high affinity to the RBD of SARS-CoV-2 S protein, with a KD value of 5.0 x 10 ‑9 M; and the monoclonal antibody has blocking activity to the binding of the RBD of SARS-CoV-2 S protein to ACEII, with an IC50 value less than 50 nM.

The invention belongs to the technical field of chemical medicines, and particularly relates to a galactorhamnoside compound as well as a preparation method and application thereof. The structural formula of the compound is selected from any one of the following formulas; ; or; in the formula, R1 ', R1 '', R1''' and R2 'are independently selected from-H, fluorine-substituted benzoyl, fluorine-substituted benzyl, nitro-substituted benzoyl or nitro-substituted benzyl. The galactorhamnoside compound is used as a galectin-3 inhibitor to be applied to preparation of anti-pulmonary fibrosis drugs and anti-chronic / acute myelogenous leukemia drugs. Particularly, the TGF-beta-induced A549 (human lung epithelial cell) fibrosis can be obviously inhibited; the obvious lethality is realized on NB4 and K562 cells.

This invention discloses a method for detecting the killing effect of multiple HLA-specific T cells, belonging to the field of biotechnology. This invention enables T2 cells to express various HLA-I molecular subtypes. After the receptor is expressed, antigen fragments loaded with various HLA-I molecules can be captured in the culture supernatant for in vitro evaluation of the killing effect of HLA-I-adapted antigen-specific T cells, thus expanding the application of T2 cells in in vitro killing effect evaluation. Simultaneously, it avoids the enormous workload and high cost of using the K562 cell line to construct a cell line for each HLA subtype-specific antigen fragment during the HLA subtype construction process.

The invention provides a method for inhibiting tumor or cancer cell growth through NK cells, and belongs to the technical field of biological medicine. According to the technical scheme, a method for separating and amplifying the NK cells is firstly constructed, the method comprises the following steps: mixing human peripheral blood from a donor with a lymphocyte separating medium, centrifuging, collecting a plasma layer, collecting a second layer of annular milk white mononuclear cells, cleaning and resuspending; and amplifying the NK cells obtained by separation in an X-VIVO 15 culture medium added with IL-2, IL-15 and K562 cells at the initial concentration of (0.5-1.5) * 10 < 6 > cells / mL for 14-15 days to obtain an NK cell finished product. On the basis, the NK cells are infused to a donor patient in an intravenous infusion mode, and a pharmaceutical composition containing rhizoma polygonati, angelica sinensis, flatstem milkvetch seeds, radix dipsaci, white hyacinth beans and other components is administered in the course of treatment, so that the inhibiting effect on tumors and cancer cells can be remarkably improved. The oral medicine disclosed by the invention is matched with an NK cell therapy, has an exact synergistic effect, and is good in safety and remarkable in technical advantage.

The invention discloses application of an NK cell expressing oncolytic virus protein in tumor treatment, and belongs to the technical field of biology. NK cells are transfected through lentivirus, and the NK-US11 cells of the US11 protein expression cassette are integrated and recombined through puromycin pressurization screening. Through Western blot detection, the US11 protein is continuously expressed along with the proliferation of the NK cells. A common NK cell and an NK-US11 cell are respectively co-cultured with a K562 cell, the killing activity of a tumor cell is detected by a lactic dehydrogenase (LDH) release method, the killing activity of the common NK cell to the K562 cell is 64.37%, the killing activity of the NK-US11 cell to the K562 cell is 87.48%, and the killing activity is improved by 35.9%. The killing activity of the NK cells expressing the oncolytic virus HSV US11 protein on tumor cells is remarkably improved, and the NK cells can play an effective role in tumor treatment.

The invention relates to the technical field of medicines, in particular to application of actinomycin V in preparation of a medicine for treating chronic myelogenous leukemia. In the aspect of cell function, the Act-V shows comprehensive anti-CML activity, not only can inhibit K562 cell proliferation in a time and dose dependent manner, but also can effectively inhibit colony forming ability, reduce the number and diameter of formed tumor spheres and lower the sphere forming frequency. Experiments prove that the Act-V further weakens the dry characteristics of tumors by down-regulating the expression of dry markers such as CD133, CD44, ALDH1A1 and the like. Besides, the Act-V can significantly inhibit the invasion and migration ability of CML cells, and western blot experiment results prove that the Act-V down-regulates the expression of interstitial markers N-Cadherin and Vimentin, up-regulates the expression of an epithelial marker E-Cadherin and inhibits the invasion and migration ability, so that the Act-V has wide application scenarios.

The application provides a method for constructing 1,2-cis fucose glycoside bond with high stereoselectivity and application in preparation of 13-sugar repeating units and 9-sugar substructures of angelica polysaccharide APS-1II, and evaluation of anti-leukemia activity of the 13-sugar and 9-sugar, and belongs to the technical field of polysaccharide preparation; the application realizes a promoter composition of 1,2-cis fucose glycosylation reaction with high stereoselectivity, which comprises iodotrimethylsilane (TMSI) and triphenyl phosphine oxide (Ph3PO), and the donor is fucosyl N-phenyl trifluoroacetyl imidate (PTFAI) with acyl substitution at O-3 and / or O-4 positions. The 13-sugar repeating units and 9-sugar substructures of angelica polysaccharide APS-1II provided by the application have significant inhibitory effect on human leukemia K562 cells and mouse leukemia L1210 cells.

This invention provides an immune cell culture kit, a method for culturing NK cells, and its applications. The immune cell culture kit includes a basal culture medium, recombinant antibodies, and cytokines. The basal culture medium includes RPMI-1640, thioglycerol, L-ascorbic acid, dexamethasone, and β-mercaptoethanol. The recombinant antibodies include two or more of CD16, CD56, NKp30, NKp44, NKp46, NKG2D, and 2B4 antibodies. The cytokines include IL-2, IL-15, IL-18, IL-21, and FLT3L. The immune cell culture kit of this invention is low-cost, and the cultured NK cells have high purity and viability, as well as good tumor-killing activity. The NK cells cultured by this method exhibit high killing activity against both A549 and K562 cells.

The invention discloses a construction method and application of a membrane-bound IL-21 K562 cell strain, a human IL-21 mature sequence is fused with an IgG4 hinge region, a human immune globulin gamma-4 chain CH2 / CH3 region and a CD4 transmembrane structural domain through genetic engineering to construct an mbIL-21 fusion gene, and the mbIL-21 fusion gene is cloned to a PT-mnud carrier by using BamH I / Xho I double enzyme cutting sites to obtain a recombinant plasmid PCDH-mbIL-21. HEK 293T cells are transfected through a lentivirus packaging system, high-titer lentivirus particles are prepared, and the cell strain stably expressing mbIL-21 is obtained through puromycin screening after K562 cells are infected. The mbIL-21 K562 cell strain constructed by the invention has the characteristics of long acting, targeting and stable expression, and provides a powerful tool for basic research and clinical transformation of immunotherapy.

The invention discloses a polypeptide targeted degradation agent based on a Bcr-Abl protein substrate binding site as well as a preparation method and application of the polypeptide targeted degradation agent, and belongs to the technical field of tumor targeted therapy. From the C end to the N end, the novel polypeptide targeted degradation agent is obtained by connecting a targeted peptide (obtained by carrying out amino acid site mutation on Abltide) with high affinity of Bcr-Abl, a connecting peptide, a degradation peptide (polypeptide ligand PMI of E3 ubiquitin ligase MDM2 and a conserved sequence LIR in autophagy receptor protein respectively exert a degradation effect) and a cell-penetrating peptide (R6 or TAT). The polypeptide targeted degradation agent has the capability of remarkably inhibiting the activity of Bcr-Abl mutant strain cells, can degrade related proteins in K562 cells, can induce apoptosis of the K562 cells, can influence the cell cycles of the K562 cells, can be used for preparing anti-tumor drugs, and has a good application prospect. The compound has a good application prospect in preparation of drugs for targeting human chronic granulocytic leukemia cells or human peripheral blood basophilic leukemia cells.

The present invention provides an application of thyroid hormones and thyroid hormone analogues to preparation of drugs for treating sickle-cell disease, particularly an application to preparation of drugs for improving the expression quantity of ζ-globin. During the differentiation of K562 cells, thyroid hormone analogue (Triac) can significantly up-regulate the expression of ζglobin gene (HBZ) by 50 folds or above. The expression of ζ-globin gene (hbae5) can also be up-regulated by 30-70 folds in zebrafish treated with thyroid hormones and thyroid hormone analogues. Therefore, according to the present invention, the expression of ζ-globin gene can be significantly activated by thyroid hormones and thyroid hormone analogues which may develop new potential therapies for patients with sickle-cell disease. It provides an economical, safe and effective method for treating sickle-cell disease and can be widely used.

The invention discloses a method for processing indigo naturalis through trichoderma fermentation, an obtained product and application. The method comprises the following steps: preparing a trichoderma seed solution; and sterilizing primary indigo naturalis, inoculating the primary indigo naturalis into the seed solution, and fermenting for 5-9 days in a dark place at 25-30 DEG C and 120-180 rpm to obtain secondarily processed indigo naturalis. Trichoderma is used for biotransformation of indigo naturalis for the first time, the content of indirubin serving as an anti-tumor key component of indigo naturalis can be specifically and remarkably increased, and the amplification reaches 150% or above. In-vitro anti-tumor experiments show that the inhibition activity of the secondarily processed indigo naturalis prepared by the invention on human leukemia K562 cells and human colon adenocarcinoma SW480 cells is obviously superior to that of common indigo naturalis. The product disclosed by the invention can be used for preparing medicines for preventing and / or treating leukemia and colon cancer, is controllable in process and suitable for industrial production, and provides a new way for developing a new efficient indigo naturalis anticancer medicine.