82 results about "Breast epithelial cell" patented technology

Studies in mice and humans indicate that membrane CD14 (mCD14) on the cell surface of monocytes, macrophages, and PMN mediates the activation of these cells by LPS. The soluble CD14 (sCD14) present in the circulation also binds to LPS and blocks LPS binding to mCD14. To determine the role of a recombinant bovine soluble CD14 polypeptide in cellular activation by LPS, a recombinant bovine soluble CD14 polypeptide, rbosCD14, was cloned and expressed in a baculovirus expression system. Results indicated that rbosCD14 inhibited the LPS-induced increase in CD18 expression and TNFα mRNA in vitro and reduced mortality in mice injected with LPS. Further, rbosCD14 sensitized mammary epithelial cells to low concentrations of LPS resulting in recruitment of white blood cells and prevention of LPS-induced infection.

The invention relates, in part, to antibodies with increased ADCC activity. Methods of producing such antibodies are also provided. The antibodies of the invention are produced in mammary epithelial cells, such as those in a non-human transgenic animal engineered to express and secrete the antibody in its milk. The antibodies or compositions comprising the antibodies can be used to treat disease in which ADCC activity provides a benefit. In one embodiment, therefore, the antibodies or compositions comprising the antibodies can be used to treat cancer, lymphoproliferative disease or autoimmune disease.

The invention discloses a primary isolated culture method for dairy cow mammary epithelial cells. The primary isolated culture method comprises steps as follows: mammary tissue of a dairy cow in the lactation period is shorn into tissue blocks, the tissue blocks are digested with collagenase / hyaluronidase digestive juice, a culture dish is inoculated with the digested chylous tissue blocks for culture, cells get free from tissue on peripheries of the tissue blocks after 1-2 days and grow along the wall of the culture dish, fusiform fibroblast is removed with a mechanical scraping method every day during culture, and initial passage is performed when a large quantity of cobblestone-like mammary epithelial cells grow in clusters and in patches and are mutually fused after about 4-5 days. During initial passage, a little fibroblast which is probably mixed is eliminated with trysin with a differential digestive method, and the dairy cow mammary epithelial cells with high purity are obtained. The mammary epithelial cells can emigrate from chylous tissue in a short time; a little mixed fibroblast is removed mainly with a mechanical scraping method, and accordingly, the obtained mammary epithelial cells are seldom damaged and are high in purity.

The invention provides a new molecular marker hsa-miR-374a of breast carcinoma, that is, non-coding RNA gene hsa-miR-374a of micromolecule as a molecular marker of breast carcinoma. Expression of the molecular marker in tissues suffering from breast carcinoma is obviously higher than that of normal mammary tissues, and is associated with clinical classification of breast carcinoma, and expression of hsa-miR-374a in breast carcinoma cell line cultured in vitro is higher than that of normal mammary epithelial cells and immortalized normal mammary epithelial cells. The invention further provides application of the molecular marker of breast carcinoma to preparing an anti-breast cancer drug. The drug comprises effective amount of blocker capable of blocking expression of non-coding RNA gene hsa-miR-374a of micromolecule. The molecular marker provides new effective way for diagnosing and treating breast carcinoma. The invention also provides certain experience and foundation for further research of function of hsa-miR-374a and relation with other tumors.

Disclosed are compositions and corresponding methods for treating fibrocystic breast disease or other breast-related disease or condition. The compositions comprise, per serving or dose, from zero to about 400 μg selenium, from about 100 mg to about 6000 mg gamma linolenic acid, and about 0.15 mg to about 5 mg iodine, with nutritional embodiments further comprising one or more of protein, fat, carbohydrate, vitamins, and minerals and providing from about 50 to about 1000 kcal of energy per severing or dose. Also disclosed are in-vitro studies showing that certain combinations of gamma linolenic acid, iodine, and / or selenium may 1) inhibit breast cancer or fibrocystic cell proliferation, 2) reinforce the function of tight junctions of endothelial cells and of mammary epithelial cells in estrogen-sensitive conditions, and 3) reduce the risk of vascular invasion by breast cancer cells.

The invention provides a primary cell culture medium for culturing primary mammary epithelial cells and containing amphiregulin, and a culture method using the primary cell culture medium. Through the culture method, the primary cell culture medium is used for culturing primary cells on a culture vessel coated with extracellular matrigel, the primary cells grow on the culture vessel coated with the extracellular matrigel, and the primary cells are rapidly proliferated under the combined action of nutritional factors contained in the primary cell culture medium and extracellular matrigel. A cell model obtained by the primary cell culture medium and the primary cell culture method can be used for curative effect evaluating and screening of medicines.

To provide a cell culture method capable of sustaining in-vivo functions for a long time by suppressing dedifferentiation. A cell culture method in accordance with the present invention is to culture cells in a multi-layered state in a minute partitioned space and to obtain a tissue structure having a function resembling an in-vivo function. When the cells are mammary gland epithelial cells, a hollow acinus-like structure can be formed. The minute partitioned space is particularly preferably a micro container in a cell culture container having a plurality of such micro containers on the surface.

The invention discloses a method for separating, purifying and culturing a mammary epithelial cell of a ruminant animal, which includes the following steps of: 1) the sampling of a mammary tissue; 2) the preparing of a culture fluid; 3) the culturing of a tissue block for obtaining a primary cultured mammary epithelial cell; 4) the purifying of the mammary epithelial cell for obtaining a purified mammary epithelial cell; and 5) the subculturing of the mammary epithelial cell. The invention simultaneously provides a preservation method for the mammary epithelial cell of the ruminant animal, which includes the following steps of: 1) preparing a freezing protection fluid which comprises the mammary epithelial cell of the ruminant animal; and 2) gradually and slowly reducing the temperature of the freezing protection fluid. The method can be adopted for prolonging the preservation time of the mammary epithelial cell of the ruminant animal.

The invention provides a method for establishing a lactation model of cow mammary gland epithelial cells for researching the milk protein gene expression regulation mechanism and providing favorable conditions for testing a mammary gland expression vector. The testing method comprises the following steps: carrying out primary culture and identification of the cow mammary gland epithelial cells; determining the secretion situation of lactose by using the HPLC method; determining the secretion situation of beta-casein protein by using the HPLC method; and carrying out cryopreservation and recovery on the cells. The method adopts the tissue block method to culturing the cow mammary gland epithelial cells in different periods, compared with other primary culture methods of the mammary gland epithelial cells, such as enzyme digestion and the like, the method does not affect the activity of the mammary gland epithelial cells, the culture cost is very low, and the operation is very simple. The method realizes the continuation of normal culture of a cow mammary gland epithelial cell system, thereby property and effectively preserving a large number of precious cow mammary gland tissue materials. The method provides the important testing materials and a technical platform for researching the lactation mechanism of the cow mammary gland during the different development periods.

The invention discloses a method for building a dairy cattle breast acinus lactation model in vitro, and relates to a method for building a lactation model in vitro. The method solves the problem of low authenticity of in-vivo environment caused by taking a mammary epithelial cell as a research carrier in the existing in-vitro study of the lactation function of the dairy cattle breast. The method comprises the following steps: 1, preparing a tissue culture plate of matrix gel; 2, digesting primary cultured dairy cattle mammary epithelial cell gestated for 4 months by adopting pancreatin and EDTA, and collecting cells after centrifugalization; 3, preparing cell suspension and diluting; and 4, adding the cell suspension into the tissue culture plate of the matrix gel, culturing for 15 days in an incubator to finish the building. The artificial building process of the dairy cattle breast acinus lactation model built by the invention is a simulation process of in-vitro simulation of a galactemia protective screen; and a three-dimensional acinus structure thereof can truly simulate in-vivo situation, has the same lactation function and biological characteristics as the breast acinus in a ruminant, and has high authenticity of in-vivo environment simulation.

The present invention relates to a three-dimensional continuous culture method of porcine mammary epithelial cells and belongs to the field of cell culture methods. The three-dimensional continuous culture method comprises the following specific steps: (1) separating and extracting primary porcine mammary epithelial cells and selecting cells before 10th generation; (2) after using trypsin to conduct digestion into singe cells, conducting centrifugation, discarding supernatant, conducting re-suspending in a common culture medium, adjusting cell concentration to 1x10<6>-5x10<6> cells / mL, taking1 mL of the cells, then conducting centrifugation again, discarding supernatant, conducting re-suspending with Matrigel for experimental use, and conducting even mixing for standby application on ice;(3) drawing an appropriate amount of the above suspension, dropping 7-8 drops into each well of a six-well plate, conducting inversion after the dropping, and conducting placing in a 37 DEG C incubator for incubation for 30-40 min for coagulation; (4) adding 2 mL of a three-dimensional culture medium per well, conducting continuous culture for 12-16 days, and changing the three-dimensional culture medium once every 2-3 days according to color of the culture medium; and (5) removing the culture medium, conducting washing with D-PBS for three times, adding 2 mL of trypsin to each well, using apipette tip for blowing off, conducting culture at 37 DEG C, conducting blowing off to conducting even mixing every other 2-4 min until single cells are obtained, conducting neutralization digestion and centrifugation at 4 DEG C, removing supernatant, and conducting passage according to the step (2) to step (4).

The invention provides a method for transdifferentiation of somatic cells into mammary epithelial cells by using a small-molecule compound through in-vitro induction. Expression of TGFbeta R1 and related loci thereof is inhibited, and transdifferentiation of somatic cells into mammary epithelial cells through in-vitro induction is achieved. By adopting the method, the blank of a technique for transdifferentiation of fibroblast into mammary epithelial cells through induction of the small-molecule compound is made up, and a research platform for research of in-vitro research on mammary gland bioreactors, mammary development and differentiation, breast cancer and ransdifferentiation of fibroblast into other types of functional cells is provided.

The invention discloses a frozen stock solution for storing mammary epithelial cells of a milk goat. The frozen stock solution is prepared by mixing a basic frozen stock solution and insulin-transferrin-sodium selenate in a volume ratio of 100 to (1-2). The basic frozen stock solution is prepared by mixing 10% by volume percent of dimethyl sulfoxide, 30-50% by volume percent of a DMEM / F12 culture medium and 40-60% by volume percent of fetal calf serum. The freeze-saving frozen stock solution disclosed by the invention is few in step, simplified in process and low in cost, the resuscitated mammary epithelial cells are strong in anti-apoptosis performance, oxidization resistance and multiplication capacity, the resuscitation rate of the mammary epithelial cells can be increased, and the vitality of the resuscitated cells reaches up to above 95%, so that the cell growth quality is ensured, the proliferation cycle of cell resuscitation is shortened and the mammary epithelial cells of the milk goat can be saved for a long time.

A cell culture article includes a porous substrate having a plurality of pores and a plurality of interstices in communication with the pores. At least some of the plurality of pores and interstices are sufficiently large for two or more mammary epithelial cells to cluster within the pores or interstices. Non-malignant mammary epithelial cells or breast cancer cells may not attach strongly to the substrate surface, which may encourage cell-cell interaction. In many cases, the article is desirably free of components of unknown origin. The articles may be capable of maintaining culture of malignant and non-malignant mammary epithelial cells and allowing for development of in vivo-like morphologies or characteristics of such cells.

The invention provides a method for constructing a milk cow blood milk barrier three-dimensional model in vitro. The method comprises the steps of cultivation of mammary epithelial cells and fibroblast of a milk cow, cultivation of venous endothelial cells of the milk cow, immunofluorescent identification of the mammary epithelial cells and the venous endothelial cells, cocultivation of Transwell,preparation of a cocultivation medium by taking prolactin, insulin, an epidermal growth factor, cow growth hormone, a vascular endothelial growth factor and a fibroblast growth factor as hormone andcell factors related to growth of the mammary epithelial cells, the fibroblast and the endothelial cells; and finally the epithelial cells, the fibroblast and the endothelial cells are inoculated on atranswell chamber, and accordingly the milk cow blood milk barrier model is successfully constructed. The method can be used for researching a molecular mechanism by which nutrient substances, drugsand toxic substances enter the mammary tissue and the milk in real-time and effectively, not only fills in the gap both domestically and internationally, but also provides a research platform for research on the molecular mechanism by which lipid, vitamins, the drugs and the toxic substances pass through the blood milk barrier.

The invention discloses a method for isolating and cultivating milk cow breast cells in vitro, relating to the cell in vitro isolation and culture technology field and solving the problem that breast fibrocytes and epithelial cells of a milk cow cannot be isolated completely during the isolation and culture of the breast epithelial cells of the milk cow. The method comprises the following steps of: selecting mammary tissue of a milk cow which is killed on the very day from a local abattoir, adopting tissue explants technique and graded trypsin digestion technique to isolate and cultivate the breast epithelial cells of the milk cow according to the different sensibility of the fibroblasts and epithelial cells to trypsin and the different adhering time, purifying the breast epithelial cells of the milk cow, identifying with keratin 18 to obtain the corresponding cell system finally. The method provides the best trypsin concentration when isolating and cultivating the breast epithelial cells of a milk cow, and can completely isolate the breast fibrocytes and the epithelial cells of the milk cow from each other.

The invention relates to a method for primary isolated culture of porcine mammary epithelial cells (PMEC). The method includes the following steps: (1) performing sampling; (2) performing pretreatment; (3) performing isolation; (4) performing culture; (5) performing subculturing; and (6) performing cell purification. The beneficial effects of the method are as follows: complex environment in vivocan be avoided by establishing a porcine mammary epithelial cell model in vitro; researches on the influences of specific conditions and treatment on the growing status of cells can be carried out without being affected by individual difference; and mammary epithelial cell cultures can be taken as models to research bio-mechanism and disease mechanism in mammary gland organs.

The invention discloses an in-vitro culture method of mammary gland epithelial cells. The method comprises the following steps: treating a mammary tissue into chylific tissue blocks with a tissue homogenizer, sequentially inoculating the tissue blocks on labeled culture bottle bottom walls, and culturing, wherein after 2-3 days, fibroblasts around each tissue block are free and perform adherent culture growth; and after 4-5 days, epithelial cells start to be free and perform adherent culture growth, and the fibroblasts are driven to the periphery of the epithelial cell and grow around the epithelial cells; and at this time, adding trypsinase to digest the adherent-culture fibroblasts, and continuing culturing for 5-7 days after finishing digestion until about 80-90% of cells are adherent to the wall, thereby obtaining the higher-purity mammary gland epithelial cells. Compared with the existing culture method, the method disclosed by the invention performs digestion treatment on the fibroblasts in the early culture stage, and at this time, only small amounts of epithelial cells are free and grow (can be completely removed in the digestion treatment); and afterwards, the free epithelial cells can not be damaged by the digestion treatment, and the cell activity and purity are very high.

The invention relates to the fields of tissue and cell engineering and particularly provides a single cell cloning method for obtaining goat mammary epithetical cells. According to the method, single cell cloning preparation is performed through a micromanipulation method, tedious cell counting and repeated dilution in a traditional method are eliminated, single cell cloning operation can be accomplished within a short time, and the single cell cloning formation rate is increased. The goat mammary epithetical cells obtained through the method provided by the invention can be used for researching functions of different lineages of mammary epithelial cells and the interaction of the mammary epithelial cells during a lactation process.

The disclosure provides methods, cells and transgenic non human mammals for the production of heparin. Specifically, the method comprising providing a transgenic non human mammal or mammary epithelial cells modified to express one or more heparin biosynthesis enzymes, and harvesting heparin produced. Further provided are the cells and transgenic non human mammals thereof as well as the heparin obtained from these cells or transgenic non human mammals.

The invention belongs to the field of biological engineering, and particularly relates to a method for producing human tissue plasminogen activator (tPA) in large scale. The method is to use an expression vector composed of regulatory sequence containing a core promoter and cDNA and a BLG signal peptide to prepare a transgenic mouse; the lymphocyte or the mammary epithelial cell of the transgenic mouse is taken and fused with a tumour cell to obtain a transgenic hybridoma; a tPA transgenic hybridoma is induced by prolactin and is screened to obtain a high-expression cell strain; then the expression cell is transfected and screened again to obtain a high-expression hybridoma strain; the high-expression cell is inoculated into the abdominal cavity of the BALB / C mouse in a lactation period; then ascites is collected to obtain the recombinant human tissue plasminogen activator. The production cost thereof is remarkably reduced compared with a cell culture method; the product is restively safe in use; the production flow is simple; and complex instruments and devices are not needed.

The invention discloses a method for establishing a lipopolysaccharide (LPS)-induced milk cow mammary gland epithelial cell apoptosis model, which comprises the following steps: (1) acquiring 2nd-3rd-generation milk cow mammary gland epithelial cells; (2) detecting inhibiting action of LPS on milk cow mammary gland epithelial cell proliferation by a CCK-8 process, and primarily detecting the LPS action concentration and action time which conform to the requirement that the apoptosis rate of the established in-vitro cell trauma model is 40-50%; (3) detecting the mammary gland epithelial cell apoptosis rate by flow cytometry; (4) detecting variation conditions of apoptosis cell nucleic acids by DNA ladder; and (5) observing the interior morphological structure variation of the mammary gland epithelial cells by a transmission electron microscope. The determination method of the LPS-induced milk cow mammary gland epithelial cell apoptosis model induction conditions has the advantages of ideal effect and low cost, and is convenient to operate.

The invention relates to a method for preparing a transgenic animal capable of carrying out specific expression of a mammary gland from mammary gland epithelial cells. The method comprises the following steps of 1, transfecting mammary gland epithelial cells cultured in vitro with exogenous genes by expression vectors, and carrying out inducible expression by 3 to 8 micromole per liter of a prolactin, 2, carrying out screening to obtain a highly expressed mammary gland epithelial cell line, and 3, carrying out one-step nucleus transplant or two-step nucleus transplant of the highly expressed mammary gland epithelial cell line to obtain an individual capable of carrying out specific expression of a mammary gland. The method can improve transgenic animal preparation efficiency. Through the method, transgenic animal expression characteristics can be forecasted in a cell screening phase, and preparation efficiency of a transgenic animal capable of carrying out specific expression of a mammary gland is improved 3 times. Through an integrated position effect, an obtained transgenic individual has an expression probability of 10 to 30% and low-level expression may occur. Through the method, preparation of transgenic cells and identification of an expression potentiality of a prepared individual can be realized simultaneously, and thus more expression animals can be obtained.

The invention discloses an octopus oligopeptide capable of promoting synthesis of breast casein and a preparation method and application of the octopus oligopeptide. The octopus oligopeptide is prepared from one or more than two of the following amino acid sequences of MGDVLNF, MGLAGPR, EAPLMHV and TEAPLMHV. According to the octopus oligopeptide, the growth of HC11 in mammary epithelial cells canbe significantly promoted, the expression level of beta-casein of the HC11 in the mammary epithelial cells is increased, and compared with a blank control group, the expression level is increased by 150% or above. The results indicate that the octopus oligopeptide has good application prospects in functional nutritious foods capable of promoting lactation and improving the milk quality on postpartum women.