172 results about "Gradient centrifugation" patented technology

The invention belongs to the field of allogenic mesenchymal stem cells, and particularly relates to a preparation method of allogenic mesenchymal stem cells by CRISPR (clustered regularly interspaced short palindromic repeats) technique editing and IGF (insulin-like growth factor) optimization and application of the allogenic mesenchymal stem cells in treating myocardial infarction. The preparation method comprises the following steps: carrying out separation by density gradient centrifugation to obtain allogenic single karyocytes, and carrying out adherent culture to obtain mesenchymal stem cells; designing a mesenchymal stem cell surface antigen B2M-gRNA and an inflammatory factor TNF-alpha-gRNA; establishing recombinant slow virus particles, and transfecting the mesenchymal stem cells; optimizing the mesenchymal stem cells by using IGF-1; and preparing drugs for treating myocardial infarctions by using the modified and optimized mesenchymal stem cells. The CRISPR/Cas9 technique is utilized to remove the antigens capable of causing immunological rejection and the inflammatory factors capable of causing inflammatory reaction on the mesenchymal stem cell surface, and the IGF-1 is utilized to enhance the apoptosis resistance of the mesenchymal stem cells and promote the homing of the mesenchymal stem cells, thereby providing a new technical scheme for preparing drugs for treating cardiovascular diseases in clinic. The prepared allogenic mesenchymal stem cells can not cause immunological rejection after cell transplantation.

The invention relates to a preparation method of a small-size transition metal chalcogenide two-dimensional nanosheet. The method is characterized in that the powder of a block is treated as a raw material; a polymer is used as an additive, and polymer assistant ball milling is performed to obtain transition metal chalcogenide two-dimensional nanosheets with different sizes and different components. The method comprises three steps of weighing high polymers and polymer assistant ball milling prepared blocky powder; performing ball milling, separating, water adding, uniformly mixing and movingout; performing gradient centrifugation to obtain products with different sizes; purifying to remove excessive polymers. With the adoption of the method, the problems such as low yield, non-uniform appearance and complex processes of a current preparation method of a current small-size transition metal chalcogenide two-dimensional nanosheet can be solved; the method has the advantages of being high in yield, simple in processes, high in generality, easy to control processes,and the like; the prepared small-size transition metal chalcogenide two-dimensional nanosheet can be applied to differentfields, such as the fields of catalyzing, photoelectric and biomedicines.

The invention discloses an extraction method of a specific mesenchymal stem cell exosome. The method comprises the following steps: collecting supernatant of serum-free mesenchymal stem cells: firstly, culturing mesenchymal stem cells, changing a culture medium for culturing, centrifuging, and removing cell debrises to obtain initial supernatant; taking the initial supernatant for carrying out further gradient centrifugation, transferring the centrifuged supernatant into a sterile centrifugal tube, centrifuging and collecting supernatant containing micro vesicles; centrifuging the supernatantcontaining the micro vesicles to obtain precipitate containing an exosome, washing and then centrifuging to obtain exosome precipitate; adding the obtained exosome precipitate into sterile PBS and filtering to obtain the specific mesenchymal stem cell exosome. The exosome collected in the extraction method disclosed by the invention adopts a filter membrane for filtration, so the extracted exosomeis enabled to be nanoscale; meanwhile, the rotational speed is reduced and optimized, high-speed mechanical damage is reduced, separation efficiency is high and the cost is low.

The invention relates to a liquid-based cell smear manufacturing and dyeing machine utilizing principles of injection and pipetting, which is characterized by structurally comprising a machine base, a machine body, a worktable, a horizontal slide rail, a moving arm, a horizontal motor moving device, an up-down motor moving device and a microcomputer motion control system, wherein four groups of smear manufacturing and dyeing units and a kit are mounted on the worktable board; and 24 samples can be automatically manufactured into smears and dyed when the machine runs once. The liquid-based cell smear manufacturing and dyeing machine does not need a tedious specific gravity liquid gradient centrifugation step, a micropipe type liquid drawing system of a liquid-based cytology test (LCT) machine, or processes of vacuum negative pressure drawing and positive pressure cell transfer of a microporous filter membrane of a Thinprep cytology test (TCT) machine. Cell samples are drawn, evenly mixed and scattered by using a pipetting injector and sample reagents are transferred by utilizing an accurate quantitative liquid drawing and injecting function of the pipetting injector. Cells in a settling and smear-manufacturing bin are naturally settled on a sticky glass slide by relying on the gravity of the cells and then automatically dyed. The liquid-based cell smear manufacturing and dyeing machine has the advantages that the operation is simple, the cost is relatively low, and high quality liquid-based cytological smears can be rapidly manufactured.

The invention belongs to the technical field of adsorption and separation, and specifically, relates to a resin ball capable of preferential adsorption of an aromatic hydrocarbon component of a hydrocarbon mixture, and a preparation method thereof. The resin ball is a styrene resin ball which has a certain crosslinking degree and is subjected to copolymerization modification by polar monomers. Through the difference of swelling adsorption capacities of the resin ball on aromatic hydrocarbons and non-aromatic hydrocarbons, an aromatic hydrocarbon component of a hydrocarbon mixture can be adsorbed conveniently by the resin ball so that an aromatic hydrocarbon component separation effect is realized. The resin ball is a random copolymer, wherein styrene is utilized as a main monomer, and various polar monomers and different cross-linking agents are utilized to modify styrene. The resin ball is prepared from easily available raw materials, is easy for synthesis, can be recycled, is convenient for operation and aftertreatment, simplifies a process flow, and reduces an operation cost and energy consumption. In application, through a gradient centrifugation method, the resin ball provides a novel and simple approach for high-efficiency separation of aromatic hydrocarbons and non-aromatic hydrocarbons.

The invention belongs to the field of biological chemistry and specifically relates to extraction method of chloroplast protein of xylophyta ginkgo leaf. The method comprises the following steps of: firstly, preparing coarse chloroplast particles from the ginkgo leaves, performing gradient centrifugation through Percoll, so as to obtain complete chloroplast by a specific centrifugal force; then, dissolving protein by using a protein extraction buffer solution, adding the same volume of Tris-balanced phenol for extraction, and precipitating protein by using a ammonium acetate methanol solution. The extraction method is capable of effectively increase extraction efficiency and purity of chloroplast protein. The ginkgo leaf chloroplast protein obtained by the method can be used for proteomics analysis of ginkgo leaf and other xylophyta materials rich in interfering substances, so that solubility and purity of protein are improved; a 2-DE map of the protein has a clear background and many protein spots are distinguishable.

The invention relates to the technical field of biology, in particular to an application of exosomes from mesenchymal stem cell in preparation medicine of promoting vascularization or inducing angiogenesis. The exosomes, processed with gradient centrifugation, filtering and ultracentrifugation, is with high purity. The exosomes has the advantages of easy preparation and storage, high safety coefficient, easy absorption, wide medicine route, has targeting ability, and makes the endothelial cells of the blood vessels come together and create new vessels, so that the microcirculation is reconstructed to increase blood flow perfusion. The deficiency of other functions is not caused, and the exosomes has a wide application prospect.

An embodiment of the invention discloses a pig circovirus III-type virus-like particle. A preparation method of the virus-like particle includes the steps: amplifying pig circovirus III-type Cap protein genes; constructing recombinant shuttle-plasmid pFB-Cap by the aid of the genes; constructing rB-Cap recombinant rod granules by the aid of the recombinant shuttle-plasmid pFB-Cap; transfecting therB-Cap recombinant rod granules into SF9 cells to obtain recombinant baculovirus rBV-PCV3 Cap expressing pig circovirus III-type Cap genes; enabling the recombinant baculovirus rBV-PCV3 Cap to infectHigh Five cells, and purifying the recombinant baculovirus rBV-PCV3 Cap infected by the High Five cells to obtain the pig circovirus III-type virus-like particl PCV3 VLP. A nucleotide sequence of theCap protein gene is as shown in SEQ ID NO.1. According to the particle, based on a baculovirus-insect cell expression system, preparation is implemented by the aid of High Five cell expression of serum-free suspension culture, and the PCV3 virus-like particle is acquired by combining sucrose cushion ultracentrifugation and sucrose density gradient centrifugation purification. The virus-like particle is good in immunogenicity and high in safety and has good development and application prospects.

The invention belongs to the technical field of regenerative medicine and biology and particularly relates to preparation and application of exosome with an efficacy of hair growth promoting. The exosome with the efficacy of hair growth promoting is prepared by steps of selecting human umbilical cord mesenchymal stem cells as an exosome source, culturing, collecting cell culture fluid and performing gradient centrifugal extraction and is rich in various physiological activators such as RNA and various proteins and capable of effectively improving hair loss of patients by subcutaneous injection.

The invention discloses a preparation method of leukocyte extract. Neonate cord blood is used directly as a raw material subjected to human lymphocyte separation density-gradient centrifugation to obtain PBMCs (peripheral blood mononuclear cells); amplification culture is performed via cord serum separated from the cord blood; centrifugal separating is performed to obtain supernate; leukocyte extract is acquired by ultra-filtration. The preparation method has no need for bovine serum, no heterogeneous animal proteins or stimulating factors are involved, and safety of application in human bodies is good. The leukocyte extract prepared via the method has rich natural human cellular factors, wherein bFGF (basic fibroblast growth factor) reaches 60 pg/ml and above, the yield is high, and the production cost is low. The invention also provides application of the leukocyte extract in the activation and restoration of cells. The leukocyte extract may be added to cosmetics to provide synergy with other components in cosmetic formulations; the leukocyte extract can cooperate to improve cell mitochondrial functionality, complete nutrients for cell metabolism are provided, and the cell activating function of the leukocyte extract is significantly improved.

The invention provides a blood cell separation tube and a method for separating a mononuclear cell by using the blood cell separation tube. The blood cell separation tube consists of a separation tube body, a rotary cover as well as a cell separation membrane and a sample adding slide plate (an auxiliary sampling device) which are arranged in the tube body. According to the method, a conventional complicated sample feeding mode is changed to an easily operated mode by changing sample-adding and sampling modes on the basis of a principle that the cell is separated through cell density gradient centrifugation. According to the blood cell separation tube and the method provided by the invention, a cell separation process can be more simplified and the operation can be easier in clinical medical examination and cell biology research.

The invention discloses umbilical cord blood mesenchymal stem cell separation liquid and an umbilical cord blood mesenchymal stem cell separation flow. The separation liquid is prepared by mixing saccharosan of which the concentration is 9 percent and meglumine diatrizoate of which the concentration is 33.9 percent in the ratio of 26.88:10, wherein the concentration of the separation liquid is 1.073+/-0.01g/L. A two-step method for separating umbilical cord blood mesenchymal stem cells comprises the following steps of: 1, precipitating red cells by a hydroxyethyl starch; and 2, separating the mesenchymal stem cells by the separation liquid of which the concentration is1.073g/L. The mesenchymal stem cell with the highest proportion in umbilical cord blood can be obtained by the separation liquid and the separation flow, the proportions of lymphocytes and mononuclear cells are very low, and the obtained cells are good in state and high in activity. The used materials and reagents are medical products, are non-toxic, do not have heat sources, can be industrially produced and are convenient to store. The used method is an optimized flow, is high in stability and repeatability, can be also used for separating human marrow and peripheral blood mesenchymal stem cells, is the best method for separating and extracting the mesenchymal stem cells in the current density gradient centrifugation and is better than other products and methods.

The invention relates to a monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and the monoclonal antibody is secreted by hybridoma cell of which the name is hybridoma cell strain Crab, the preservation number is CCTCC NO: C2012196, the preservation unit is China Typical Culture Preservation Center, and the preservation data is Jan 7th, 2013. The monoclonal antibody has a characteristic of being specifically combined with blue crab particle hemocyte 26.7kDa protein. The preparation method comprises the steps of: obtaining particle hemocyte by continuous density gradient centrifugation of Percoll medium; purifying the particle hemocyte 26.7kDa protein as antigen to immunize mice by electrophoresis rubber cutting and recycling; preparing hybridoma cells by a cell fusion method; screening out the monoclonal antibody of the anti-blue crab particle hemocyte 26.7kDa protein by an immunological detection method. The monoclonal antibody disclosed by the invention provides an important tool for researching functions of particle hemocyte in nonspecific immune response of blue crab and in other physiological function aspects.

The invention provides eye drops containing exosomes for dry eye treatment and a preparation method thereof. The eye drops are prepared from the following components: 2.5% of exosomes, an auxiliary ingredient and the balance water for injection, wherein the total content of the raw materials is 100%, the auxiliary ingredient is prepared from the following components in percentage by mass: 0.2% ofvitamin E, 0.2% of carbomer and 2% of an osmotic pressure regulator, the exosomes are extracted from human umbilical cord mesenchymal stem cells, the vitamin E can inhibit the reaction of lipid peroxidase in lens of eyes, enable peripheral blood vessels top be expanded and improve blood circulation, the carbomer enables the eye drops to achieve proper viscosity, and the osmotic pressure regulatoris NaCl. The method comprises the following steps of extracting the exosomes in a gradient centrifugation method, preparing a sealed container, preparing the auxiliary ingredient, and finally carryingout split packaging to obtain a sterile preparation convenient to use so as to safely and effectively treat dry eyes.

The invention belongs to the field of assisted reproduction, and particularly relates to sperm gradient centrifugation liquid and a preparation method thereof. According to the sperm gradient centrifugation liquid, pH (potential of hydrogen) of the sperm gradient centrifugation liquid is adjusted and controlled by HEPES+MOPS double-buffering systems, the pH is more stable, a key raw material lecithin is added into the sperm gradient centrifugation liquid, sperm activity is excited, and damage to sperms is repaired and reduced. Sperms selected by the sperm gradient centrifugation liquid are high in activity and higher in normal from rate, and full preparation is made for subsequent in-vitro fertilization.

The invention belongs to the technical field of film preparation and separation and purification, and discloses a vermiculite-based ion screening film and a preparation method thereof. The preparationmethod comprises the following steps: 1, mixing expanded vermiculite and a saturated NaCl solution, heating at high temperature, performing suction filtration and washed, and preparing powder A; 2, mixing the powder A with a LiCl solution, heating at high temperature, and performing suction filtration and washing to obtain powder B; 3, mixing the powder B with deionized water, stirring and stripping to obtain a turbid liquid C; 4, carrying out low-speed centrifugation on the turbid liquid C, and then carrying out gradient centrifugation so as to prepare nanosheet dispersion liquid under different rotating speed ranges; and 5, stacking the nanosheet dispersion liquid on the surface of a porous substrate, and drying to obtain the vermiculite-based ion screening film. The film has quicker and more efficient selective screening capability on monovalent/multivalent metal ions; the method is simple, low in cost, free of acid and alkali, wide in applicability and suitable for industrial production.

The invention relates to a biological agent for treating habitual abortion in the medicine technological field, which comprises the following steps: extracting peripheral blood of healthy adult men; diluting the blood to 1. 5 times through glass fibers from the ball and Hanks liquid to separate lymphocyte to collect single core cells; washing by Hanks liquid to culture and flow and count with absolute culture liquid of RPMI-1640; adjusting the cell concentration to 2X106/ml to add phaescolosaxin to culture at 37 Deg. C for 72hours; collecting supernatant to produce T cells through centrifuging of centrifugal ultrafiltration and cane sugar density gradient centrifugation; filtering by sterile filter membrane to be split charging to produce completed product at 80 Deg. C below zero. The invention evokes immune tolerance of body effectively and overcomes the more faults of evoking immune tolerance through cell adoptive immunotherapy to make easier clinical application, which can be produced in a large industrialization scale to get more stabilizing mass control and higher controllability for treating the habitual abortion with good curative effect.

The invention relates to an animal DNA extraction method, in particular to a soil nematode DNA extraction method. The soil nematode DNA extraction method comprises the following steps: adopting a washing-screening-magnesium sulfate gradient centrifugation method to extract nematodes from a soil sample and killing the nematodes by a heating method to obtain a nematode suspension; then, firstly adding a cell lysis buffer solution to the nematode suspension for freeze thawing treatment and then adding protease for heat preservation and digestion; and respectively adopting chloroform and isoamyl alcohol to extract DNA, NaAc and isopropanol sediment DNA and naturally drying the DNA, the NaAc and the isopropanol sediment DNA at a room temperature so as to obtain soil nematode DNA. The invention combines physical, chemical and enzyme methods to crack nematode cells, improves the extraction rate of the nematode DNA, has lower cost and is suitable for extracting the DNA of soil nematode communities.

The invention discloses a nucleus pulposus-cartilage extracellular matrix scaffold and a preparing method thereof and belongs to the biological tissue engineering technology. The preparing method comprises the steps that nucleus pulposuses and bony articular surface cartilage are smashed, decellularized, subjected to gradient centrifugation to collect extracellular matrix microfilament suspension, and freeze-dried to obtain a freeze-dried decellularized nucleus pulposus matrix and a freeze-dried decellularized cartilage matrix, the two matrixes are taken in certain proportion and dissolved in ultrapure water to prepare mixed matrix suspension with certain concentration, and the mixed matrix suspension is freeze-dried and subjected to ultraviolet irradiation crosslinking and chemical reagent crosslinking in sequence so that the nucleus pulposus-cartilage extracellular matrix scaffold can be obtained. In this way, the defect that the nucleus pulposuses are hard to obtain due to the small number is overcome; the preparing technology is simple, and decellularizing can be achieved thoroughly; the matrix scaffold is degradable and high in biocompatibility; the matrix scaffold is of a three-dimensional structure, is high in mechanical property and plasticity, and can facilitate the distribution, growth and differentiation of seed cells; the freeze-dried extracellular matrixes are convenient to store and prepare and can be used for constructing a tissue engineered intervertebral disc to repair partial defects of the intervertebral disc, and broad clinical application prospects are realized.

The invention provides a preparation method of apoptotic vesicle. The preparation method comprises the following steps: 1) culturing a mouse mononuclear macrophage leukemia cell line in vitro; (2) when the confluence degree of the cells reaches 90%-100%, adding STS (staurosporin) to induce cell apoptosis; and (3) collecting the supernatant of the culture solution, and then separating the supernatant by virtue of a gradient centrifugation method, so as to obtain the apoptotic vesicle (apoVs). The invention also provides the apoptotic vesicle and application of the apoptotic vesicle in preparation of a preparation for promoting adipogenic differentiation of mesenchymal stem cells. The apoptotic vesicle provided by the invention can promote adipogenic differentiation of mesenchymal stem cells, can be used for repairing soft tissue defects or recesses, and have no obvious side effects.

The invention discloses an exosome for mass spectrometric flow cytometry detection and a preparation method and application thereof, and belongs to the field of nanobiological materials. The exosome comprises a heavy metal tag compound which can be used for mass spectrometric flow cytometry detection, wherein and the heavy metal tag compound is distributed in the lumen of the exosome. The preparation method comprises the following steps: 1) extracting the exosome: obtaining the exosome by gradient centrifugation, concentration and precipitation; or obtaining the exosome by ultra-high speed centrifugation; 2) introducing a heavy metal tag: introducing the heavy metal tag compound into the exosome by electroporation and incubation of the exosome obtained in the step 1) and the heavy metal tag compound; 3) obtaining the exosome with the heavy metal tag: removing the free heavy metal tag compound which is not encapsulated by the exosome by using a sedimentation kit or ultra-high speed centrifugation so as to obtain the exosome for mass spectrometric flow cytometry detection. The present application also provides the application of the exosome to realize high-throughput single cell tracing exosome uptake.

The invention relates to a purification method of recombinant herpes simplex virus. The method comprises: adding 5% of PEG 8000 to the recombinant herpes simplex virus supernatant obtained through amplification in Vero cells, and carrying out centrifugation; dissolving the precipitate with a TNMC buffer solution, and adding n-dodecyl-beta-D-maltoside to the solution to promote the precipitate dissolving; and adding the concentrated mtHSV solution to the sucrose density gradient top layer, and carrying out centrifugation identification analysis. The method of the present invention combines the precipitation method and the sucrose density gradient centrifugation method, has characteristics of low cost, no damage on virus, good purification effect, and the like, and is the effective virus purification method capable of be promoted.

The invention provides a separation method of autotrophic bacteria and heterotrophic bacteria in activated sludge of a sewage treatment plant, which belongs to the technical field of biological sewage treatment. According to the method, DNAs of the autotrophic bacteria and the heterotrophic bacteria with different demands for types of carbon sources in the activated sludge of the sewage treatment plant can be separated, and the characteristics of the two types of bacteria can be analyzed at a gene level. The activated sludge of the sewage treatment plant is cultivated in situ by adopting 13C-marked inorganic matters or organic matters as carbon sources, and DNA solutions at different levels are obtained by adopting a superspeed density gradient centrifugation method of cesium chloride with high stability and good separation effect. The densities of the solutions at all levels after separation are cut down from big to small correspondingly in the bottom-to-top direction of a centrifugal tube. The characteristics of microbial communities of DNA samples at 5-9 levels are analyzed by adopting a real-time quantitative PCR (polymerase chain reaction) technology and a high throughput sequencing technology. The invention provides the economic, simple, convenient and reliable separation method of the autotrophic bacteria and the heterotrophic bacteria in the activated sludge of the sewage treatment plant.