70 results about "Ovarian Granulosa Cell" patented technology

The invention provides an ovarian cell microcapsule, which adopts alginate-poly-lysine- alginate (APA) to encapsulate ovarian cells and prepare a microcapsule containing ovarian cells inside. The ovarian cells in the microcapsule consists of ovarian granulosa cells and theca cells, which has endocrine function; undifferentiated ovary inner mesenchymal cells and connective tissue cells. If allogenously plant the microcapsule of the invention, the endocrine cells in the microcapsule can constantly secret sex hormone in body, and the endocrine function can be under the control of neuroendorine of the body to reach the aim of personalized supplementing the inefficiency of sex hormone endocrine. The allogenous plant of ovarian cell microcapsule has positive prevention and treatment effects to body degenerative lesion. Experiment shows that the invention can alleviate the loss of bone quantity and presents positive effect to the prevention and treatment of osteoporosis; meanwhile, the invention can affect the biological activity of islet Alpha cell and Beta cell, and regulate the endocrine of insulin and glucagons.

The invention discloses an application of tri-methylation of histone H3lysine 4( H3K4me3) to granulosa cells of pigs, and belongs to the technical field of cell engineering and genetic engineering. H3K4me3 is used as a penetration point, and a cell biology method is used for researching the influence of the H3K4me3 to the functions of the granulosa cells of pigs. The technical scheme is detailed in design, and reliable in results. In order to confirm the influence of H3K4me3 to the functions of the granulosa cells of the pigs, verification is performed from multiple levels and multiple angles,and verification is performed from the cell level and the protein level. The invention explains that the H3K4me3 has influence on the functions of the granulosa cells of the pigs, the H3K4me3 can promote proliferation of the granulosa cells of the pigs, can restrain the apoptosis of the granulosa cells of the pigs, and can reduce the proportion of the granulosa cells of the pigs, blocked in the GO / G1 period. The application has a good application value of research of histone methylation on an influence mechanism on the development of granulosa cells and the reproductive performance of sows.

The invention discloses a construction method of a Rosa26 gene Mir3061 fixed-point knock-in heterozygote mouse model and an application thereof. The constructed mouse gene is Rosa26LSL / +, and the construction method is provided for the first time and is not reported in any domestic or foreign literature reports. And the model provides an effective experimental animal model for the research on thepathogenesis of POF, the research and development of medicines and the evaluation of drug effect. The invention also provides the application of the mouse model in screening and preparing medicines for detecting / treating reproductive development diseases and medicines for detecting / treating aging and apoptosis diseases of ovarian granulosa cells. The model provides an effective experimental animalmodel for reproductive development genetics, preparation of medicines for detecting / treating ovarian diseases and screening of related medicines, and has good practical value, good medical clinical application prospect and great social benefit.

The invention discloses an application of an RSPO3 gene in sow ovarian granulosa cells. Two kinds of follicles with diameters of 3-5 mm and 7-10 mm respectively are used as experimental materials, andthe relationship between H3K4me3 and the expression level of genes related to ovarian follicle development is studied by a sequencing technology. The enrichment degree of H3K4me3 in a promoter regionof the RSPO3 gene is found to be significantly different in the follicles with different size by ChIP-Seq; the expression quantity of the RSPO3 gene in the follicles with different size is found to be significantly different by qRT-PCR; promotion of the degree of the H3K4me3 is found to promote transcription of the RSPO3 gene by promoting or inhibiting the degree of H3K4me3 in the sow ovarian granulosa cells, and inhibition of the degree of H3K4me3 is found to inhibit transcription of the RSPO3 gene; the RSPO3 is found to promote the proliferation of the sow ovarian granulosa cells and inhibit the apoptosis of the cells through overexpression or interference of RSPO3.

The invention provides a preparation method of oyster bioactive peptides. The method comprises the following steps: carrying out high speed homogenate on fresh oyster meat, centrifuging, and adjusting the pH to 7 in order to obtain crude oyster proteins; centrifuging the obtained oyster protein suspension, homogenizing through a high speed disperser, adding Papain, maintaining the pH value of the obtained solution unchanged, heating reactants for enzyme killing, and cooling to obtain freeze-dried oyster protein powder; and preparing a solution, stirring, centrifuging, determining the content of amino acids in the above obtained supernatant which is a high temperature enzyme-killed oyster enzymatic hydrolysate through a biosensor, separating the oyster enzymatic hydrolysate through a SephadexG-25 chromatography column, eluting, freeze-drying, and carrying out gel column desalination to obtain the oyster bioactive peptides. The oyster enzymatic hydrolysate obtained in the invention contains high content of total amino acids, so the activity of biological components is effectively protected, and the ovarian granulosa cell function interference effect caused by bisphenol A can be substantially antagonized; and the oyster bioactive peptides can be applied in the preparation of medicines for treating the ovarian function disorder caused by the exposure of chemical environment interferent bisphenol A.

The invention discloses application of PIK3R2 in pig ovarian granular cells. A PIK3R2 interference small fragment is synthesized with PIK3R2 as a research object, ovarian granular cells are transfected, it is found that PIK3R2 promotes granular cell apoptosis and inhibits cell proliferation, and research is performed by regulating the functions of miR-126-3p cells expressed by PIK3R2 in granular cells. After exogenous disturbance PIK3R2 is supplemented, cell function phenotype caused by miR-126-3p can be restored through the verification, it is shown that PIK3R2 is an important functional target of miR-126-3p in granular cells, and miR-126-3p can regulate cell development of the granular cells by means of PIK3R2. By means of PIK3R2 and application of targeted miRNA of miR-126-3p in ovarian granular cells, PIK3R2 has good application value for researching an ovarian follicles locking mechanism.

The invention discloses an application of STAT3 in porcine ovarian granulosa cells. STAT3 is taken as a research object, and a porcine STAT3 gene promoter double-luciferase reporter gene recombinationplasmid is constructed, and the core promoter region of the STAT3 is found through the expression activity of the STAT3 gene promoter in the porcine ovarian granulosa cell; and then the interaction between the transcription factor C / EBP beta and the core promoter region of the STAT3 is verified; then C / EBP beta superexpression vector is constructed and small interfering RNA (C / EBP beta-siRNA) issynthesized, the effect of C / EBP beta on STAT3 is detected, finally, the expression vector of C / EBP beta and C / EBP beta-siRNA were transfected respectively to the granulosa cells in order to detect the apoptosis and proliferation of the cells. The application of STAT3 in porcine ovarian granulosa cells finds the application of the transcription factor C / EBP beta in the ovarian granulosa cell by finding the transcription factor C / EBP beta in the STAT3 promoter region, and has good application value for researching the ovarian follicular atresia mechanism.

The invention discloses an application of a DNMT1 gene in porcine ovarian granulosa cells, belonging to the technical fields of cell engineering and gene engineering. According to the invention, the influence of DNMT1 on pGCs proliferation, apoptosis and E2 secretion is explored from a cellular level and a molecular level by taking a catalytic enzyme DNMT1 formed by DNA methylation as an entry point. Results show that the DNMT1 gene participates in promotion of proliferation of granulosa cells and E2 secretion and inhibition of apoptosis and maturation of the granulosa cells. The invention provides an important value for researching the influence mechanism of DNA methylation on ovarian follicle development and sow reproductive performance.

The invention discloses new application of an extract of Chinese angelica and szechuan lovage rhizome composition to the preparation of a medicament for promoting ovary granular cell proliferation. Intensive study is performed on a large quantity of ancient and modern prescriptions and traditional Chinese medicine application data, traditional Chinese medicine resources are fully utilized according to a traditional Chinese medicine theory and the pathogenesis of ovarian functional disturbance and insufficient growth of ovary granular cells and a large number of pharmacological tests are performed based on the current researches of Chinese angelica and szechuan lovage rhizome, so that new application of the Chinese angelica and szechuan lovage rhizome to the treatment of ovarian functional disturbance and the promotion of ovary granular cell proliferation; moreover, the weight ratio of the Chinese angelica and szechuan lovage rhizome composition with the highest activity is screened through a large quantity of experiments. When clinically taken as a medicament for treating ovarian functional disturbance and promoting ovary granular cell proliferation, the extract has the advantages of good curative effect, definite active ingredient, small side effect, convenience of taking and the like.

The invention discloses a method for constructing a heterozygote mouse model of an Amh gene fixed-point knock-in 2A-Cre and an application thereof. According to the method, the heterozygote mouse model can be obtained, and an effective experimental animal model is provided for the research of POF pathogenesis, the research and development of drugs and the evaluation of drug effect. The invention provides the application of the heterozygote mouse model in screening and preparing drugs for detecting / treating reproductive development diseases, drugs for detecting / treating ovarian diseases and drugs for aging and apoptosis diseases of ovarian granulosa cells. The invention provides the method for constructing the heterozygote mouse model for the first time, and no domestic and foreign literature reports are found before. The model has good medical clinical application prospect, has great application value in preparing medicaments for detecting / treating ovarian diseases, and has great social benefit.

The invention discloses a porcine ovarian granulosa cell primary culture method. The method comprises steps as follows: ovaries are collected, follicular fluids are sucked, and cells are centrifugally collected; the cells are re-suspended and centrifugally collected to be dispersed, and the process is repeated 2-3 times; the cells are placed in an incubator for culture; solution replacement is performed for the cells after 24 h of culture, a solution, anchorage-independent cells and oocytes are sucked, rinsing is performed once, a fresh solution is added for continuous culture, and the solution is replaced once after 24 h of culture of cells. The method has the benefits as follows: (1) in the granulosa cell collecting process, the step of picking of oocytes with a mouth pipette under a stereomicroscope is omitted, the oocytes suspend to grow in the culture solution, a few of oocytes in a culture dish are sucked away with solution replacement after 24 h of culture, and adherent granulosa cells are pure; (2) the in-vitro operation time is shortened, and the survival rate of the granulosa cells is increased; (3) the obtained granulosa cells is purer.

The invention provides a method for separating and culturing mouse ovarian granule cells. The method comprises (a) intraperitoneally injecting pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) into a female mouse, (b) killing the mouse through a cervical dislocation method, separating mouse ovarian, carrying out PBS cleaning and removing fat and tunica around the mouse ovarian through a stereoscopic microscope, (c) puncturing follicles in a mixed base culture medium through a syringe needle to release granule cells and oocytes, adding a mixed enzyme into the culture medium, carrying out digestion, carrying out filtration through a screen, centrifuging the filtrate and removing the supernatant and (d) carrying out inoculation with completely mixed medium resuspended cell precipitates. The method realizes simple, efficient and stable separation and culture of ovarian granule cells.

The invention discloses application of a CTNNB1 gene in porcine ovarian granulosa cells, and belongs to the technical field of cell engineering and gene engineering. According to the invention, the relative expression quantity of CTNNB1 mRNA in ovarian tissues, follicles with different sizes and granulosa cells of pigs in different periods, and the proliferation and apoptosis of the granulosa cells after overexpression and inhibition of CTNNB1 and the secretion of steroid hormones are detected. Results show that the CTNNB1 gene participates in promoting the proliferation of granulosa cells andthe secretion of estradiol and inhibiting the apoptosis of granulosa cells and the secretion of testosterone and progesterone, which indicates that the CTNNB1 gene participates in the development andmaturation of ovarian follicles of sows. Materials are accumulated for molecular mechanism research in the sow ovarian follicle development process.

The invention belongs to the field of medicaments and veterinary medicines and discloses application of trichostatin A (TSA) to preparation of a medicament for inhibiting activity of swine ovary granular cells. In the invention, the influence of the TSA to the activities of the swine ovary granular cells is systemically studied, and a result shows that the TSA has no influence to the growth of the swine ovary granular cells at low concentration (below 10ng / mL), with the increase of the concentration, the growth of the swine ovary granular cells is inhibited (P<0.05), and the inhibition effect is dose-dependent. After the granular cells are treated by using 200ng / mL TSA, the cell cycle is retardant within a period G0 / G1, and the marked decrease of gene expression of Cyclin D2 and CDK4 (Cyclin-Dependent Kinase 4) is possible to be an important reason for the cycle retardance. Accordingly, the TSA can be applied to preparation of the medicament for inhibiting the activities of the swine ovary granular cells.

Studies show that miR-26b participates in the reproductive regulation of animals, miR-26b can regulate apoptosis of porcine ovarian granulosa cells by targeting Smad4 gene, miR-26a may further has thesimilar function, but studies on the miR-26a in germ cells are not reported. The invention discloses an application of the miR-26a in regulating germ cell apoptosis. The invention discloses that DHCR24 and Smad4 are target genes of the miR-26a. In porcine ovarian granulosa cells, the miR-26a negatively regulates the expression of the DHCR24 and the Smad4 at transcription level and translation level; miR-26a promotes apoptosis of ovarian granulosa cells by interfering with expression of the DHCR24 and the Smad4, and at the same time controls secretion of estradiol and progesterone by regulating steroid synthesis pathway and affecting hormone secretion.

The invention discloses a sheep ovarian granular cell separation, culture and identification method, and belongs to the technical field of animal cell culture. Ovarian granular cells are obtained through special treatment modes of collection, disinfection, cutting and the like. The method has the advantages of being simple, convenient, labor-saving, low in pollution rate, high in follicular fluid obtaining rate and the like, and a foundation is laid for research on development of ovarian granular cells, oocytes and follicles.

The invention discloses a mouse ovarian granulosa cell extraction and in-vitro chemotherapy injury model construction method. The method mainly comprises the following steps of 1) injecting PMSG to promote ovulation of a young mouse with age of 3-4 weeks; 2) performing mouse ovary separation and follicle breakage: dissecting the abdomen of the mouse subjected to ovulation induction for 48 hours, taking out ovaries on two sides, stripping fat tissues and connective tissues around the ovaries, puncturing follicles by using a 1 mL syringe needle under a microscope to release ovarian granulosa cells, sieving the cells with a 200-mesh sieve, and then putting the cells into a 15 mL centrifuge tube for later use; 3) digesting the ovaries with 0.25% pancreatin: putting ovary tissues after folliclebreakage into the 0.25% pancreatin, digesting theovary tissues in a water bath kettle of 37 DEG C for 30 minutes, sieving the ovary tissues with the 200-mesh sieve, putting the ovary tissues into the15 mL centrifuge tube, and centrifuging the ovary tissues to obtain a large amount of ovarian granulosa cells with higher purity; and 4) constructing an ovarian granulosa cell in-vitro chemotherapy injury model: treating the ovarian granulosa cells with cyclophosphamide (CTX) with the concentration of 5 mg / mL for 24 hours to obtain the ovarian granulosa cell chemotherapy injury model.

The invention discloses application of an RSPO2 gene to porcine ovarian granulosa cells, and belongs to the technical field of cell engineering and gene engineering. According to the invention, RSPO2 is taken as a research object, an RSPO2 overexpression vector or RSPO2-siRNA are respectively transfected into ovarian granulosa cells by constructing the RSPO2 overexpression vector and synthesizing small interfering RNA (RSPO2-siRNA); then, the changes of signal channel gene mRNA and protein level related to ovarian granulosa cell apoptosis, proliferation and E2 secretion are detected; and finally, the phenotypic change of the ovarian granulosa cells is changed. The results show that RSPO2 can inhibit apoptosis of ovarian granulosa cells and promote proliferation and E2 secretion. By exploring the application of the RSPO2 to the ovarian granulosa cells, the invention has very good application value on researching an ovarian follicle atresia mechanism, initial estrus initiation and the like.

The invention provides a circRNA biomarker for diagnosing a polycystic ovarian syndrome, wherein the circRNA biomarker is hsa-circ-0000284. The expression conditions of the hsa-circ-0000284 in ovarian granular cells of PCOS patients and normal people are detected, and it is found that the expression level of the hsa-circ-0000284 is significantly reduced; by silencing the expression of hsa-circ-0000284 in human ovarian granular cells, the proliferation of the granular cells can be inhibited, and the generation of estrogen is reduced. The research shows that the hsa-circ-0000284 can be used as a biomarker of the polycystic ovarian syndrome, a target is provided for clinical diagnosis and treatment, and a theoretical basis is provided for pharmacy.

The invention discloses an analytical method of utilizing adherent cell-human ovarian granular cell tumor cell strain (KGN) to evaluate in-vitro biological activity of human follicle stimulating hormone. The method comprises the following steps: using the human follicle stimulating hormone to stimulate the human ovarian granular cell tumor cell, measuring the content of generated progesterone, so as to reflect the activity of the human follicle stimulating hormone. The analytical method of using the human ovarian granular cell tumor adherent cell to evaluate the in-vitro biological activity of the human follicle stimulating hormone, disclosed by the invention, is effective, stable and quick, and avoids the limitations of long time consumption, complicated operation and poor result repeatability in the animal in-vivo ovarian weight increment method; meanwhile, the progesterone content is determined by a commercial enzyme linked immunosorbent assay (ELISA) kit, so that the biological activity of the human follicle stimulating hormone can be reflected truly and more accurately.

The invention relates to a traditional Chinese medicine composition used for anti-premature ovarian failure, a preparation method of the traditional Chinese medicine composition and a paste agent. Based on a traditional treatment principle of reinforcing kidney to replenish essence, and invigorating spleen and activating blood, a treatment idea for freeing channels and stimulating circulation of the blood is firstly provided by an inventor, through a lot of pharmacological experiments, two traditional Chinese medicines of Chinese starjasmine stem and Japanese honeysuckle stem are screened to combine with the traditional Chinese medicines (epimeddium, Radix Rehmanniae Preparata, astragalus root, bighead atractylodes rhizome, Chinese angelica and licorice) capable of reinforcing kidney to replenish essence, and invigorating spleen and activating blood to obtain the traditional Chinese medicine composition. The experiment research shows that the traditional Chinese medicine composition is capable of effectively adjusting hormones content in serum (increasing E2 level and reducing FSH level), increasing InhB level, increasing ovary and uterus wet weight, and promoting ovary particle cells proliferation, and can be used for treating premature ovarian failure. In addition, two traditional Chinese medicines of Chinese starjasmine stem and Japanese honeysuckle stem are added to obviously increase the promotion effect of other medicinal materials in the formula to the ovary particle cells proliferation, and especially have synergistic interaction under a specific ratio of 50: 15.

The invention belongs to the field of traditional Chinese medicine, and discloses a traditional Chinese medicine composition for resisting ovarian failure and application thereof. The traditional Chinese medicine composition for resisting ovarian failure mainly comprises 30 to 70% of cuscuta chinensis, 10 to 50% of fructus lycii, 4 to 20% of mulberry, 10 to 20% of radix rehmanniae recen, and 1 to 10% of flowers carthami. The Chinese patent medicine for resisting ovarian failure prepared from such composition can be applied to preparing the medicine for resisting ovarian failure; the ovarian failure symptoms are preferably shown as polycystic ovarian syndrome, functional abnormal menstruation, premature ovarian failure or climacteric syndrome. As the results shown, the traditional Chinese medicine composition for resisting ovarian failure is obvious in effect of generating on separated ovarian granular cells, and has the capacity of obviously improving progestogen secretion level of the separated ovarian granular cells, obviously improving the anti-apoptosis effect of the separated ovarian granular cells, reducing the levels of FSH (Follicle-Stimulating Hormone), LH (Luteinizing Hormone) and T (Triiodothyronine) of a model mice with the polycystic ovarian syndrome, and adjusting the sex hormone of a rate subjected to premature ovarian failure caused by cyclophosphamide.

The invention belongs to a cell biology technology, and particularly relates to an extraction and culture method of mouse ovarian granular cells. According to the technical scheme, the method comprises the following specific steps: (1) injecting pregnant mare serum gonadotropin PMSG into the abdominal cavity of a female mouse; (2) killing the mouse by using a cervical vertebra dislocation method, separating the ovary of the mouse, cleaning with PBS (Phosphate Buffer Solution), and removing a fat envelope around the mouse under a microscope; (3) puncturing follicles in the mixed operation solution by using a syringe needle to release granular cells and oocytes, filtering by using a screen, centrifuging the filtrate, and discarding the supernatant; and (4) mixing the complete culture medium, resuspending the cells, precipitating and inoculating, wherein the operation liquid includes DME / F-12:1(1X), 3% of fetal calf serum, 2% of mycillin mixed liquid and 2% of mycoplasma scavenger. Furthermore, the mouse is an immature female mouse which is 3 weeks old and 10-13g in weight, the injection amount of the PMSG is 5-10IU, the injection method is intraperitoneal injection, the injection amount of the PMSG is preferably 8IU, and the time for killing the mouse after cervical vertebra dislocation is 46-48h after injection. The mechanical method provided by the invention is simple and convenient to operate and short in time consumption, and the obtained ovarian granular cells are high in yield.

The invention discloses a composition for improving ovarian function. The composition comprises umbilical cord mesenchymal stem cells and umbilical cord blood CGF. The functions of stem cells and CGFare maximized through an ovary multi-site injection administration mode, so that the ovary weight, the follicle number of each stage and the E2 level of an ovarian premature senility model rat are increased, the FSH level and the ovarian granulocyte apoptosis rate are reduced, the Bax protein is remarkably reduced, and the Bcl2 protein is increased. The invention provides a powerful tool for clinically treating the premature ovarian failure and improving the ovarian function.

The invention relates to the technical field of isolated culture of ovarian granulosa cells, in particular to an isolated culture method of duck ovarian granulosa cells. The isolated culture method comprises the following steps: (1) bleeding necks of female ducks to kill the female ducks, and taking out complete ovaries; (2) shearing complete follicles before ovulation to obtain single follicles before ovulation; (3) cutting off the single washed follicles before ovulation, scraping off yolk, and washing for 5 times; (4) respectively digesting cleaned granulosa cell layers by adopting hyaluronidase and trypsin; (5) resuspending the digested granulosa cell layers, conveying an obtained cell suspension into a culture medium, and repeatedly blowing and beating by using a pipettor to dispersecells; and (6) carrying out incubator culture on the resuspended cell suspension. The duck ovarian granulosa cells obtained by the isolated culture method provided by the invention are high in purityand high in activity, and the survival rate of the duck ovarian granulosa cells is high.

The invention provides a method for adjusting and controlling ovarian granulosa cells based on circ-Atp5BP. The method comprises the following steps: (1) carrying out inoculation and subculture on nest granulosa cells; and (2) when the cell density is cultured to be 35 to 45 percent, transfecting the circ-Atp5BP gene or the pik3 gene into the nest granular cells. The application of circ-Atp5BP and pik3 in porcine ovarian granular cell proliferation adjustment and the targeting adjustment relation between circ-Atp5BP and pi3k are proved for the first time. The proliferation of the ovarian granular cells can be remarkably adjusted and controlled.

The invention discloses a method of converting ovarian granular cells serving as precursor cells into multipotential stem cells. After the ovarian granular cells are separated and attached, measures such as reprogramming factors or small molecule compounds are adopted to convert the ovarian granular cells into the multipotential stem cells. In the experiment of mice, an operator successfully converts the ovarian granular cells of the mice into the true multipotential stem cells efficiently. In the experiment of pigs, the operator also obtains a good multipotential stem cell system in a high-efficiency manner. The ovarian granular cells have the advantages of wide source, high reprogramming efficiency and the like, and the obtained multipotential stem cells made from ovarian granular cells have the higher safety and great potential application to the clinical medicines of people. The method is efficient, economical, good in safety and wide in application prospect.

The invention discloses application of a mistletoe extract in preparing a traditional Chinese medicinal product for improving the activity of ovarian granular cells. According to the invention, novel application of the mistletoe extract in treating ovarian dysfunction and improving the activity of the ovarian granular cells is developed through pharmacological experiments on the basis of preparing the mistletoe extract by fully utilizing traditional Chinese medicinal resources according to the pathogenesis of the ovarian dysfunction and hypotrophy of the ovarian granular cells; and the extract can be added into products of different dosage forms and reaches the activity of nourishing ovary by promoting the proliferation of the ovarian granular cells and improving the estrogen and progesterone secretion levels, is clinically used for treating the ovarian dysfunction, and has the advantages of good curative effects, small side effects, convenience in use and the like.

The invention discloses application of a transcription factor CEBP alpha serving as a transcription factor of a Kiss1 promoter region, and belongs to the technical fields of gene engineering and cellengineering. According to the invention, the transcription factor CEBP alpha is taken as a research object, a potentially combined transcription factor CEBP alpha is predicated through bioinformatics,and the influence on the activity of the Kiss1 promoter region by the transcription factor CEBP in ovarian granulosa cell is researched, so as to achieve research on expression regulation of gene inthe ovarian granulosa cell. According to the invention, expression regulation to the Kiss1 gene by the transcription factor CEBP in porcine ovarian granulosa cell and research on functions of the transcription factor CEBP in the ovarian granulosa cell are verified for the first time. The research is rich in experiment content, and results are complete and accurate.