139 results about "Granular leucocyte" patented technology

Methods and cell culture medium for the generation of human pluripotent embryonic stem cells are disclosed. Human embryonic stem cells are cultured with human granulosa feeder cells, muscle cells, Fallopian ductal epithelial cells, bone marrow stromal cells, and skin fibroblasts and the embryonic stem cells maintain their pluripotent phenotype. The human pluripotent embryonic stem cells can be cultured without feeder cells, and in the presence of supplemental growth factors. The human pluripotent embryonic stem cells can be alternatively cultured with conditioned medium obtained from a cell culture capable of maintaining human embryonic stem cells in a pluripotent state, wherein the cell culture is a human granulosa cell culture.

The invention discloses an in-vitro recombined human skin epidermis model.A layer of hemidesmosome protein is arranged on the lower surface of a basal cell layer, the upper surface of the basal cell layer is covered with 6-8 spinous cell layers containing short protruding spinous cells, the upper surface of the spinous cell layers is covered with 3-4 granular cell layers containing transparent granular cells, the upper surface of the granular cell layers is covered with a keratinocyte layer, all the cell layers are obviously separated, and the overall barrier function of a lipid composition is approximate to that of human skin.A preparation method comprises the steps of preparation of matrigel particles, screening and amplification of epidermal stem cells, preparation of the basal cell layer, preparation of the spinous cell layers, preparation of the granular cell layers, and maturation and keratinization of the in-vitro recombined epidermis model.The model is applied to evaluation of cosmetics with the human skin barrier repair effect, screening of plant extract with the human skin barrier repair effect, evaluation and detection of irritation of medical apparatus materials on the human skin and detection of irritation of chemicals on the skin.

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 a method for non-invasive preimplantation hereditary detection of embryos, and belongs to the technical field of biological detection. The method comprises the following steps:carrying out whole genome amplification on a blastocyst culture solution sample by using a kit, carrying out short tandem repeat sequence analysis on an amplification product and DNA samples of parents to detect maternal pollution, carrying out library preparation and next-generation sequencing detection on the amplification product to determine whether the number of chromosomes is abnormal or not; and optimizing a pre-amplification mixed solution and an amplification mixed solution by the provided kit. According to the method provided by the invention, the blastocyst culture solution can besubjected to parent source pollution detection, so that whether the chromosome aneuploidy detection result of the culture solution is accurate and reliable or not is judged. The invention provides a detection method for judging whether granular cells are completely removed or not, and the inhibition effect of components in the culture solution on amplification is effectively avoided through optimization of the kit, so that amplification uniformity is good, and the single cell amplification yield is high. The detection method is simple, the result is accurate, and data quality is improved.

A method for evaluating the quality of mammalian oocytes comprises determining the expression level of one or more of the genes ACPP, AQP11, CCDC126, CLU, CYP11 A1, CYP19A1, EGR3, FN1, FOSL2, GMNN, HRAS, HSD3B2, HS-D17B1, HSD11B2, HSDL1, IGF1, IGFBP4, IGFBP5, IRS1, KCNK3, KLF6, NEK6, SMAD7 or STC1 in a test sample derived from a cumulus cell or granulosa cell associated with the oocyte, and comparing the expression level of said at least one marker gene expression in the sample with the expression level in a control. Differential expression of the gene between the sample and the control is indicative of the quality of the oocyte.

A novel growth factor specific for vascular endothelial cells has been identified in conditioned medium of bovine pituitary derived folliculo stellate cells. This factor, named folliculo stellate derived growth facto (FSdGF) or vascular endothelial growth factor (VEGF), was purified to homogeneity by a combination of heparin sepharose affinity chromatography, Bio Gel P-60 exclusion chromatography, Mono S ion exchange chromatography and hydrophobic chromatography on a C4 reverse phase HPLC column. The factor is also found in the murine AtT-20 cell line. Alternatively, the growth factor is purified by a first reverse phase HPLC using acetonitrile gradient followed by a second reverse phase HPLC using an isopropanol gradient. FSdGF, having a molecular weight of about 43,000 da, was characterized as a glycoprotein composed of two homologous sub units with MW of about 23 kDa. FSdGF was a potent mitogen for vascular endothelial cells with activity detectable at 10 pg / ml and saturation at 500 pg / ml. It did not stimulate the proliferation of other cell types such as bovine corneal endothelial cells, adrenal cortex cells, granulosa cells, BALB / MK cells or BHK-21 cells. Microsequencing revealed an amino terminal sequence containing no significant homology to any known protein. The release of FSdGF by pituitary cells and its unique target cell specificity indicate that FSdGF is useful in angiogenesis.

The present invention addresses the problem of providing a method for differentiating primordial germ cells into functionally mature GV stage oocytes by in vitro culture. The present invention pertains to a method for differentiating primordial germ cells into functional GV stage oocytes in vitro including (a) a step for forming secondary follicles by culturing primordial germ cells and feeder cells adjacent to the primordial germ cells under conditions that eliminate the effects of estrogen or factors having a similar function to estrogen, (b) a step for partially cleaving the bonds between the granulosa cell layer and the thecal cell layer among the oocyte, granulosa cell layer, and thecal cell layer that constitute the formed secondary follicles, and (c) a step for differentiating the oocytes into functional GV stage oocytes by culturing the oocytes and granulosa cell layer that constitute the secondary follicles and the thecal cell layer in medium including a polymer compound.

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.

Nucleic acid compositions encoding a pro-apoptotic protein, Bok (Bcl-2-related ovarian killer) are identified. Bok has conserved Bcl-2 homology domains 1, 2 and 3 and a C-terminal transmembrane region present in other Bcl-2 related proteins, but lacks the BH4 domain found only in anti-apoptotic Bcl-2 proteins. Over-expression of Bok induces apoptosis. Cell killing induced by Bok is suppressed by co-expression with selective anti-apoptotic Bcl-2 proteins. Bok is highly expressed in the ovary, testis and uterus, particularly in granulosa cells, the cell type that undergoes apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic protein with wide tissue distribution and hetero-dimerization properties facilitates elucidation of apoptosis mechanisms in reproductive and other tissues, and provides a means for manipulating apoptosis.

The invention relates to a biomarker of granulosa cells used for polycystic ovary syndrome (PCOS) diagnosis as well as a screening method and a diagnostic kit thereof. The novel diagnostic biomarker is discovered by screening PCOS-related biomarkers in the whole genome by adopting combined analysis of RNA-seq, miRNA-seq and MBD-seq. The biomarker comprises miR-429, miR-141-3p and miR-126-3p, and / or XIAP gene, BRD3 gene, MAPK14 gene and SLC7A5 gene. Compared with the prior art, the biomarker of granulosa cells used for PCOS diagnosis has the advantages of being high in detection sensitivity, high in accuracy and so on.

The invention provides 1ncRNA SFR1 as well as application thereof and a product and a method for regulating and controlling follicular development and relates to the technical field of biologics. Theinvention provides novel long-chain non-encoding RNA SFR1, wherein the 1ncRNA SFR1 is related to apoptosis of granular cells, and by regulating and controlling expression of 1ncRNA SFR1, correspondingregulation and control functions on channels, genes and hormones related to follicular development can be also achieved. Therefore, by adopting the 1ncRNA SFR1 and a corresponding product provided bythe invention, effective in-vivo / in-vitro regulation and control on follicular development can be achieved, and powerful guarantee can be provided for research on follicular development and physiology and biochemical functions. In addition, through the regulation and control functions of the 1ncRNA SFR1 on follicular development, the population fertility can be improved while excellent propertiesof genetic resources of mammals, particularly sheep, are maintained, and thus the economic benefits of breeding can be increased.

The invention relates to a method for bovine in-vitro fertilization embryo culture. The method comprises the following steps: collection of an oocyte and in-vitro maturity; in-vitro fertilization; embryo in-vitro culture and preservation. During the in-vitro fertilization, mature COCs are cultured in a fertilization culture solution of the invention, during embryo in-vitro culture, granule cells around an embryo are completely removed by using an egg peeling needle, the granule cells are placed into the embryo culture solution to be cultured, and after the culture is finished, the usable embryo is subjected to liquid nitrogen cryopreservation in refrigerating fluid. The method has many excellent technical effects as described in the description.

The invention provides oocyte culture fluid and a culture method of the oocyte culture fluid. The oocyte culture fluid is easy to prepare and low in cost and comprises G-IVF culture fluid, follicular fluid and granular cells. According to the oocyte culture fluid and the culture method of the oocyte culture fluid, the follicular fluid extracted from the follicle of a patient and granular cells around the oocyte are treated specially and added into the culture fluid, so that in-vitro maturity culturing is carried out on the immature egg of the remaining ICSI period, then fertilization, embryonic development and pregnancy results are analyzed, a simple, convenient and effective in-vitro maturity culture scheme is set up, the effective utilization rate of the immature egg is increased, and more pregnancy opportunities can be provided for the infertility patient.

The invention discloses a separation, primary culture and subculture method for granulosa cells in follicles in a pig ovary GV period. The method comprises the following steps: collecting a cyst-free healthy pig ovary, and cleaning the ovary; separating granular cells; carrying out primary culture on the granular cells for 48 hours; changing liquid in a culture bottle for primary culture; after changing the liquid, continuously culturing the cells for 36 hours, and cleaning, digesting and terminating the cultured cells; and finally, sub-packaging and subculturing the cells for 48 hours. The invention provides a pollution-free ovary collection method, optimizes a granulosa cell acquisition and culture method, and further provides a primary granulosa cell subculture method. Granular cells obtained by the method are almost free of pollution, high in cell uniformity, high in consistency in the growth and development period, high in cell survival rate, high in success rate of making oxidative stress models and the like, and high in result consistency, practicability, stability and feasibility. In addition, the number of obtained cells is large, many subsequent experiments can be carried out, the number of times of going to a slaughter house is reduced, and the efficiency is high.

The invention discloses a method applicable for blood cell high-flux fast classification analysis of common fishes such as crucian and carps. By aiming at the problem of white blood cell classification and counting interference by removing-incapable nucleated red blood cells in two aspects of the quantity and the form, an automatic measurement and analysis strategy of combining the fluorochrome with the multi-channel images is utilized; the interference red blood cell sampling points are separated from the white blood cells according to the differences of particles in the cells, cell nucleus forms, positions and the like; different white blood cell class group range of lymphocyte, monocyte, particle cells and the like undertaking important immunologic functions is divided; the automatic analysis is completed. The difficulty that the nucleated red blood cells cannot be effectively removed troubles the automatic analysis of hemogram of various kinds of animals for a long time. The fluorochrome is combined with the imaging analysis; a high-flux and high-sensitivity measuring module of a multidimensional panoramic analyzer is used for accurately recognizing the red blood cell class groups; the interference is eliminated; the important technical support is provided for the focused analysis of the white blood cell class groups; wide application prospects are realized.

The invention belongs to the technical filed of fat transplantation, and particularly relates to a fat graft rich in high activity fatty granule cells and adipose-derived stem cells and a preparationmethod and application of the fat graft. The method comprises the following steps of performing manual negative-pressure liposuction with an injector, performing centrifugation on obtained fat to obtain a fat layer, enabling the lower layer of the fat layer to be digested to prepare cell suspension, pushing the upper layer to obtain an intermediate layer for transplantation, and finally, uniformlymixing the cell suspension with the intermediate layer to obtain the fat graft rich in high activity fatty granule cells and adipose-derived stem cells. The fat graft has the advantages that comparedwith conventional SVF-gel, the fat graft prepared by the method is higher in yield, and contains massive high-activity granule adipocyte, SVF-cells and adipose-derived stem cells, the concentration of the cells is 5-10 times of that of normal fat tissues, the survival rate of the transplantation fat can achieve 70%, the transplantation fat is soft and uniform in texture, and the probability of adverse reactions of oily tumor masses, tumor tubercles and the like is notably reduced.

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 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 diagnostic kit and a diagnostic method. The composition comprises the following components: anti-mullerian hormone (AMH), assisted reproduction (ART), ovarian granuloma (GCT)and sinus follicle count (AFC), and the anti-mullerian hormone (AMH) is one of members of a transforming growth factor beta superfamily and is generated by recruitment of granulocytes of presinus follicles and small sinus follicles. The invention relates to the technical field of diagnostic kits. According to the diagnostic kit and the diagnostic method, AMH is not influenced by the menstrual cycle, the serum AMH level does not fluctuate obviously in the whole menstrual cycle, the kit can detect and measure the AMH level at any time and can relatively truly reflect the stock condition of original follicles and avoids the troubles and personal errors of sinus follicles (AFC) counting under B-ultrasonic waves and is more objective and accurate in detection result and is not affected by hormone contraceptives or exogenous hormones and is convenient for clinical use.

The invention discloses a hair care essence spray. The hair care essence spray comprises a polygonum multiflorum extract, acetyl tetrapeptide-3, a willow herb flower / leaf / stem extract, a preservative,a humectant, a red clover flower extract, an eclipta prostrate extract, a rehmannia root extract, a boxthorn root extract, adenosine, dimethyl sulfone, EDTA-2NA and water. According to the hair careessence spray, multiple traditional Chinese medicine extracts are matched, and peptides, the adenosine and the dimethyl sulfone are taken as effective substances; the traditional Chinese medicine extracts can improve the activity of tyrosinase, increase the synthetic amount of melanin, promote the increase of melanin granular cells in a basal layer, promote melanin synthesis, up-regulate the expression of the tyrosinase and provide nutritional factors; meanwhile, the dimethyl sulfone can provide organic sulfur for hair; and the acetyl tetrapeptide-3 and the adenosine can provide nutrition forhair. The hair care essence spray has the effects of turning white hair into black hair, preventing hair loss and promoting hair growth through reasonable matching and multi-target action.

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 belongs to the technical field of medical imaging contrast agents, and particularly relates to a follicular granular cells near-infrared fluorescent probe Nirova-2 and a preparation method and application thereof. The fluorescent probe provided by the invention is a fluorescent dye-human follicle stimulating hormone coupling complex formed by coupling an organic small molecule near-infrared dye IR800CW and human follicle stimulating hormone (FSH); according to the complex, the fluorescent dye can specifically target granular cells forming follicle walls in ovaries, so that near-infrared fluorescent labeling of all follicles is realized. The fluorescent probe can be used as a near-infrared region fluorescent contrast agent for the follicles in the ovaries by combining the characteristic of fluorescence emission of the IR800CW in a near-infrared region with the property of specific targeted binding of the FSH to the follicle granular cells in the ovaries, so that the numberand the development stage of the follicles in the ovaries of women can be accurately and effectively detected and evaluated.

The invention discloses an in-vitro recombined human skin epidermis model.A layer of hemidesmosome protein is arranged on the lower surface of a basal cell layer, the upper surface of the basal cell layer is covered with 6-8 spinous cell layers containing short protruding spinous cells, the upper surface of the spinous cell layers is covered with 3-4 granular cell layers containing transparent granular cells, the upper surface of the granular cell layers is covered with a keratinocyte layer, all the cell layers are obviously separated, and the overall barrier function of a lipid composition is approximate to that of human skin.A preparation method comprises the steps of preparation of matrigel particles, screening and amplification of epidermal stem cells, preparation of the basal cell layer, preparation of the spinous cell layers, preparation of the granular cell layers, and maturation and keratinization of the in-vitro recombined epidermis model.The model is applied to evaluation of cosmetics with the human skin barrier repair effect, screening of plant extract with the human skin barrier repair effect, evaluation and detection of irritation of medical apparatus materials on the human skin and detection of irritation of chemicals on the skin.

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 belongs to the technical field of traditional Chinese medicines, and particularly discloses a traditional Chinese medicine composition for treating premature ovarian failure. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 8-20 parts of radix rehmanniae preparata, 6-10 parts of fructus corni, 10-30 parts of Chinese yam, 10-15 parts of fructus lycii, 6-10 parts of tortoise-plastron glue, 6-10 parts of deer-horn glue, 10-25 parts of semen cuscutae, 6-10 parts of radix cyathulae, 10-15 parts of fructus psoraleae, 10-15 parts of herba taxilli, 10-15 parts of radix dipsaci, 6-10 parts of ginseng, 10-30 parts of radix astragali, 6-10 parts of rhizoma atractylodis macrocephalae, 10-15 parts of radix angelicae sinensis, 10-15 parts of radix paeoniae alba, 6-10 parts of pericarpium citri reticulatae and 6-10 parts of radix glycyrrhizae preparata. The traditional Chinese medicine composition for treating premature ovarian failure can effectively inhibit apoptosis of granulosa cells, promote follicular maturation and improve ovarian reserve capacity, and is high in safety.

The invention belongs to the technical field of livestock molecular biology, and particularly relates to microRNA (miRNA) related to pig antral follicle atresia and an application of the microRNA. The miRNA is miR-9820-5p, and the linear nucleotide sequence of the miRNA is as shown in SEQ ID NO: 1. According to the invention, a specific hybridization probe is designed aiming at the miR-9820-5p, and real existence of the miR-9820-5p is verified through fluorescence in situ hybridization. Specific amplification primers are designed for the miR-9820-5p, it is detected through fluorescent quantitative PCR that the expression of the miR-9820-5p in healthy cavity follicle tissues in pig ovaries is lower than that of atresia follicles, and treatment with corresponding analogues and inhibitors proves that the miR-9820-5p has a remarkable promoting effect on granular cell apoptosis. Besides, the invention provides nucleotide sequences of an miR-9820-5 analogue and an miR-9820-5 inhibitor respectively. The miR-9820-5p disclosed by the invention is expected to be used as a molecular marker related to sow reproduction, and provides a new theoretical basis and a potential molecular target for evaluation of porcine reproduction traits, improvement of reproductive capacity and occurrence and prevention of human follicular abnormality related diseases.

The invention relates to a fertility protection and preservation technology. The invention discloses a method for improving vitrification freezing efficiency of ovarian tissues. The method comprises the following steps: 1) freezing the ovarian tissues: melatonin is added into a basic solution, an equilibrium solution and a freezing solution respectively, and the concentration of the melatonin reaches 0.01 mM; then, the ovarian tissues taken from a human body are immersed in the basic solution, the equilibrium solution and the freezing solution respectively for 5-20 min in sequence, and then the ovarian tissues are put into freezing tubes respectively to be sealed and directly put into liquid nitrogen to be frozen and stored; and (2) unfreezing the ovarian tissues, namely respectively adding melatonin into a 1mol / L sucrose solution, a 0.5 mol / L sucrose solution and a 0.25 mol / L sucrose solution to obtain three resuscitation solutions with the melatonin concentration of 0.01 mM, and preheating at 37 DEG C. The method has the characteristics that apoptosis of granular cells in the unfrozen ovarian tissues can be reduced, and the vitrification freezing efficiency of the ovarian tissuesis improved.