42 results about "Sertoli cells" patented technology

The invention relates to the technical field of reproductive biology, in particular to a method for efficiently constructing pig testis organs in vitro. Comprising the following steps: S1, enzymolysis of testicular tissues of piglets; and S2, testicular cell resuspension counting: uniformly suspending the testicular cells obtained after enzymolysis in the step S1, sequentially collecting the cells into a 96-well plate with a U-shaped bottom, and adding a DMEM base culture medium containing 5% of FBS, 10% of KSR and 1% of P / S into each well to complete resuspension culture of the cells. S3, testicular organ in-vitro culture: separating testicular cell masses, transferring the separated cell masses into a 96-well plate with a U-shaped bottom, and adding the cell masses into a DMEM base culture medium containing 5% of FBS, 10% of KSR and 1% of P / S for culture; and the quantity of cells in the 96-well plate with the U-shaped bottom is 5 * 104 cells per well. After the culture system is optimized, the experimental process is simplified, the testicular organs can be directly cultured in the 96-well plate with the U-shaped bottom, the replacement frequency is reduced, the pollution risk is reduced, the number of adopted cells is remarkably reduced, and the culture efficiency is improved.

The invention discloses application of mangiferin in preparation of a protective agent for testicular sertoli cells and preparation of a medicine for treating male infertility diseases, and relates to the technical field of medicine application. Wherein the mangiferin can play a role in protecting the sertoli cells, and the protection role is reflected in ways of improving the cell viability of the sertoli cells, inhibiting the apoptosis of the sertoli cells, inhibiting the oxidative stress of the sertoli cells, inhibiting the inflammatory response of the sertoli cells and the like; meanwhile, mangiferin can be used for treating male infertility diseases in ways of relieving weight loss, repairing testis, improving endocrine system functions, inhibiting oxidative stress of testis tissues, inhibiting inflammatory response of the testis tissues and the like. Wherein the mangiferin can inhibit the inflammatory reaction mainly by inhibiting the activation of NLRP3 inflammasome to inhibit the NLRP3 pathway, so that the inflammation and even pyroptosis of testicular sertoli cells are remarkably reduced to improve spermatogenesis, and the male infertility disease is well treated.

The invention discloses a method for generating haploid sperm-like cells through in-vitro differentiation of pluripotent stem cells and application of the haploid sperm-like cells. Human pluripotent stem cells are firstly differentiated into ectoderm-like cells, then the ectoderm-like cells are spontaneously gathered and converted into primordial germ cell-like cells under the induction of ectoderm induction factors, and then the primordial germ cell-like cells and testicular sertoli cells are subjected to mixed culture to form recombinant testicular tissues; the recombinant testis tissue enters meiosis under the induction of nutrition limitation and retinoic acid, then meiosis is completed under the stimulation of meiosis second-stage induction factors such as testosterone, follicle-stimulating hormone and pituitary extract, and interaction between primordial germ cell-like cells and testis supporting cells is enhanced by using small molecules SB431542, so that the meiosis of the testis is enhanced. And finally inducing to generate haploid sperm-like cells. According to the invention, a new tool can be provided for researching a molecular mechanism of male reproduction, and an important thought can be provided for research and transformation of asthenospermia, oligospermia, drug screening and the like.

The invention discloses novel application of a GJB5 gene, a construction method of a GJB5 gene overexpression vector and a method for improving high temperature resistance of economic animals, and relates to the technical field of molecular biology. The preparation for overexpressing the GJB5 gene improves the heat resistance of the economic animal sertoli cells through overexpression of the GJB5 gene. Meanwhile, the invention also discloses a construction method of the GJB5 gene overexpression vector and a method for improving the high temperature resistance of economic animals. The effect of the GJB5 gene in heat stress is found for the first time, the gene is expected to be overexpressed in pigs or genetically modified into economic animals such as cattle and sheep in the future, so that the heat resistance of the pigs is improved, the semen quality of the pigs under the heat stress condition is further improved, and the GJB5 gene has important social benefits and economic benefits.

This invention provides a biomarker for spermatogenesis disorders and its application, wherein the biomarker is the MNDA molecule. This application characterizes the functional abnormalities or spermatogenesis disorders of Sertoli cells by detecting the expression level of MNDA molecules on cells in testicular tissue sections, and can therefore be used for the clinical diagnosis of non-obstructive azoospermia.

The invention belongs to the technical field of medicines, and particularly discloses application of afzelin in preparation of an anti-testis aging medicament, and testis aging resistance is particularly improvement or recovery of an aged testis tissue structure. The invention also provides a medicament for resisting testis aging. The medicament contains afzelin. The invention provides a new application of afzelin, the afzelin can be used for saving senescence testicular tissue structure disorder, remarkably reducing the epididymal sperm deformation rate and increasing the number of supporting cells and undifferentiated spermatogonium cells, the new application value of afzelin is developed, and a theoretical basis is provided for developing afzelin into anti-reproductive aging drugs.

The invention relates to the technical field of biomedicine, in particular to a construction method and application of a testis organ capable of differentiating sperms in vitro. The polypeptide hydrogel is used as a three-dimensional scaffold for testicular cell growth, and meanwhile, the testicular cell proliferation promoting liquid and the testicular cell differentiation promoting liquid are added in different culture stages, so that the construction of testicular organs is effectively realized. The obtained testis organs comprise testis seminiferous tubule structures consistent with those in vivo and various testis cells with the same arrangement mode; and in addition, spermatogonium can be successfully induced to differentiate deformed sperms in vitro. The testicular organ construction method provided by the invention is short in period, high in sperm differentiation success rate, simple and convenient to operate and low in cost. The constructed testis organ can be applied to screening of drugs or toxicants, testis ectogenesis models and clinical diagnosis and treatment, and has a wide application prospect and an important scientific value.

The invention discloses a reagent for editing a pig INSL3 gene and application of the reagent. The reagent for editing the pig INSL3 gene contains an INSL3-sgRNA A and an INSL3-sgRNA B, wherein the INSL3-sgRNA A and the INSL3-sgRNA The target sequence of the INSL3-sgRNA A is as shown in SEQ ID No.5; the target sequence of the INSL3-sgRNA B is shown in SEQ ID No. 6. Experiments prove that the INSL3-sgRNA A and the INSL3-sgRNA B are combined with a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 system, so that the pig INSL3 gene can be efficiently edited, and the editing efficiency of the INSL3 gene of pig testicular cells and the editing efficiency of the INSL3 gene of pig fetal fibroblasts can reach 8.47% and 11.45% respectively. The invention has important significance on the function research of the INSL3 gene of pig cells or individuals and the breeding of pig genetic disease resistance.

The invention discloses a pilose antler active peptide and application thereof in anti-inflammation, anti-oxidation and anti-aging, and belongs to the technical field of biology. According to the invention, the brand-new active peptide SPLASDLDL screened out from pilose antler for the first time is utilized, and competitive inhibition on TNFR1 is realized through the characteristic that the brand-new active peptide SPLASDLDL is specifically combined with a TNFR1 receptor. A constructed sertoli cell senescence model proves that the polypeptide has anti-inflammatory, anti-oxidation and anti-senescence effects. The invention successfully solves the technical problem that in the prior art, the competitive inhibition TNFR1 targeting peptide cannot be found from pilose antler and the action mechanism of the TNFR1 targeting peptide cannot be clarified, and provides the action mechanism and scientific basis for developing novel anti-inflammatory and anti-aging drugs derived from natural products.

The invention discloses a testis Leydig cell senescence marker and application thereof, the senescence marker is CCN5 protein, the senescence condition of the Leydig cell can be specifically reflected by detecting the CCN5 level of testis tissue, the senescence marker can be used as an index for evaluating Leydig cell senescence and testis senescence, and meanwhile, the senescence marker is an effective supplement for a diagnostic scheme of late onset hypogonadism (LOH). Furthermore, the invention also provides application of a preparation for inhibiting CCN5 protein expression in preparation of a product for improving testis Leydig cell senescence or treating diseases caused by testis Leydig cell senescence, the testis Leydig cell senescence can be improved and the LOH symptom can be treated by reducing CCN5 in a targeted manner, and treatment can be performed aiming at the core phenotypes of'lipid droplet loss' and'fibrosis' of testis Leydig cell senescence. The method is a treatment scheme different from exogenous testosterone supplement, stem cell injection, Senlytics drugs and exosome therapy.

The present invention discloses a thermosensitive hydrogel of porcine testicular acellular extracellular matrix and a preparation method thereof, including: using freeze-thaw cycles to disrupt cell membranes, and performing decellularization treatment with a mild detergent TritonX-100. While effectively removing testicular cells, the biological composition and structural scaffold of the testis are highly retained. This hydrogel can rapidly solidify and form after being injected into the testis, adapt to the complex environment of the testis, recruit testicular interstitial stem cells at the same time, regulate their differentiation into testicular interstitial cells, and then secrete testosterone. In addition, the hydrogel can also degrade under the action of in vivo enzymes, release cytokines, and reduce the inflammatory reaction of testicular interstitium. The hydrogel also has high biological safety and low immunogenicity.

The invention discloses a testis organ as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing printing ink and carrying out photocuring 3D printing, wherein the printing ink comprises photocuring hydrogel and testicular cells. The testicular organ has similar cell type composition, morphology and expression of a cell specific marker as the testicular organ. The method for preparing the testicular organ is easy to operate, rapid and convenient, and the 3D cultured testicular organ is formed through the 3D photocuring printing technology. The testicular organ can be used for evaluating the reproductive toxicity of a compound such as a medicine to male, and has reliability and wide application prospect.

The application provides application of an ACOT12 gene in regulation of proliferation of bovine testis Sertoli cells and belongs to the technical field of biotechnology.The application observes the proliferation of bovine testis Sertoli cells by interfering with ACOT12 gene expression.The results show that interference with the ACOT12 gene can significantly inhibit the proliferation of bovine testis Sertoli cells, which indicates that the ACOT12 gene has a positive regulation effect on the proliferation of bovine testis Sertoli cells.

The present invention relates to in vitro methods for production of Sertoli cells and related organoids. The Sertoli cells and testis like organoids may be used for therapeutic purposes including facilitation of the production of spermatogonia from pro-spermatogonia stem cells.

This invention discloses a method for the isolation, culture, and identification of chicken sexual gonadal cells, belonging to the field of biotechnology. The invention selects chicken embryos hatched to 18.5 days old, isolates the gonads after PCR sex identification, and uses 0.25% trypsin-EDTA digestion followed by filtration through a 70μm filter to obtain a single-cell suspension with a survival rate >90% and free from PGC contamination. A sex-specific culture system is established, allowing cells to be stably passaged and maintain a sex-specific phenotype. Through morphological, qRT-PCR, immunofluorescence, and Western blot multidimensional identification, accurate identification of male testicular Sertoli cells and female ovarian granulosa cells is achieved. This method is simple to operate, has good reproducibility, and produces high cell viability and purity. It can provide a stable gonadal microenvironment for the in vitro directed differentiation of chicken primordial germ cells, significantly improving gamete induction efficiency. It is suitable for research on avian reproductive and developmental mechanisms, transgenic breeding, and germplasm resource preservation.

The invention discloses an application of miR-486-3p targeting ACSL4 (acyltransferase) in regulation of testicular ferroptosis. The invention is based on the discovery that ACSL4 is a target gene of miR-486-3p in plateau zokor and participates in androgen regulated seasonal spermatogenesis. According to the invention, the functions of the miR-486-3p and the ACSL4 are researched by using a mouse. A dual-luciferase verification report shows that the expression of the miR-486-3p in a mouse is regulated and controlled by combining 3UTR of the ACSL4 gene. In vitro, testicular sertoli cells regulated by androgen signals are used as research objects, and the fact that the proliferation, oxidative stress and ferroptosis of the cells are regulated by miR-486-3p targeting ACSL4 is found. In vivo, oxidative stress and ferroptosis of mouse testis are caused by inhibition of miR-486-3p expression, and sperms are abnormal. MiR-486-3p is overexpressed in testis with ferroptosis, so that accumulation of peroxide in tissues is relieved, and sensitivity to ferroptosis is reduced.

The present invention discloses a method for screening key functional proteins of different cells in bovine testes and its application. The method is based on single-cell RNA sequencing technology, which details five different germ cell types and eight somatic cell types in bovine testicular cells, and provides gene markers specific to testicular cell types, namely key functional protein genes. Through the expression of these proteins, cell types can be quickly and accurately identified. In addition, these proteins can also help us develop targeted intervention measures to improve the fertility of livestock.

The application discloses a method for extracting mitochondria-associated membranes (MAM) and other subcellular components from podocytes, which comprises the following steps: pretreating the podocytes, crushing the podocytes by low-power ultrasonic, separating the crude mitochondrial components by differential centrifugation, and then purifying the high-purity MAM and other subcellular components by density gradient centrifugation and ultracentrifugation. The method replaces the traditional homogenizer grinding with low-power ultrasonic crushing, improves the MAM extraction yield by controlling the ultrasonic parameters, appropriately simplifies the differential centrifugation steps, increases the yield while ensuring the purity, sequentially separates the unbroken cells, the crude mitochondria, the cytoplasm and the endoplasmic reticulum components, and finally obtains the high-purity MAM and other subcellular components by density gradient centrifugation and ultracentrifugation purification. The method effectively improves the yield of the podocyte separation and extraction of the MAM and other subcellular components, reduces the cost, and provides technical support for the research on the podocyte MAM proteomics.

The invention relates to the technical field of microorganisms, in particular to application of Bacteroides fragilis in preparation of a product for preventing or treating chicken testis blood testis barrier injury. The invention discloses application of Bacteroides fragilis in preparation of products for preventing or treating chicken testis blood-testis barrier injury for the first time, and experiments prove that the Bacteroides fragilis can improve the weight of chicken testis, the diameter and area of the testis, the number of spermatogonial stem cells in the testis and the expression level of testis blood-testis barrier related protein, and can be used for preparing a product for preventing or treating chicken testis blood-testis barrier injury. The completeness and functions of chicken testis cells can be maintained, and the normal development of the testis can be promoted. The application of the Bacteroides fragilis in the field of reproductive development of chickens is beneficial for promoting the breeding industry to develop towards a more efficient and sustainable direction.

The invention discloses novel application of a CLIC3 gene, a construction method of a CLIC3 gene overexpression vector and a method for improving high temperature resistance of economic animals, and relates to the technical field of molecular biology. The preparation for overexpressing the CLIC3 gene improves the heat resistance of the economic animal sertoli cells through overexpression of the CLIC3 gene. Meanwhile, the invention also discloses a construction method of the CLIC3 gene overexpression vector and a method for improving the high temperature resistance of economic animals. The effect of the CLIC3 gene in heat stress is found for the first time, the gene is expected to be overexpressed in pigs or genetically modified into economic animals such as cattle and sheep in the future so as to improve the heat resistance of the pigs and further improve the semen quality of the pigs under the condition of heat stress, and the CLIC3 gene has important social benefits and economic benefits.

The present invention relates to in vitro methods for production of Sertoli cells and related organoids. The Sertoli cells and testis like organoids may be used for therapeutic purposes including facilitation of the production of spermatogonia from pro-spermatogonia stem cells.

The invention discloses a yak sertoli cell separation method which comprises the steps of yak testis disinfection, yak testis tissue separation, cell digestion, cell purification and the like, so that the whole process of the yak sertoli cell separation method is realized. The separation method of the yak sertoli cells is simple and convenient to operate, the separated yak sertoli cells are relatively high in purity, other purification measures are not needed, the subsequent culture stability is good, and the separation efficiency is high; meanwhile, the aspects of cell pollution, separation purification and identification, separation method cost and the like can be effectively controlled, and popularization and application are facilitated.

The invention discloses a gonad skin chip as well as a preparation method and application thereof, based on a micro-fluidic technology and a three-dimensional cell culture system, firstly, a culture unit containing skin tissue and gonad tissue is constructed in the chip, and nutrient substances and hormones are supplied through micro-fluidic channels respectively; the dynamic interaction of the skin and the gonad tissue is realized by accurately controlling the flow velocity of the culture medium and the hormone concentration gradient. The skin culture room of the chip supports growth and differentiation of skin cells; and the gonad culture room supports the culture of ovarian or testicular cells and the secretion of hormones. The obtained gonad skin chip can effectively simulate the skin-gonad interaction process, and provides an experimental platform for hormone research, dermatology and drug screening.

The invention provides application of an ACOT12 gene in regulation and control of bovine sertoli cell proliferation, and belongs to the technical field of biology. According to the invention, the proliferation condition of the bovine sertoli cells is observed by interfering the expression of the ACOT12 gene. Results show that the ACOT12 interference gene can significantly inhibit the proliferation of the bovine sertoli cells, which prompts that the ACOT12 gene has a positive regulation effect in the proliferation of the bovine sertoli cells.

The invention belongs to the technical field of genetic engineering and viral vectors, and particularly relates to a recombinant adeno-associated virus (AAV) vector capable of specifically infecting mouse sertoli cells, and the virus is characterized in that a polypeptide sequence DGCRPPR is inserted into the 590th amino acid site of AAV8. Compared with a wild-type AAV8 vector, the mutant has higher specificity to the sertoli cells, and after seminiferous tubules are injected, the infection ability to the sertoli cells in the testis is higher, and the specificity is better.

The invention relates to application of ginsenoside Rg4 as a unique active ingredient in preparation of a medicine for improving male delayed gonad hypofunction, belonging to the field of biological medicine. According to the invention, the action mechanism of the Rg4 for improving LOH is defined for the first time: the Rg4 can specifically up-regulate the mRNA expression quantity of StAR and StARD7 in a testis tissue, and promote the transport of cholesterol to the inner mitochondrial membrane of a testis Leydig cell from the transcriptional level; the Rg4 improves the mRNA expression quantity of P450scc, 3 beta-HSD in testicular tissues by promoting the efficient transport of cholesterol, enhances the conversion of cholesterol to pregnenolone and the catalytic synthesis of subsequent steroids, and improves the endogenous testosterone synthesis capability; the Rg4 can recover the negative feedback regulation function of an HPG axis, regulate secretion of pituitary LH and FSH to physiological steady state, and avoid abnormal fluctuation of hormone level. The invention fills the technical blank of Rg4 in the field of LOH gene regulation and control.

The invention belongs to the technical field of medicines, and particularly relates to application of a Chinese chive seed extract in preparation of a medicine for treating oligoasthenospermia. Based on network pharmacology prediction and animal experiment verification, it is found that by activating a PI3K / AKT signal channel, testicular cell apoptosis is inhibited, spermatogenesis disorder is improved, and therefore the sperm number and vitality of an oligoasthenospermia model rat are remarkably improved. Clinical treatment research in combination with levocarnitine oral liquid proves that the traditional Chinese medicine composition can reduce the sperm DFI level and improve the semen quality. Animal research proves that the compound can regulate the level of serum testosterone (T), reduce the levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH), repair pathological damage of testis tissues, and antagonize testis cell apoptosis induced by tripterygium glycosides by up-regulating protein expression of p-PI3K / PI3K, p-AKT / AKT and Bcl-2.

Provided is use of PGAM2 in the breeding of Blcattle black cattle, which belongs to the technical field of molecular biological breeding, in which individuals having a high PGAM2 content are screened as high-quality breeding bulls. In the present disclosure, it is proved by means of experiments that PGAM2 may promote the proliferation and glycolytic metabolism of Sertoli cells to produce lactic acid, an energy substrate required for spermatogenic cell development. It regulates the formation of blood-testis barrier and facilitates the communication between Sertoli cells and germ cells, thereby promoting spermatogenesis. The present disclosure provides a new direction for the breeding of Blcattle black cattle for high quality beef production.

The present invention relates to a method for the in vitro production of Sertoli cells and related organoids. Sertoli cells and testis-like organoids can be used for therapeutic purposes, including promoting the production of spermatogonia from pre-spermatogonial stem cells.

The invention belongs to the field of biology, and provides a bovine nodular skin disease virus purification method which comprises the following steps: S1, preparing a bovine nodular skin disease virus LSDV strain and a lamb primary testis cell strain; s2, preparation of reagents and consumables; s3, preparing an LSDV antigen and determining the virus titer; s4, carrying out LSDV antigen purification; and S5, observing the concentrated and purified LSDV sample through an electron microscope. The method has the advantage of low requirements on equipment; the molecular sieve is simple in structure and high in selectivity, viruses with different diameters can be separated out by selecting fillers with proper pore sizes according to the sizes of virus molecules, and the molecular sieve can keep better immunogenicity of antigens after being purified and can be repeatedly used, so that the production cost in large-scale production is reduced, and the method is suitable for industrial production. Reference is provided for establishment of a bovine nodular skin disease virus inactivated vaccine purification process, and a molecular sieve purification method is used as an LSDV purification method and is more suitable for large-scale production of LSD vaccines.