Stable cell line of human ovarian adenocarcinoma skov-kat
SUBSTANCE: invention can be used for screening of anti-tumour preparations in vivo, in particular for screening synthetic and natural substances with potential anti-tumour activity.
EFFECT: claimed invention makes it possible to create stable cell line of human ovarian adenocarcinoma.
2 dwg, 2 ex
The invention relates to biotechnology and experimental Oncology and can be used for screening of anticancer drugs in vivo, in particular for the screening of synthetic and natural substances with potential antitumor activity.
In the selection of synthetic and natural substances with potential antitumor activity widely used test for the lines of tumor cells in vivo.
The use of panels of diverse genetic and phenotypic characteristics of cell lines provide a very effective screening of synthetic compounds, natural substances and physical effects on searching for their biological activity at the cellular and molecular levels. One of the directions in cancer therapy is the development of drugs with mechanisms for targeted delivery to cancer cells. When working with tumor models in vivo, it is extremely important to have objective methods of observation and monitoring of tumor cells. In recent years, was developed region fluorescence tomography. It allows you to observe cancers non-invasive method, which is especially important for a detailed analysis of the effectiveness of cancer drugs.
Known cancer cell lines with overexpression of HER marker/neu (Oncogene, 11; 19(53):6102-14, 2000):
- SKOV-3 cell line of human ovarian adenocarcinoma (J. Natl. Cancer Inst. 59, 221-226, 1977);
In SK-BR-3 cell line breast cancer man (New York: Plenum Publishing Corp, pp.115-145, 1975).
However, these HER2/neu positive line does not have a pronounced fluorescence, which makes them unsuitable for direct observation using fluorescent imaging.
Known cancer cell lines, modified green fluorescent protein GFP:
1) cancer cell lines human breast MF-7, MDA, MW, MDA-MB435;
2) human cell lines of prostate cancer RES, DU145;
3) cell line human glioblastoma 324;
4) cell line human lung cancer Anip-73, N;
5) cancer cell lines human colon l-205, HCT-15, WiDr;
6) cell line gastric cancer man NVGC-4;
7) cell line kidney cancer human SN12C;
8) cancer cell line of human language SCC-25;
9) cell line human melanoma LOX, SK-mel-5;
10) cancer cell line of Chinese hamster ovary Cho-K1;
11) cell line melanoma mouse 16.
Known cancer cell lines, modified red fluorescent proteins:
cell line human pancreatic cancer MIA-Race-2, modified RFP (Clinical and Experimental Metastasis, Volume 21, Number 1 7-12, 2004);
cell line alveolar rhabdomyosarcoma man is ka Rh30, modified DsRed2 (Cell Proliferation, Vol.41 Issue 2 Page 365, 2008);
cell line melanoma B16F0, modified RFP (Proceedings of the National Academy of Science, vol. 100, Issue 24, p.14259-14262, 2003);
cell line prostate cancer mouse MMT, modified RFP (Proceedings of the National Academy of Science, vol. 100, Issue 24, p.14259-14262, 2003);
cell line prostate cancer human PC-3, a modified RFP (Proceedings of the National Academy of Science, vol. 100, Issue 24, p.14259-14262, 2003);
cell line of colon cancer HCT-116, modified RFP (Proceedings of the National Academy of Science, vol. 100, Issue 24, p.14259-14262, 2003).
Known closest to the claimed cancer cell line human breast MDA-MB-231, modified tdTomato (Neoplasia, 8(10): 796-806, 2006).
Known fluorescent lines do not have the overexpression of this gene marker HER2/neu, and therefore they cannot be used for screening synthetic anticancer compounds and natural substances with targeted delivery to HER2/neu or directional mechanism of action for HER2/neu.
The objective of the invention is the creation of a model cell line bearing a tumor marker HER2/neu and fluorescing in the far red region, for advanced analysis and in vivo screening of substances targeted antitumor activity and detailed research results impact on this type of cancer in vivo.
The problem is solved due to tile the offered lines of human ovarian adenocarcinoma SKOV-kat, do overexpress tumor marker HER2/neu and fluorescing in the far red spectral region from 600 to 700 nm, for use in the screening of anticancer drugs in vivo.
For specified cell line gene of the fluorescent protein Katushka contained in the vector plasmid pKatushka-N, injected with the drug Unifectin56(Unifact Groups) in a line of cancer cells SKOV-3 and perform the selection on the antibiotic gentamicin and G418 (Sigma).
It is known that animal tissues are more transparent to long-wave radiation (Neoplasia, 8(10): 796-806, 2006).
Protein Katushka fluorescent in the far red spectrum has a maximum fluorescence at 635 nm and the maximum absorption at 588 nm and is the brightest at the wavelength of 650 nm, other proteins have 40-60% brightness Katushka in this wavelength range (Nature Methods, 4, 741-746, 2007) - this makes it one of the best tools for monitoring grafted tumors in the tissues of a living organism using fluorescence imaging.
After removing the cells from cryopreservation they retain fluorescence in full. Measurement of the fluorescence produced by using a flow cytometer (Beckman-Coulter, USA) (column 1, 2).
Cells received line SKOV-kat consistently reproduce the acquired fluorescence for six months of in vitro cultivation (more than 500 generations).
Using the test system on the basis of the e immunospecific of staining (RF patent No. 2303783, 2007) indicate the presence of a HER2/neu receptor on the surface of transfected cells in number, are identical to lines SKOV-3 (see table).
Cell line SKOV-kat have intense fluorescence in the far red region of the spectrum (600-700 nm) in contrast to the original cell line SKOV-3 (column 1, 2).
Cell line SKOV-kat resistant to the antibiotic G418 in contrast to the cell source line SKOV-3.
Cell line SKOV-kat have the level of expression of a marker HER2/neu on the surface, identical to the level of expression of HER2/neu on the surface of SKOV-3.
Cell line SKOV-kat morphologically similar to cells of the parental line SKOV-3 cell adenocarcinoma of the ovary.
The culture of the adhesive type. Vials area of 25 cm2sow 1.0-9.0×105cells in 5 ml growth medium. Passedout every three days. Cell lines are not demanding in terms of cultivation and may persist attached to the tablet in the form of a monolayer.
Growth environment: the environment of a needle in a modification of Dulbecco (DMEM), L-glu, 10% serum.
Marker signs following.
- Overexpression of surface antigen HER2/neu.
Intense fluorescence in the far red region of the spectrum (600-700 nm).
Cell line SKOV-kat resuspended in serum-free medium containing
7, 5% is metilsulfate (DMSO), at a concentration of 2-10×106cells/ml, poured into plastic vials and placed in a heat insulating material (polyurethane foam) and transferred into a low-temperature refrigerator at -70°C for 24 hours, after the initial freeze was placed on long-term storage at -135°C. Thawed quickly at 37°C. Cells diluted in 10 ml serum-free medium and precipitated by centrifugation, resuspended in 5 ml of medium containing 10% fetal calf serum, and transferred into a culture flask. Cell viability assessed by adhesion on plastic for 20 to 50 hours.
Thus, the obtained transfusiona genome Katushka line SKOV-kat has a number of qualities which distinguish it from the original line SKOV-3, as well as other cancer cell lines:
- cell line SKOV-kat resistant to the antibiotic G418 in contrast to the cell source line SKOV-3;
- cell line SKOV-kat have significant fluorescence in the far red region of the spectrum (600-700 nm) in contrast to the original cell line SKOV-3 (column 1, 2);
cells lines have the overexpression of tumor marker HER2/neu unlike other fluorescent cell lines.
The invention is illustrated graphic materials
Figure 1 - histogram 1.
Comparative characteristic fluorescence cell lines SKOV-kat and SKOV-3 620 nm.
Data flowing qi is fluorimeter (Beckman-Coulter,USA) is presented in the form of a histogram of the number of registrations (cells) from the logarithm of the luminosity, presented in arbitrary units luminosity (Cup). In parentheses are shown the average value of the fluorescence, normalized to 100 for SKOV-kat, averaging was performed over 10300 registrations.
Figure 2 - histogram 2.
Comparative characteristic fluorescence cell lines SKOV-kat and SKOV-3 675 nm.
Data from the flow cytometer (Beckman-Coulter,US) presents the histogram of the number of registrations (cells) from the logarithm of the luminosity presented in arbitrary units luminosity (Cup). In parentheses are shown the average value of the fluorescence, normalized to 100 for SKOV-kat, averaging was performed over 10300 registrations.
The invention is illustrated by the examples.
Obtaining cell line.
Transfection make as follows: 200 ng of plasmid DNA (gene fluorescent protein Katushka contained in the vector plasmid pKatushka-N) in 10 µl environment Dulbecco''s Modified Eagle's Medium (DMEM) (paneco) without serum mixed with 1 ál Unifectin56(Unifact Groups) 40 µl (medium without serum), and incubated for 15 minutes and add to the cells SKOV-3 (calculation of 60 mm Cup, 70% of the monolayer). One day after transfection the medium was added to the antibiotic G418 (Sigma)
in the amount of 500 μg/ml and continue to add in the same amount every 5 days, together with the change of environment. After 20 days in selective conditions on the cups are formed of fluorescent colonies, which is s selected using a platinum loop in wells with medium and an antibiotic. Then after the formation of the colony re-selection in the wells with medium without antibiotic. Within 50 days see a stable level of fluorescence. Numerical analysis of fluorescence produced using flow cytofluorometry (column 1, 2). To do this, cells are removed with a solution of Versene (Hyclone) and using centrifugation with a frequency of 800 rpm, the cells are transferred into a solution of PBS (KN2RHO41.7 mM; Na2HPO45.2 mm; NaCl 150 mm) for monitoring on the device. Data from the flow cytometer (Beckman-Coulter, USA) treated for 30,000 registrations.
Demonstration of the presence of cell marker.
Cells are placed in a solution of PBS with 0.5 million cells/ml with molecular structures 4D5-barnase-4D5+barstar-EGFP 4 μg/ml and incubated for 45 minutes at +4°C. as a negative control non-specific binding using the cell line HeLa (Cancer Res., 12: 264-265, 1952), and as positive control - the original cell line SKOV-3. For the numerical analysis on flow cytometer cell is removed from the tablet in a solution of PBS (table). Data from the flow cytometer (Beckman-Coulter, USA) treated for 30,000 registrations. The number of HER2/neu on the surface of cells is directly proportional to the incremental signal from the dye minus nonspecific staining.
Cell line SKOV-kat HER2/neu-positive adenocarcinoma of the ovary of man, fluorescent in the far red region of the spectrum (600-700 nm), obtained by transfection of the cell line SKOV-3 gene of the protein Katushka. Stable cell line SKOV-kat fully preserves the phenotype of HER2/neu-positive adenocarcinoma of the ovary and produces intracellular fluorescent protein Katushka. Cells obtained fluorescent lines reproduce the acquired characteristic for at least six months of in vitro cultivation.
The above properties and evaluation results of these properties allow the use of cell line SKOV-kat of human ovarian adenocarcinoma for screening of anticancer drugs in vivo targeted delivery mechanism to a surface marker of cells of HER2/neu.
Stable cell line human ovarian adenocarcinoma SKOV-kat resistant to the antibiotic G418, do overexpress tumor marker HER2/neu and fluorescing in the far red spectral region from 600 to 700 nm, obtained by a process comprising the following stages:
A. providing the plasmid containing the gene of the fluorescent protein Katushka;
b. transfection of cells SKOV-3 plasmid containing the gene of the fluorescent protein Katushka;
C. selection of fluorescent colonies in the environment in the presence of the antibiotic G418;
d. repeated selection of fluorescent colonies in the environment in the absence of the antibiotic G418.
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention relates to molecular biology and can be used in designing agent and methods of modulating body functions associated with HGF/c-met signalling pathway. The invention discloses HGF/c-met polypeptide-antagonists which are mutant forms of HGF which contain a mutation in the N-terminal part of the β-chain and/or in its dimerisation part. The disclosed polypeptides have lower biological activity compared to wild type HGG and can be used in modulating activity of c-met, cell proliferation, cell migration and angiogenic cell activity.
EFFECT: invention describes a method of obtaining HGF muteins using DNA recombinant technology and agents which are necessary for its existence.
22 cl, 8 dwg, 1 ex
SUBSTANCE: purified RNA synthesised in vitro by nonviral transfection is introduced into placental and cord cells. RNA comprises at least one sequence providing cell transition to a pluripotent state.
EFFECT: method allows increasing efficiency of preparing selected somatic pluripotent cells, reducing probability of oncogenesis and other negative consequences of cell reprogramming, and eliminating invasiveness at the stage of preparation of the cell material.
10 dwg, 3 ex
SUBSTANCE: nucleotide sequences, which code polypeptides of resistance to glyphosate are used in structures of DNA or expression cassettes for transformation or expression in organisms, including microorganisms and plants.
EFFECT: transformed organisms become resistant to herbicide.
17 cl, 5 dwg, 11 tbl, 23 ex
SUBSTANCE: method facilitates linkage of sequences, coding immunoglobulin variable regions, T-cells receptors or B-cells receptors. Method is instrument of higher effectivity for making sequence data libraries. Capability of multiple RT-PCR with chain extension by interruption with employment of matrix, derived from single cell, provides highly effective creation of sister pairs libraries.
EFFECT: method is effective for linkage of two or few nucleotide sequences, coding domens or subunits of heteromeric protein as a result of single reaction performance.
51 cl, 25 dwg, 27 tbl, 14 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically obtaining hemopoietic cells from blood, and may be used in medicine. The homopoietic cell CD34+ is extracted from peripheral blood of cancer patients undergone a growth factor treatment course. The obtained cell is transduced by a ligand which induces apoptosis with participation of the tumour necrosis factor, and is used to treat tumours.
EFFECT: invention enables to obtain a hemopoietic cell CD34+ which has anti-tumuor activity.
6 cl, 3 dwg, 8 tbl, 3 ex
SUBSTANCE: invention refers to medicine, particularly to a method of treating ophthalmopathies. The method of treating an ophthalmopathy in a mammal by recovery from mammal's bone marrow of a population of haematopoietic stem cells of a negative lineage which includes precursor endotheliocytes, transfection of the prepared population with a gene which functionally codes a fragment of antiangiogenic protein T2 of human tryptophanyl-tRNA-synthetase, and the following introduction in a vitreous body of the population of transfected cells in amount sufficient for symptomatic relief. The method of transgene delivery in a retinal vasculature of the mammal by introducing in the mammal's vitreous body of the population of haematopoietic stem cells of the negative lineage including precursor endotheliocytes where the cells are transfected with the antiangiogenic fragment of protein T2 of human tryptophanyl-tRNA-synthetase.
EFFECT: method of treating the ophthalmopathy is novel and highly effective.
2 cl, 27 dwg, 2 tbl, 10 ex
SUBSTANCE: invention includes compositions, methods and cell lines related to insect control. Substance of invention includes compositions that contains vegetable essences purposely acting at least at one receptor of insects selected from the following - tyramine receptor , olfactory receptor Or83b or olfactory receptor Or43a, as a result of which intracellular levels cAMP, Ca+2 or both vary in insects. Also invention includes method of insect control, line of insects cells including sequence of nucleic acid of above mentioned receptors and methods for selection of composition by activity in respect to insects with application of specified line of insect cells.
EFFECT: development of softer sparing compositions that are not toxic for mammals and other types.
20 cl, 34 dwg, 1 tbl, 33 ex
FIELD: food industry.
SUBSTANCE: strain Streptococcus thermophilus which produces lactic acid is described. Sequence of nucleic acids made of the strain producing polysaccharides are also described as well as food or pharmaceutical composition and milk product containing such strain.
EFFECT: strain has strong structural properties.
16 cl, 4 dwg, 6 tbl, 5 ex
SUBSTANCE: this invention is related to the field of biotechnology and may be used in production of various protein products with the help of recombinant DNA technology. New sequences of DNA are defined and generated, which are related to matrix attachment region, which are characterised by ability to improve producing of protein in eukaryotic cells.
EFFECT: methods are suggested for transfection of eukaryotic master cells, including new method of multiple transfection, based on use of active sequenes of DNA MAR according to invention and providing for substantial increase of recombinant protein expression level compared to similar cells transfected by traditional methods.
11 cl, 21 dwg, 9 tbl, 17 ex
SUBSTANCE: invention relates to field of genetic engineering and medicine. Described is animal, non-human, having sequence of nucleic acid encoding presenilin 1, carrying mutations, corresponding to M233T and L235P mutations in PS1 protein of mouse. Animal also contains sequence of nucleic acid, encoding whole gene or part of gene, encoding APP. APP protein represents APP751, originates from human and carries mutations Swedish and London. Animal is intended for application in fight against Alzheimer's disease. Also described are PS1 protein and encoding it nucleic acid.
EFFECT: invention can be used in medicine for discovering compounds intended for Alzheimer's disease treatment.
20 cl, 50 dwg, 1 tbl, 8 ex
SUBSTANCE: cell line of human skin melanoma mel Cher possesses stable culture and morphological characteristics, is stored in Specialised collection of cell cultures of Institute of CYTOLOGY RAS under number PKKK ("П") 704"Д". Ctll line of human skin melanoma mel Cher expresses growth factors, such as VEGF, of VEGF receptors of I-st and II-nd types, markers of endothelial cells, such as VIII as.ag., Laminin 5 gamma 2.
EFFECT: obtained cell line is able in stable manner to reproduce process of vasculogenic mimicry in vitro and in vivo.
4 dwg, 3 tbl, 6 ex
FIELD: veterinary science.
SUBSTANCE: multipotent mesenchymal stromal cells (MMSC), previously cultivated under the following gas conditions, are added to the area of fracture: 5% O2, 5% CO2, 90% N2.
EFFECT: invention makes it possible to accelerate process of callus formation.
1 dwg, 1 tbl, 1 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.
EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.
41 cl, 3 dwg, 8 ex
SUBSTANCE: cell line of humans melanoma mel H has stable cultural and morphological characteristics, is stored in Specialised collection of cell cultures of vertebrates of Russian collection of cell cultures of Institute of Cytology RAS by number "РККК (П) 715 Д".
EFFECT: invention allows to extend arsenal of human melanoma lines, which are used for creation of antitumour vaccine, applied for treatment of melanoma and other malignant neoplasms.
1 tbl, 2 ex
SUBSTANCE: cell line of human melanoma mel Me has stable cultural and morphological characteristics, is stored in Specialised collection of cell cultures of vertebrates of Russian collection of cell cultures of Institute of Cytology RAS by number "РККК (П) 712 Д".
EFFECT: invention allows to extend arsenal of cell lines of human melanoma, applied for creation of antitumour vaccine, which are applied for treatment of melanoma and other malignant neoplasms.
1 tbl, 2 ex
SUBSTANCE: cell line of human melanoma mel Rac has stable cultural and morphological characteristics, is stored in Specialised collection of cell cultures of Institute of Cytology RAS by number "РККК (П) 710 Д".
EFFECT: invention allows to extend arsenal of cell lines of human melanoma, used for creation of antitumour vaccines, which are applied for treatment of melanoma and other malignant neoplasms.
1 tbl, 2 ex
SUBSTANCE: invention refers to medicine and biology, more specifically to technology and methods for recovery of leukaemia cell suspension from spleens of high-leukaemia mice, and can be used in experimental oncological haematology for storage of low-frequent subtypes of murine leukaemia. There is offered a method for preparing a leukaemia cell suspension from spleens of inbred mice AKR/JY involving skin preparation with an antiseptic, splenectomy, decapsulation and homogenisation of spleen, filtration of the prepared suspension to be diluted in a sterile cooled artificial extracellular medium to the required concentration of leukaemia cells, prepackaging of the prepared suspension in ampoules for storage and transportation with decapsulation, grinding and homogenisation of fragmented spleen pulp performed in polyglucin solution containing ozone 25.72 mg/l and cooled to 24.0±1.0°C, leukaemia cells are filtered and diluted in polyglucin solution containing ozone 25.72 mg/l and cooled to 15.0±2.0°C.
EFFECT: invention provides maximum safety of specific functional adequacy of the transplanted leukaemia cells.
1 ex, 1 tbl
SUBSTANCE: invention relates to biotechnology and can be used in treatment of diseases, namely oncologic ones. Method is based on sampling from patient immature dendrite cells, their cultivation ex vivo for maturing and formation of allostimulating activity. Cells are additionally collected with antigen and are introduced to the same patient for formation of adaptive immunity. During cultivation ex vivo of immature dendrite cells fragmented allogenic double-stranded genomic DNA with fragments with size 200-6000 bp is introduced into culture medium in amount 5 mcg/ml of medium.
EFFECT: invention allows to extend field of obtained product application with increase of general immune state of patients.
SUBSTANCE: there is produced a new CT-B1/F8 hybrid cell clone producing monoclonal cholera toxin antibody (MCAB) in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises MCAB specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:20000 in the cultural fluid, 1:2000000 in ascitic.
EFFECT: antibodies can be used for designing immunobiological systems of cholera toxin detection exceeding available analogues in sensitivity.
2 dwg, 4 ex
SUBSTANCE: there is offered a method of precursor recovery from a human body, including all cells having stem cell characteristics or similar, in particular closely active or multiple active precursors, and such cells are recovered either directly, or indirectly from human breast secretion sampled from the specified human body. This secret can be colostrum, ripened milk or secretions in males and females in a lactation interval, during at least one of the following periods: absence of pregnancy, pregnancy period, lactation period, involution period. Further, the present invention refers to preferable applications of such recovered cells.
EFFECT: improved clinical effectiveness.
24 cl, 8 dwg
FIELD: organic chemistry, natural compounds, medicine, oncology.
SUBSTANCE: invention represents new saponin mixtures used for inhibition of initiation and activation of mammalian epithelial cell in pre-malignant or malignant state, for stimulation of apoptosis of mammalian malignant cell, prophylaxis of anomalous proliferation of mammalian epithelial cell, for treatment of inflammatory and regulation of angiogenesis in mammal. These mixtures are isolated form plants of species Acacia victoriae. Also, invention relates to methods for their applying. These compounds can comprise triterpene component, such as acacic or oleanolic acid to which oligosaccharides and monoterpenoid components are joined. Mixtures and compounds elicit properties associated with regulation of apoptosis and cytotoxicity of cells and strong anti-tumor effect with respect to different tumor cells.
EFFECT: valuable medicinal properties of compositions.
43 cl, 53 tbl, 50 dwg, 44 ex