192 results about "K562 cells" patented technology

The invention discloses a method for amplifying NK cells of human beings under the condition of in vitro culture, which is characterized in that a peripheral blood mononuclear cell (PBMC) is taken asthe original culturing material, and the PBMC is cultured together with an engineering cell which is built by adopting the genetic engineering method and is used for stimulating the growth of the NK cell. The built engineering cell which is used for stimulating the growth of the NK cell expresses several cytokines (IL-2, IL-12, IL-15, IL-18, 4-1BB) which can promote the growth of the NK cell on the surface of the K562 cell; after irradiation and inactivation with gamma-rays, the engineering cell is cultured with the PBMC in vitro, as a result, the amplification effect of the stimulation methodin the invention is hundreds of times stronger than that of the currently universal method in which soluble cytokines are added purely to the culture solution; and after cultured for 3 weeks, the non-NK cells in the PBMC are mainly dead and disappeared, the NK cells are proliferated in great quantity, the purity of the NK cells reaches over 96% and the total number of the NK cells is amplified byover 1500 times.

The invention relates to a method for in-vitro amplification of NK cells, and in particular relates to a method for massive in-vitro amplification of NK cells, wherein the method comprises the following steps of: a, inoculating a peripheral blood mononuclear cell in a CD3McAb and CD226McAb pre-coated culture bottle for coculture; b, adding 1L-2 and 1L-18, coculturing for 72hours to stimulate amplification of NK cells; c, transferring the NK cells, K562 cells after lethal treatment and a serum-free medium containing 1L-2 and 1L-18 in a cell culture bag for coculture; and d, collecting the NK cells. According to the method for in-vitro amplification of the NK cells, two antibodies CD3McAb and CD226McAb are simultaneously coated, so the cell factor synthesis and ADCC effect are promoted, and killing toxicity of the NK cells is remarkably improved; the activation and amplification on the NK cells are achieved just by the 1L-2 and 1L-18 cell factors, so the amplification multiple and cell toxicity of the NK cells are guaranteed, and the cost of cell culture is reduced.

The invention belongs to the field of cell engineering, in particular to a method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and an application of the method. According to the invention, fusion genes are subjected to gene recombination to an AAVS1 (Adeno-Associated Virus Integration Site 1) transposon through a plasmid, and the freezing/thawing step is introduced to the NK cell expansion step taking a modified K562 cell line as a basis in the intermediate stage of cell expansion; finally, the NK cells are expanded and saved. A K562-mbIL15-41BBL-mbIL21 cell line developed by the integration technology of site specific genes is used, and the activity, purity and cytotoxicity of the NK cells are high to realize high expansion efficiency of the NK cells to improve NK expansion. The method for the high-efficient expansion and cryopreservation of the NK (Natural Killer) cells and the application of the method for the high-efficient expansion and cryopreservation of the NK (Natural Killer) cells disclosed by the invention have great market prospects and economic values.

The invention relates to the field of immunology, in particular to a method for amplifying and activating a natural killer (NK) cell into a lymphokine-activated killer (LAK) cell by using a CD8 alpha-interleukin 21-CD137 compound. The method disclosed by the invention comprises the following steps of: forming the CD8 alpha-interleukin 21-CD137 compound by using CD8 alpha, interleukin 21 and a CD137 functional polypeptide, making an exogenous expression vector enter a host K562 cell, then activating a promoter and culturing a cell to obtain a cell for expressing a transmembrane interleukin 21-CD137 compound; and purifying the compound in the conventional way, and amplifying and activating a lymphocyte by using the purified compound to generate the LAK cell. The method has the advantage that: the LAK cell cultured and amplified by using the transmembrane interleukin 21-CD137 compound and a small dose of interleukin 2 is used for enhancing the immunity of a patient to help the patient resist tumors, viruses and bacteria. The method has a wide clinical using prospect.

Patients who develop increased numbers of γδ+ cytotoxic T lymphocytes 2–6 months after allogeneic bone marrow transplantation are less likely to relapse than those who do not. The γδ+ T cells isolated from blood of patients with increased γδ+ T cells are CD3+CD4−CD8−CD57+, cytolytic to K562 cells, and express the Vδ1 T cell receptor phenotype. Similar γδ+ T cells can be generated in vitro by culture of donor mononuclear cells which are enriched for γδ+ T cells by immunomagnetic depletion of depleted of CD4+ and CD8+ cells. This γδ-enriched cell preparation was cultured on a combination of immobilized pan-δ monoclonal antibody and irradiated recipient B cell leukemia. After four weeks, the cultures were almost exclusively Vδ1+CD3+CD4−CD8− cells that co-expressed activation-associated antigens CD69, CD25, and HLA-DR. Furthermore, they were cytolytic against the primary leukemia obtained from the recipient and lymphoblastic leukemia cell lines, yet had minimal cytotoxicity against normal donor-derived mononuclear cells or myeloid leulemia cell lines. These observations suggest that donor-derived cytotoxic γδ+ T cells can be generated in vitro, and may provide therapeutic potential for prevention of disease relapse.

The invention relates to a long-chain non-coding RNA IncRNA-BcrAR and application thereof in cell canceration resistance. IncRNA-BcrAR is in low-level expression in a human chronic myelogenous leukemia cell line K562 with positive Bcr-Abl, the K562 cell line with overexpressed IncRNA-BcrAR is constructed, the action of IncRNA-BcrAR on Bcr-Abl induced cell neoplastic transformation is observed, experiments prove that the IncRNA-BcrAR overexpression can obviously promote K562 cell apoptosis induced by Imatinib (therapeutic drug of Abl positive leukemia patients) and can remarkably inhibit tumor growth induced by K562 cells in a naked mouse body; and besides, the IncRNA-BcrAR overexpression can remarkably promote A-MuLV transformed mouse leukemia cell BC44 apoptosis induced by Imatinib. The IncRNA-BcrAR has important effect on Bcr-Abl and v-Abl mediated cell canceration resistance, and the long-chain non-coding RNA IncRNA-BcrAR provides new molecular marker and drug target for diagnosis and treatment of Abl induced leukemia.

The invention discloses a new medicament application of cucurbitacins, namely an application of mixing effective monomers of cucurbitacins in medicament for treating leukaemia or an application of effective monomers of cucurbitacins to independently treating leukaemia, belonging to the technical field of medicament. The cucurbitacins with nano-mole concentration can obviously inhibit multiplication of leukaemia HL60 and K562 cells; the cucurbitacins have no cell toxic effect to human leukaemia cells in a multiplication inhibiting concentration range; and the multiplication inhibiting effect to leukaemia cells in vitro is stronger than that to solid tumour cells. Further researches suggest that the cucurbitacins with micro-mole concentration can induce apoptosis of leukaemia HL60 cells by increasing cucurbitacin concentration. Anti-tumour researches in vivo suggest that the cucurbitacins can prolong survival time of a small mouse P388 leukaemia animal model. The effective monomers of cucurbitacins in resisting leukaemia have obvious effect, low toxicity, long lasting drug administration, can be used for both chemotherapy and radiotherapy, and are promising to be further developed into the medicament for treating leukaemia.

The invention discloses an artificial antigen submitting cell and preparing method, which comprises the following steps: choosing human chronic granular leukocyte leukemia bacterial strain K562 cell as carrier; transfer-dying and expressing eucaryon expressing carrier of CD32a; expressing CD32 molecular on the surface of K562 cell stably through G418 screening and flowed cell sorting device sorting; forming K32 cell; producing eucaryon expressing carrier of double expressing CD86 and 4-1BBL co-simulating signal; proceeding homomycin screen and sort with flowed cell device for the K32 cell; getting stable expression K32 cell of CD86 and 4-1BBL molecule; getting the end product. This invention possesses low cost and good repeatability, which can inhibit die effectively.

The invention relates to an application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities. The Trichoderma pseudokoningii extracellular polysaccharides having the anticancer activities can reduce the vitality of K562 cells in vitro when they are applied to tumor growth inhibition (auxiliary) medicines, so intracellular active oxygen and free calcium ion concentrations are improved, the expression abundance of mRNA of p53 and Bax genes is improved, the expression of the Bc1-2 transcription level is reduced, the ectropion of cell membrane phosphatidylserine of the human chronic granulocyte leukemia cells K562 is induced, and the nuclear polycondensation and the fragmentation of K562 are induced to generate apoptotic bodies and apoptosis; and in-vivo administration can increase weights of tumor bearing mice and alleviate growth speeds and weights of tumors of the tumor bearing mice, thereby tumor cell killing and tumor growth inhibiting purposes are reached.

The invention provides an immunoliposome of a CD19 monoclonal antibody. First, a film dispersion method is adopted to prepare the cubical space stable norcantharidin immunoliposome, wherein the mole ratio of soy bean lecithin, cholesterol, methoxyl-polyethylene glycol 2000-phosphatidyl ethanolamine is 2:1:0.01, the mass ratio of norcantharidin immunoliposome and soy bean lecithin is 1:20, and entrapment efficiency of high-performance liquid detection NCTD is 46.5+/-2.21%. The research proves that 2E8-SL-PE can be combined with Nalm-6 and Raji cell specifically, the positive rates thereof are respectively 89.44% and 88.73%, the 2E8-SL-PE is almost not combined with Molt-3 and K562 cell with the positive rates being only 1.95% and 1.39%, which shows that the immunoliposome has obvious target recognition effect. The norcantharidin immunoliposome can be applied in preparing specific target killer B lymphocyte leukemia and stem cell drugs thereof.

The invention provides a preparation method of autologous natural killer cell proliferation and culture. The preparation method of the autologous natural killer cell proliferation and culture is characterized in that mononuclear cells of a cancer patient are amplified and activated to obtain natural killer cells after the mononuclear cells are transfected by 3- phosphoinositide-dependent protein kinase-1 and CD122 and under the effect of K562 cells which are expressed stably serve as trophoblast cells and interleukin 2, proliferation times of the natural killer cells are above 2500, phenotype of CD16+/CD56+ is above 90%, and in-vivo and in-vitro tests show that the natural killer cells have an anti-tumor and have quite high killer toxicity. The invention also provides a kit which is used for autologous natural killer cell proliferation and culture and is obtained by using the preparation method. The kit has an efficient anti-tumor function.

The invention relates to a tyrosine kinase inhibitor and a preparation method and use thereof, and belongs to the technical field of pharmaceutical chemistry. The tyrosine kinase inhibitor having the structural features shown in the general formula I, or its pharmaceutically acceptable salts or stereoisomers can effectively inhibit tyrosine kinase activity, and can inhibit kinases such as DDR1, DDR2, Abl, Src, Btk and Kit. Compared with a positive contrast dasatinib, the tyrosine kinase inhibitor has higher half inhibitory concentration or the same half inhibitory concentration, and especially, aiming at DDR1, DDR2, Src, Btk and Kit, the compound 8j has lower K562 cell half inhibitory concentration. The tyrosine kinase inhibitor has good enzyme inhibitory activity and cell activity and has a large application prospect.

The invention provides a tetrahydropyridopyridone derivative as shown in a general formula (I), and a preparation method thereof. The method comprises the following steps of: preparing N,N-bi(methoxycarbonyl group) substituted benzylamine (b) by carrying out Michael addition on substituted benzylamine (a) and methyl acrylate, carrying out Dieckmann condensation on (b) under the action of sodium alcoholate, subsequently carrying out hydrolysis and decarboxylation under the action of acid so as to obtain N-substituted benzyl-4-piperidone (d), carrying out an aldol condensation reaction on (d) and bimolecular aromatic aldehyde so as to obtain N-substituted-3,5-bi(substituted benzylidene piperidine-4-ketone (e), and refluxing and heating (e) malononitrile and ammonium acetate in ethanol so as to obtain a final product as shown in the general formula (I). The tetrahydropyridopyridone derivative is simple in process and convenient to produce in scale, and the compound (I) has a good inhibition effect on multiplication of leukemia K562 cells, ovarian cancer HO-8910 cells and liver cancer SMMC-7721 cells. Therefore, the invention further provides an application of the compound as shown in the general formula (I) in preparing medicaments for preventing multiplication of the leukemia K562 cells, the ovarian cancer HO-8910 cells and the liver cancer SMMC-7721 cells.

The invention discloses a method for identifying a differential marker of IC50 dose of vitamin C for RAW264.7 and K562 cells by applying 1H NMR technology and metabonomic methodology. The method comprises the following steps: (1) determining the IC50 dosage of vitamin C for RAW264.7 or K562 cells; (2) preparing a cell extract; (3) performing nuclear magnetic resonance pretreatment; (4) performing 1H nuclear magnetic resonance detection; (5) screening differential metabolites; and (6) performing metabolism pathway analysis. The method studies influence of different cell differential metabolites and metabolic pathway by applying H-nuclear magnetic resonance technology in combination with metabonomic methodology, and the basic research provides theoretical basis for studying the method for analyzing sub-toxicity of vitamin C for RAW264.7 or K562 cells.

The invention relates to phenylbutyryl curcumin derivate and application thereof in anti-tumor drug preparation, in particular to 4-(di(2-chloroethyl) amino) phenylbutyryl curcumin, 4,4'-(di(2-chloroethyl)amino) diphenylbutyryl curcumin, pharmaceutically acceptable salts and a preparation method thereof, and applications of 4-(di(2-chloroethyl) amino) phenylbutyryl curcumin, and 4,4'-(di(2-chloroethyl)amino) di phenylbutyryl curcumin in anti-tumor drug preparation. The phenylbutyryl curcumin derivate can be used for (but not limited to) preparing drugs for treating leukemia, skin cancer, gastric cancer, colon cancer, liver cancer, breast cancer or prostatic cancer. The derivate has obvious inhibition on a plurality of animal tumor cell transplanting modules, especially for the human chronic granulocytic leukemia model constructed by NOD-SCID mice inoculated with the human chronic granulocytic leukemia K562 cell stain, the life prolonging rate is obviously enhanced, and the serious toxicity to mice does not exist.

Disclosed are a hydroxyproline peptoid derivative and a preparation method and application thereof. With biphenyl pyridine as a hinge region binding fragment, by adopting a design strategy of a fragment drug and introducing L-hydroxyproline as a flexible Linker, a peptoid micromolecule-like compound library with kinase inhibition activity is constructed, and through screening of ADP-Glo and otheractivity tests, a peptoid tyrosine-like kinase inhibitor which has Bcr-Abl kinase inhibition activity and prevents the proliferation of tumor cells is found. The compound can be used for preparing anti-tumor drugs and has the activity of inhibiting Bcr-Abl and Bcr-AblT315I kinases and activity of inhibiting the cell proliferation of K562 cells. By introducing the L-hydroxyproline, the structural diversity of the Bcr-Abl inhibitor is expanded, the activity result shows that the introduction of proline has a certain effect on the inhibition activity of the compound, and the proline can serve asa novel pharmacological-effect fragment of the Bcr-Abl tyrosine kinase inhibitor.

The invention provides a method for amplifying NK cells by utilizing gene modified K562 trophoblast cells. IL-21, IL-27 and CD70 genes are transfected into K562 cells through a lentivirus reprint system, so that K562 engineering cells capable of stably expressing IL-21, IL-27 and CD70 protein can be obtained; the K562 engineering cells are mixed with PBMC to perform cultivation after being inactivated, so that the NK cells can be amplified in a serum-free medium under the presence of IL-2, and the amplification multiples can be about 800-1,100; the effective purity of the NK cells can reach 91%, and the secretion volume of IFN-gamma can be five times that of the NK cells cultivated by common methods; and the killing rate of the NK cells on hepatoma carcinoma cell HepG2 can reach 88.5% under the condition that effector-target ratio is 10: 1, which is about 20% higher than that of the NK cells cultivated under normal conditions, and therefore, the NK cells cultivated by the method have stronger killing abilities.

The invention relates to specifically relates to a genetically engineered cell and a method for efficiently amplifying NK cells in vitro by using the genetically engineered cell, belonging to the field of immunology. According to the invention, a Piggybac transposon system is utilized to construct a K562 engineered cell that realizes transmembrane stable expression of IL-15, IL-18 and 4-1BBL; andthe method for amplifying and activating NK cells based on the genetically engineered K562 cell is provided in virtue of an in-vitro cell stimulation technology. The genetically engineered cell can realize simultaneous transmembrane high-expression of IL-15, IL-18 and 4-1BBL. The NK cell amplification method of the invention is simple to operate and low in cost; the amplified NK cells have the advantages of high purity, large quantity, good killing activity, etc.; and the NK cell amplification method is suitable for large-scale production of NK cells and lays a good foundation for the clinicalapplication of adoptive immunotherapy of NK cells.