74 results about "Canine kidney" patented technology

The invention discloses a serum-free medium for Madin-Darby canine kidney (MDCK) cell full suspension culture. The serum-free medium for MDCK cell full suspension culture comprises an amino acid part, a vitamin part, an inorganic salt part and other additive parts. The serum-free medium has the beneficial effects that the serum-free medium for MDCK cell full suspension culture, provided by the invention, is high in cell culture density and clear in composition and does not contain animal serum, a downstream product is purified, the product quality is improved, and the serum-free medium is convenient to prepare and use and suitable for large-scale production of influenza vaccines and avian influenza vaccines.

The invention discloses a quantum-dot-based method for carrying out in-situ and real-time detection on heavy metal ions in cells. The method comprises the following steps of: A. preparing a carboxymethyl chitosan-CdTe quantum dot solution with certain concentration; B. digesting canine kidney cells overgrowing in a T25 cell culture flask bottle into a unicellular suspension, inoculating into a cell culture dish, and culturing; C. adding the carboxymethyl chitosan-CdTe quantum dot solution into the culture dish, and hatching for a period of time, thereby obtaining the canine kidney cells marked with fluorescence; and D. adding a DMEM (dulbecco's modified eagle medium) nutrient solution containing heavy metal ions with gradient concentration and unknown concentration respectively in washed cells in the culture dish, hatching together with the cells, detecting the average fluorescent intensity of the cells respectively by a flow cytometry, and drawing a standard curve through the changes of the cell fluorescent intensity, so that the concentration of the heavy metal ions in the cells can be calculated. The method is simple and convenient to operate; the fluorescence of marked cells can be quenched regularly as the increase of heavy metal ions, and the real-time and in-situ detection on the outer source heavy metal ions in the cells is realized.

The present invention relates to a commercial-scale process for the production of influenza virus or antigens for prophylactic, diagnostic, immunotherapeutic or therapeutic purposes. Particularly, the invention provides a Madin-Darby Canine Kidney (MDCK)-derived, cell line and a cell culture-based process for the production of an influenza vaccine and more particularly, a human vaccine comprising influenza types A and B.

The invention discloses a method for preparing an MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and the MDCK cell line. The method includes steps of 1), discarding culture media for cultivating MDCK cells, cleaning cell layers by the aid of pancreatin solution, then discarding the pancreatin solution, adding pancreatin solution into the MDCK cells again, digesting the MDCK cells, stopping digesting the MDCK cells after the cells are rounded, centrifuging cell suspension and then discarding supernatant to obtain cell clusters; 2), re-suspending the cell clusters obtained at the step 1) by the aid of serum-free culture media to obtain cell re-suspension; 3), arranging the cell re-suspension obtained at the step 2) in a shaking table, cultivating the cell re-suspension in culture tanks, diluting the cell re-suspension by the aid of the serum-free culture media and carrying out passage on the cell re-suspension to obtain the MDCK cell line. The method and the MDCK cell line have the advantages that the MDCK cell line is high in cell density and activity and uniform in size, individually grows in a dispersion manner, is plump in form and is suitable for serum-free suspension culture by the aid of bioreactors.

The invention provides a serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells. The culture medium is prepared from a DMEM/F12 culture medium containing 5g/L of NaCl and the following components: sodium stannite, recombinant human epidermal growth factors, hydrocortisone, recombinant full-chain insulin, prostaglandin E1, human transferrin, thyroxine (T3), vitamin E, cholesterol, ethanol amine, beta-mercaptoethanol, Tween-80, Hypep1510, recombinant human serum albumin ACF and mannitol. With the adoption of the culture medium provided by the invention, the full-suspension culture of the MDCK cells can be carried out very well under the condition that serum is not added; after the MDCK cells are continuously cultured for 20 generations, the average proliferation concentration of the MDCK cells is 2.308*10<6>/mL, the average cell viability is 97.4 percent and the average doubling time is 34.48 hours, so that the serum-free culture medium provides technical supports for developing influenza vaccines of cell matrixes of mammals in China.

The invention discloses application of curcumin to preparation of medicaments for preventing and/or treating autosomal dominant polycystic kidney disease. A madin-darby canine kidney (MDCK) vesicle model is used for screening to obtain the curcumin which inhibits formation and growth of vesicles. An experimental result shows that: the curcumin has an obvious inhibiting effect on the formation and growth of MDCK vesicles and the effect of the curcumin is in dose response relationship; the curcumin has no cytotoxicity to MDCK cells, so that the vesicle inhibiting effect of the curcumin is independent of the cytotoxicity; the curcumin does not obviously induce MDCK cell apoptosis, so that the vesicle inhibiting effect of the curcumin is independent of cell apoptosis promotion of the curcumin; the curcumin can promote the MDCK cells or vesicles to form tubular structures; and the effect is in dose response relationship; and the curcumin has an inhibiting effect on the growth of the embryonic kidney vesicles. The curcumin is expected to be developed into a specific medicament for preventing and/or treating autosomal dominant polycystic kidney disease.

The invention discloses a cryopreservation method for protecting cell junction. The method comprises the following steps of: a) culturing Madin Darby canine kidney (MDCK) cells; b) dividing the MDCK cells in the step a) into a normal control group, a cryopreservation group, a trehalose group, an Hsp70 high expression group and an Hsp70+ trehalose group; c) performing filter culture on the normal control group, the cryopreservation group and the trehalose group respectively, performing filter culture and gene transfection on the Hsp70 high expression group, and performing filter culture and gene transfection on the Hsp70+ trehalose group; d) performing cryopreservation on the normal control group, the cryopreservation group, the trehalose group, the Hsp70 high expression group and the Hsp70+ trehalose group; e) growing cells and performing morphologic observation (the cultured cells are observed by a microscope and are subjected to HE staining); f) testing the survival rate of the cells; g) observing and analyzing the cell junction morphology and structure; and h) performing a fluorescein experiment. The method is simple, and convenient to use; and the structure and function of the cell junction after tissues and organs are subjected to cryopreservation can be kept complete, and the damage of the cell junction can be effectively avoided.

The invention discloses a series of urea transporter inhibitors of which the structural formula is disclosed in the specification. An erythrocyte model is used for screening to obtain compounds for inhibiting urea transporters. The experimental result indicates that the compounds (such as Youti) can inhibit the permeation of urea transporter UT-B mediated erythrocyte membranes for urea, and the action forms a dosage dependency relationship; Youti within effective dose range has no cytotoxic action on the MDCK (Madin-Darby Canine Kidney) cells, which indicates that the action of Youti for inhibiting cell permeable urea is irrelevant to cytotoxicity; the inhibiting action of Youti on the urea transporter UT-B gradually increases; the inhibiting action of Youti on the urea transporter UT-B is reversible; and the in-vivo test result proves that Youti can obviously increase the uresis amount of a rat, lower the concentration of urea in the urine of the rat, and lower the osmotic pressure, which indicates that Youti has selective diuresis action on urea in vivo.

The invention belongs to the technical field of animal infectious diseases, and in particular relates to a method for separating and identifying influenza A viruses H1 and stably proliferating the viruses on Madin-Darby canine kidney (MDCK) cells. An influenza A viruses H1 strain A-Influ/ JML-F9 is obtained by separation and identification, and the collection number of the strain is CCTCC NO: V201105. The influenza A viruses H1 are stably proliferated on the MDCK cells by an optimal method, an optimal culture medium and optical culture conditions, so that the average blood congeal titer of the viruses reaches 7log2 by large-scale proliferation, and the highest blood congeal titer of the viruses reaches 9log2.

The invention discloses canine recombinant interferon alpha, and also provides a preparation method of the canine recombinant interferon alpha. Recombinant genes of canine interferon alpha are expressed in escherichia coli, and canine interferon alpha with a purity of more than 98% is obtained through processes of renaturation and purification. The biological activity is determined by a MDCK (canine kidney cell)-VSV (vesicular stomatitis virus) virus system, and the specific activity reaches 2-3*107 IU/mg.

The invention belongs to the technical field of biology, and particularly relates to an MDCK (Madin-Darby canine kidney) cell line capable of stably expressing a human-derived TIGAR (TP53-induced glycolysis and apoptosis regulator) gene. The cell line is an MDCK cell line TG-418-E5 capable of stably expressing the human-derived TIGAR gene and contains a TIGAR coding gene sequence, and the preservation number of the cell line is CGMCC NO:12983. Through stable expression of the TIGAR gene in MDCK cells, anti-apoptosis capacity of the cells is improved, and the survival time of recombinant cells is prolonged. The method not only lays the foundation for research of application of MDCK serum-free fully-suspended culture in mass production of cell culture vaccines, but also increases breeding titer of avian influenza vaccine strains and saves the cost of a production process. Besides, the cell line TG-418-E5 has better application prospect in aspects of screening of anti-avian influenza virus drugs, screening of vaccine strains and production of the cell culture vaccines.

The invention discloses a method for marking canine kidney cells through a chitosan-quantum dot fluorescent probe. The method comprises the following steps of: A, preparing solution of a carboxymethyl chitosan-CdTe quantum dot; transferring the solution of the carboxymethyl chitosan-CdTe quantum dot to a sterilized volumetric flask by using a pipette according to the concentration of the carboxymethyl chitosan-CdTe quantum dot, and adding high-purity water for constant volume; B, digesting the canine kidney cells overgrown in a T25 cell culture flask by using ethylene diamine tetraacetic acid (EDTA)-trypsin so as to form unicellular suspension, counting cells, inoculating the canine kidney cells to a cell culture dish, and adding nutrient solution for culturing; and C, after the canine kidney cells grow adherently, adding the chitosan-quantum dot fluorescent probe into the culture dish, and incubating the probe and the canine kidney cells together to obtain canine kidney cells marked with fluorescence. The method is easy and convenient to operate, has high fluorescent efficiency, good biocompatibility and long stable time, and can be used for observing the cells for a long time; and the fluorescent probe does not have cytotoxicity.

The invention relates to herbal medicine health wine, which is prepared by grinding and wine steeping the following raw materials in parts by weight: the fruit of Chinese wolfberry, dried longan pulp,yam, flos caryophyllata, cortex cinnamomi, Chinese date, fructus amomi, dark plum fruit, poria cocos, canine kidney, crystal sugar and pink grapefruit. The herbal medicine health wine belongs to pureChinese traditional secret recipes, which takes traditional medicinal materials and high-quality liquor as raw materials and is prepared through infusing by adopting a scientific proportioning, thusbeing applicable to the majority of the middle-aged and aged persons, and being capable of regulating yin and yang, soothing fatigue, protecting health and nourishing. The herbal medicine health wineis also characterized by pure naturalness and no toxic and side effects.

The invention provides construction of a cell model for cotransfection of CYP3A4 and MDR1. The method comprises the following steps of: cloning P-gp gene to pcDNA3.1(+), transfecting Madin-Darby canine kidney (MDCK) cells, and screening monoclonal cells by using G418, thus obtaining MDCK-MDR1 cell strains; and cloning CYP3A4 to a carrier pcDNA3.1(+)/Hygro which is provided with a hygromycin B selection marker, transfecting MDCK-MDR1 cells, and obtaining the cell model for cotransfection of CYP3A4 and MDR1 through hygromycin B selection. The MDCK-MDR1/CYP3A4 was collected in China Center for Type Culture Collection (CCTCC) on Dec.15, 2011, with the collection number of CCTCC2011119. The model constructed can be used for simulating the absorption and metabolic process of a medicine in the intestinal tract so as to screen the common substrate of P-gp and CYP3A4.

The invention provides a construction method and applications of a cell model expressing human organic cation transporter-1. A hOCT1 wild-type gene segment is obtained from a hepatic tissue; two mutant gene segments P341L and M420del can be obtained by specific point mutation; the two segments are connected with a plasmid vector; darby canine kidney cells (MDCK) are transfected; G418 resistance screening is carried out to obtain wild-type cells expressing hOCT1 and cells of two mutants; mRNA level verification is carried out on the cells; and functional verification is carried out by utilizing a hOCT1 substrate and an inhibitor. The cell model provided by the invention can be used for screening the hOCT1 classic substrate and the inhibitor, and predicting the transportation of medicament in human bodies and medicament interaction which possibly happen; and by utilizing the cell model, the influence of gene polymorphism of the transporter on the medicament transportation function can be predicted; and the cell model provides a standard for clinical reasonable medicament administration and individualized medicament administration, and has reasonable design and good repetitiveness.

The invention discloses a spinner-flask culture method for H9N2 subtype of avian influenza virus. The spinner-flask culture method comprises a step of culturing MDCK (Madin-Darby canine kidney)-HA cells, namely culturing the MDCK-HA cells in a spinner flask; a step of inoculating the MDCK-HA cells with the H9N2 subtype of avian influenza virus; particularly, in the inoculating step, when monolayer cells in the cells growing in the spinner flask are more than 90%, the H9N2 subtype of avian influenza virus is inoculated and incubated for 1-2 hours in a cell incubator, and 50ml of 2% NCS (new-born calf serum) DMEM (Dulbecco modified eagle medium) maintenance solution is added after the incubation is ended, and the cells inoculated with the H9N2 subtype of avian influenza virus are cultured in the cell incubator, and finally cell supernatant is collected. According to the spinner-flask culture method disclosed by the invention, the MDCK-HA cell system is adopted, and the conditions of spinner-flask culture are optimized, thus the H9N2 subtype of avian influenza virus is rapidly proliferated to reach a high virus titer and obtain a good immune effect.

The invention provides a method for culturing MDCK (medin-darby canine kidney) cell proliferation recombinant H5N1 subtype avian influenza virus comprises the following steps: preparing a chip carrier, pretreating a paper scrap carrier, sterilizing a bioreactor, carrying out preculture, inoculating cells, adsorbing on the paper scrap carrier, continuously culturing cells, inoculating avian influenza virus and harvesting influenza virus bulk. The method for culturing the MDCK cell proliferation recombinant H5N1 subtype avian influenza virus has the beneficial effects that carrier cost is low, hematic coagulating rate of obtained influenza virus bulk is high, the paper scrap carrier is adopted for carrying out bioreactor large-scale culture of MDCK cell proliferation recombinant H5N1 subtype avian influenza virus, large-scale culture can be realized more easily, occupying space of a production line is small, production efficiency is high, and the adopted equipment has the advantages of strong stability, good repeatability and the like.

The invention discloses a hybridoma cell strain 2C1 capable of secreting CAV-2 monoclonal antibodies, the monoclonal antibodies secreted by the cell strain and application. The preservation number of the hybridoma cell strain 2C1 is CGMCC No. 10878. An indirect immunofluorescence result shows that the monoclonal antibodies secreted by the hybridoma cell strain 2C1 can specifically recognize CAV-2 viruses and do not react with MDCK (madin-darby canine kidney) cells. A result of a superposition experiment of the hybridoma cell strain 2C1 and two monoclonal antibodies secreted by another hybridoma cell strain shows that the two monoclonal antibodies are used for different antigenic epitopes of the CAV-2 viruses. The monoclonal antibody prepared by the invention can react with CAV-2 well and can be used for identifying a clinic isolated strain. The 2C1 monoclonal antibody and the 7D7 monoclonal antibody which are prepared by the invention are used for recognizing two different antigen determinants of the CAV-2, so that the monoclonal antibodies can be used for detecting preparation of CAV-2 pathogenic colloidal gold and establishment of a sandwiched ELISA (enzyme-linked immuno sorbent assay) method.

The invention discloses a serum-free suspension culture type MDCK cell (Madin-Daby Canine Kidney Cell) strain and an application thereof. The serum-free suspension culture type MDCK cell strain is preserved in the China Center for Type Culture Collection, the classification designation is madin-daby canine kidney cell MDCK-XF04, and the preservation number is CCTCC NO:C2018192. The serum-free suspension culture type MDCK cell strain can be applied to production of influenza vaccines and avian influenza vaccines. Through serum-free adapted culture and suspension culture domestication, the serum-free suspension culture type MDCK cell strain is obtained, the madin-daby canine kidney cell MDCK-XF04 cells are high in suspension culture vitality on serum-free mediums, seldom to aggregate and good in uniformity, and completely adapt to suspension culture on the serum-free mediums, and the serum-free suspension culture type cell strains are provided for relevant research and production of development of influenza and avian influenza vaccines in China.