230results about How to "Promote amplification" patented technology

The invention discloses a method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells. The method comprises the steps as follows: the concentration of separated PBMC (peripheral blood mononuclear cells) is adjusted by a serum-free medium containing autologous plasma, an Anti-CD16 antibody, IL-2 and IL-15 are added, and then the mixture is transferred into a T175 culture flask for culture; an Anti-CD3 antibody and an Anti-CD137 antibody are added; a serum-free medium containing the autologous plasma, IL-2 and IL-15 is supplemented every two days according to the cell growth condition; the cell concentration is controlled to be about 1.5*10<6>/ml; and after culture is performed for 14-21 days, large quantities of high-purity CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells can be obtained simultaneously, and the total cell quantity can reach an effective value of the cell quantity required for adoptive cellular immunotherapy clinically for tumor. The method for simultaneous and efficient amplification of the CD<3+>CD<56+>CIK cells and the CD<3->CD<56+>NK cells is simple, convenient, effective and high in cell killing activity.

The invention discloses an expansion method of various lymphocyte subpopulations and application of the expansion method to preparation of an adoptive immunotherapy medicine for cancers. The expansion method comprises the following steps: S1, adding IFN gamma and zoledronic acid into a culture medium of PBMC collected in the blood of a tumor patient, and re-adding an OKT3 antibody and recombinant human interleukin-2 after culture to stimulate PBMC so as to finish preliminary expansion; S2, after finish of preliminary expansion, mixing immune lymphocytes with feeder layer cells, and after adding zoledronic acid, an OKT3 antibody and recombinant human interleukin-2, continually performing expansion. By adoption of the expansion method, after twice expansion, the sum of the immune cells can be 10,000 to 80,000 times by expansion, the expanded immune cells include alpha beta T cells, gamma delta T cells, NKT cells and NK cells, and tumor cells can be directly killed, or a cellular immunotherapy drug is prepared to kill the tumor cells.

The invention discloses a method and a kit for simultaneously constructing a sequencing library by DNA and RNA. The method comprises the following steps: (1) carrying out reverse transcription on RNAin a reaction substrate to form RNA/cDNA composite double strands; (2) directly adding Tn5 transposase and a fragmentation buffer solution into a mixture of a reverse transcription product cDNA/RNA composite double strand and a DNA double strand for fragmentation, and adding a part of linker sequence to the 5' end of the double strands during fragmentation; (3) strand displacement and library amplification: adding an amplification buffer solution and a strand displacement and amplification enzyme mixture into the obtained product, and carrying out PCR amplification; and (4) carrying out magnetic bead purification to obtain a sequencing library of the original DNA/RNA. According to the method, the library building steps are greatly reduced, the library building time is shortened, the library output and offline data quality is improved, and more real information is helped to be obtained.

The invention discloses a method and kit for realizing multiple detection of nucleic acid through a colloidal gold chromatographic technology, and belongs to the field of medical biotechnology. The invention provides a nucleic acid detection test strip, a universal probe is marked with colloidal gold particles to be fixed on a glass cellulose film, the universal probe is designed into a universal sequence, an NC film on the test strip have one or more detection lines and a quality control line, each detection line is coated with a section of nucleic acid sequence which can be in specific hybridization combination with a corresponding specific probe B series; the quality control line is coated with a section of nucleic acid sequence which can in specific hybridization combination with a specific probe A series; the specific probe A series is in complementary pairing hybridization with the universal probe; the probe marked with gold and a nucleic acid amplified fragment are combined in series by virtue of the specific probe A series and the specific probe B series, so that the specific detection of the nucleic acid fragment can be realized. The method has the advantages that the technical requirements of experimenters are low, the required detection time is short, special instruments are not required, and the method is easy to popularize towards grass-roots and remote countryside medical institutions.

The invention provides an erythrocyte lysis buffer. The erythrocyte lysis buffer comprises the following components: 4.5-5.5 mmol/L of sodium chloride, 0.8-1.3% (v/v) of triton 100, 300-340mmol/L of glucose and 0.008-0.012 mol/L of trishydroxymethyl aminomethane. The erythrocyte lysis buffer can effectively lyse the erythrocyte in a whole blood sample, separate and enrich leucocyte in the blood, and facilitate the subsequent nucleic acid extraction operation of the leucocyte, so as to obtain the leucocyte DNA in the whole blood sample and the DNA of virus in the leucocyte.

The invention discloses a KIR and ligand genetic typing experimental method. By the technology, the time and labor are saved, and meanwhile, DNA sample capacity is further saved. A multi-PCR technology is adopted, meanwhile, 36 pairs of primers are further combined according to the sizes of PCR products, and finally, amplified reaction is finished in 12 reaction holes. The KIR and ligand genetic typing experimental method is conveniently applied to amplification of 96 pore plates, conventional Taq enzyme is used, typing of all KIR genes and ligands thereof can be finished at a time under the same reaction conditions, and the practicality of the KIR and ligand genetic typing experimental method is greatly improved. Two pairs of primers are used for a KIR gene, the accuracy is improved, anda false negative result is avoided. The KIR gene can be combined to MHC-I type ligand molecules on the surfaces of targeting cells, inhibiting or activating signals are transmitted to regulate the activity of NK cells and T cells, and the KIR and ligand genetic typing experimental method plays an important regulation role in hematopoietic stem cell transplantation, feto-matemal tolerance, anti-infectious immunity, tumor immunity and autoimmune diseases. Therefore, KIR genetic typing facilitates understanding of influences of KIR to tumor immunity, hematopoietic stem cell transplantation and autoimmune diseases.

The invention relates to the field of cells, and discloses a culture medium for mesenchymal stem cells and a large-scale culture method thereof. The culture medium for mesenchymal stem cells disclosed by the invention comprises a serum-free culture medium, EGF and PDGF, wherein the concentration of the EGF is 5-10ng/mL, and the concentration of the PDGF is 5-10ng/mL. The large-scale culture method for mesenchymal stem cells disclosed by the invention comprises the following steps: taking P1-generation mesenchymal stem cells which are primarily isolated and cultured, adjusting an inoculum density to be 2*10<4>/mL to 3*10<5>/mL by the culture medium for mesenchymal stem cells, culturing to a cell fusion degree of 80-90% and then recording as P2-generation, and repeatedly passing to P10-generation cells. The culture medium disclosed by the invention reduces a cell pollution probability, promotes MSC amplification, and delays the ageing speed of MSC in in-vitro culture. The large-scale culture method disclosed by the invention is capable of obtaining lots of high-purity mesenchymal stem cells, and keeping the good stem cell characteristics of the mesenchymal stem cells.

The invention discloses a method for co-culturing cord blood stem cells and mesenchymal stem cells, belonging to the biotechnology and tissue engineering technical field. The method is characterized by adding no blood serum but only cytokine and trophocyte; wrapping the trophocyte calcium alginate-chitosan gel beads; using microcarrier and rotating wall vessel to implement the co-culture of cord blood stem cells and mesenchymal stem cells; and separating and obtaining the cord blood-derived stem cells respectively. The invention has the advantages that: the microcarrier provides a surface for the cord blood mesenchymal stem cells to adhere to, so the adherent, dynamic, suspension culture of the cord blood mesenchymal stem cells can be implemented. The rotating wall vessel provides an environment for hemopoietic stem cell to suspend and growth in, and also reduces the damages caused by fluid shear stress to the hemopoietic stem cells. The throphocyte can nourish the cord blood stem cells outside the gel beads, replace partially blood serum and contribute to the separation and harvest of different cells.

The invention discloses a 3D printed Ti-PDA-PLGA microsphere bone defect repair stent. A 3D printed Ti stent is prepared by a laser sintering technology. Then, under certain conditions, dopamine is self-polymerized on the fiber surface of the 3D printed Ti stent to form a PDA coating, thereby preparing a 3D printed Ti-PDA stent; then PLGA microspheres carrying VEGF are prepared by a double emulsion-solvent evaporation method, and finally, BMP-2 and PLGA microspheres carrying VEGF are adsorbed and immobilized on the surface of the stent by an adsorption method, and finally the 3D printed Ti-PDA-PLGA microsphere bone defect repair stent is formed. The bone defect repair tissue engineering stent disclosed by the invention has the advantages of reliable mechanical property, high biological activity and safety, convenient implantation, small trauma and low cost, and can be used for the repair treatment of bone defect after bone traumas, bone tumors and bone infection.

The invention relates to a serum-free cryopreservation medium and method for peripheral blood monouclear cells. The serum-free cryopreservation medium comprises recombinant human interleukin-2, humanserum albumin, polyethylene glycol, trehalose and a basic culture medium or sodium chloride for injection. The serum-free cryopreservation medium has the advantages that the recombinant human interleukin-2 (IL-2) is added into the cryopreservation medium, so that the activity stability of immune cells cultured by thawed cells can be increased greatly, the high activity of original cells can be kept, the immune cells can well keep the physiological function and biological feature of the thawed cells, and the problem of direct large-scale amplification of the thawed cells can be solved effectively; the polyethylene glycol and the trehalose are added into the cryopreservation medium, the cryopreservation medium can replace an existing dimethyl sulfoxide-based (DMSO-based) cryopreservation medium, the toxicity of the cryopreservation medium to the cells is lowered effectively, cell stability is maintained, the direct application safety of the thawed cells is increased, and the thawed cellscan be directly used.

The invention discloses a Taq DNA polymerase mutant and application thereof. The Taq DNA polymerase mutant has amino acid substitutions at one or more of the following amino acid positions in the sequence shown as SEQ ID NO. 1; and the various amino acid substitutions are represented in triplets as 'letters-numbers-letters', wherein the numbers reflect position of an amino acid mutation, the letters before the numbers are corresponding to an amino acid involved in the mutation, and the letters after the numbers reflect an amino acids used to replace the amino acid corresponding to the lettersbefore the numbers. The Taq DNA polymerase mutant disclosed by the invention is capable of changing configuration of an enzyme, thereby improving tolerance of the enzyme; and thus, the mutant can be very well applied in blood sample amplification.

The invention discloses a 3D printed PCL-Mg bone tissue engineering scaffold and a preparation method thereof. The 3D printed PCL-Mg bone tissue engineering scaffold is prepared from poly-epsilon-caprolactone PCL and magnesium chloride as raw materials through a 3D printing technology. After being implanted into the human body, PCL as a bioabsorbable material is gradually degraded and releases Mgto promote osteochondral generation. The porous structure of the scaffold can induce bone ingrowth and finally repair bone defects, tumors and bone defects after infection. The 3D printed PCL-Mg bonetissue engineering scaffold has the advantages of simple and reliable structure, controllable shape and microstructure, reliable mechanical property, controllable ion release performance, convenient implantation, small trauma and low cost.

The invention relates to the technical field of cell culture, in particular to a building method of NK (natural killer) cell and gamma delta T cell co-culture capable of realizing the in vitro commonmultiplication culture on NK cells and gamma delta T cells. The method mainly comprises the following steps of S1, reagent selection; S2, performing PBMC culture by a culture medium for 3 days; S3, supplementing the culture medium; maintaining the concentration; performing culture for 4 days; S4, after the seventh day, removing supernatant through centrifugation; transferring the cells into a T25cell culture bottle; then, replacing an EX culture medium; S5, after the tenth day, supplementing the culture medium for maintaining the concentration; S6, after the twelfth day, continuously supplementing the culture medium for maintaining the concentration; S7, after the fourteenth day, detecting the NK cell and gamma delta T cell proportion and the cell total number. The co-culture building method has the advantages that two kinds of anti-tumor immune cells with similar properties are cultured in one step; the cell culture efficiency is improved; the culture cost can be greatly reduced through being compared with that of independent culture of various immune cells for obtaining NK cells and gamma delta T cells; the culture technical difficulty is also reduced.