CN105055450A - Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment - Google Patents
Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment Download PDFInfo
- Publication number
- CN105055450A CN105055450A CN201510079968.2A CN201510079968A CN105055450A CN 105055450 A CN105055450 A CN 105055450A CN 201510079968 A CN201510079968 A CN 201510079968A CN 105055450 A CN105055450 A CN 105055450A
- Authority
- CN
- China
- Prior art keywords
- cell
- stem cells
- mice
- people
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Abstract
The present invention relates to a new method for premature ovarian failure treatment, wherein human endometrium stem cells can promote ovarian function recovery. Particularly the present invention discloses applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment, specifically in preparation of cell drugs for premature ovarian failure treatment, and/or medical technologies.
Description
Technical field
The present invention relates to a kind of new method for the treatment of premature ovarian failure: people's endometrial stem cells helps lend some impetus to the recovery of ovarian function.
Background technology
Premature ovarian failure (PrematureOvarianFailure, POF) refers to that women stopped with front menstrual phase of exhausting because of follicle in ovary or cause because of iatrogenic injury at 40 years old; The class gynecological common disease that main manifestations is estrogen deficiency, follicule-stimulating hormone (FSH) raises; It is the main cause [1] causing female acyesis.Research display, the probability that premature ovarian failure occurs in 30 ~ 40 years old women is about 1-2%, and the probability occurring in less than 30 years old is 0.1%, and the probability occurring in less than 20 years old is 0.01%, premature ovarian failure has rejuvenation and the high feature [2,3] of sickness rate as can be seen here.The exhaustion ahead of time of ovarian function makes patient lose fertility prematurely, and the decline of estrogen level, cause the increase of osteoporosis and risk of cardiovascular diseases, the physical and mental health and the family that have a strong impact on patient stablize.
At present, the known factor of premature ovarian failure that causes mainly comprises: inherited genetic factors, autoimmunity destroying in overy, postoperative iatrogenic injury and such environmental effects etc.The use of chemotherapeutics is the important means of clinical treatment malignant tumor and disease of immune system, but because the selectivity of chemotherapeutics to target cell is poor, while killing tumor cell the cell of normal tissue especially sexual cell also there is comparatively significantly cytotoxic effect.Because ovary is to inherent and environmental stimuli and sensitivity thereof, physics and chemical factor all can cause destroying in overy [4,5].In today of the continuous rising of gynaecopathia and cancer morbidity, although the use of chemotherapeutics can reach good therapeutic effect and improve the survival rate of patient, it too increases the probability that female patient premature ovarian failure occurs.Therefore, suitable effective Therapeutic Method is found to be our problem demanding prompt solution.
The treatment of premature ovarian failure is very difficult, up to the present, does not still have definite effective method can protect or recover the function of ovary.Hormone replacement therapy is the method for POF patient's extensive use, can alleviate perimenopausal symptom, improvement function; Reduce the Developmental and Metabolic Disorder that estrogen deficiency causes, as there is the risk of osteoporosis, fracture, Alzheimer, cardiovascular disease, slow down aging; But reproductive function cannot improve.For the POF patient having fertility to require, think that solution is still donor oocyte and the embryo transfer in hormone replacement cycle most effectively at present, and to use the oocyte of donations to carry out external fertilization and embryo transfer be high, the most best gravidity assisting method of success rate in POF patient, supplementary reproduction Medical Technology both domestic and external achieves successfully [6] all in this respect.But donor oocyte exists the dispute about the aspect such as ethics and law, and the source problem that comes of ovum is still its matter of utmost importance developed of restriction; Can embryo and Endometrium development the synchronous raising also affecting success rate of becoming pregnant, and this is a difficult problem for current reproductive medicine circle facing.Ovarian freezing is the treatment means developed at present with transplanting.Nowadays autologous ovary transplantation technology is comparatively ripe, patient's successful pregnancy of existing Orthotopic autotransplantation freeze thawing ovary tissue the report of healthy babies of giving a birth [7,8], but its application is very limited.Allosome ovary transplantation then faces a lot of problem, and as donor source difficulty, rejection, after transplanting, long term immunosuppressant applies problems [9] such as impairing one's health.
The stem cell of derived from bone marrow studies adult stem cell the most widely at present, has the advantages such as stronger differentiation potential and autotransplantation, be thus considered to the optimum stem cell of clinical treatment.2005; Johnson etc. report that the ovum of mammal may derive from the medullary cell come by Peripheral Circulation; and confirm that medullary cell expresses the molecular marker of some sexual celies, and then confirm bone marrow cell transplantation can make chemotherapy after sterillization wild-type mice model or normally recover ovarian function [10] because of the mouse model of ataxia genetic flaw development of ovary exception.2007, after this research group discovery bone marrow cell transplantation can make chemotherapy, the mice of ovarian failure recovered long-term reproductive function, and produces the filial generation of non-donor source, has found the immature egg cell [11] of donor source from the ovary transplanting Mus simultaneously.But these achievements in research receive a series of challenge.Can not arrive ovary by blood circulation and form ovum after having report to adopt disjunctor mouse model to find bone marrow cell transplantation, the cell arriving ovary be only some leukocyte.Nearest Santiquet report thinks that bone marrow cell transplantation can not produce new fertilizable ovum in the mice after chemotherapy sterillization, but the ovarian function [12] of sterillization mice after really improving chemotherapy.Regardless of the arguement of bone marrow cell transplantation for the development of ovary, but have any to reach common understanding, namely bone marrow transplantation can improve the reproductive function of mice after chemotherapy.
But the Clinical practice of bone marrow stem cell has certain limitation, such as cell derived limitation, multiplication capacity is limited, and acquiring way has traumatic etc.All the time, scientist concentrates on studies and attempts to find the stem cell of more extensively originating.
2008, people's reported first people endometrial stem cells such as Patel can separatedly from menstrual blood obtain [19].Scientists has carried out large quantity research to it in various preclinical animal model subsequently, mainly comprises the diseases [20-23] such as cerebral infarction, posterior-limb ischemia and myocardial infarction.Draw materials conveniently because people's endometrial stem cells has, non-invasive and autologous comparatively advantages of higher, have a wide range of applications in the treatment of disease clinically.
The list of references related in the present invention is specific as follows:
1.WaxmanJ (1983) Chemotherapyandtheadultgonad:areview.JRSocMed76:144-148 (WaxmanJ (1983) chemotherapy and adult's gonad: examination .JRSocMed76:144-148);
2.CoulamCB, AdamsonSC, AnnegersJF (1986) Incidenceofprematureovarianfailure.ObstetGynecol67:604-6 06 (CoulamCB, the sickness rate .ObstetGynecol67:604-606 of AdamsonSC, AnnegersJF (1986) premature ovarian failure);
3.SadeghiMR (2013) Newhopesforthetreatmentofprimaryovarianinsufficiency/pre matureovarianfailure.JReprodInfertil14:1-2 (newly the wishing .JReprodInfertil14:1-2 of SadeghiMR (2013) Turner syndrome/premature ovarian failure treatment);
4.Kalich-PhilosophL, RonessH, CarmelyA, Fishel-BartalM, LigumskyH, etal. (2013) Cyclophosphamidetriggersfollicleactivationand " burnout "; AS101preventsfolliclelossandpreservesfertility.SciTransl Med5:185ra162 (Kalich-PhilosophL, RonessH, CarmelyA, Fishel-BartalM, LigumskyH, etal. (2013) cyclophosphamide causes hair follicle to activate and " asthenia "; AS101 prevents follicle from losing and retains .SciTranslMed5:185ra162 fertility);
5.MeirowD, BiedermanH, AndersonRA, WallaceWH (2010) Toxicityofchemotherapyandradiationonfemalereproduction.C linObstetGynecol53:727-739 (MeirowD, BiedermanH, AndersonRA, WallaceWH (2010) chemotherapy and radiation is to female genital toxic effect .ClinObstetGynecol53:727-739);
6.GenerosoWM, StoutSK, HuffSW (1971) Effectsofalkylatingchemicalsonreproductivecapacityofadul tfemalemice.MutatRes13:172-184 (GenerosoWM, StoutSK, HuffSW (1971) alkylating agent chemical substance affects .MutatRes13:172-184 to the reproductive performance of adult female mice);
7.SakuradaY, KudoS, IwasakiS, MiyataY, NishiM, etal. (2009) Collaborativeworkonevaluationofovariantoxicity.5) Two-orfour-weekrepeated-dosestudiesandfertilitystudyofbu sulfaninfemalerats.JToxicolSci34Suppl1:SP65-72 (SakuradaY, KudoS, IwasakiS, MiyataY, NishiM, etal. (2009) Collaborative Evaluation ovarian toxicity .5) the busulfan repeated doses of 2 weeks or 4 weeks is to the research .JToxicolSci34Suppl1:SP65-72 of female rats fertility),
8.ShirotaM, SodaS, KatohC, AsaiS, SatoM, etal. (2003) Effectsofreductionofthenumberofprimordialfolliclesonfoll iculardevelopmenttoachievepubertyinfemalerats.Reproducti on125:85-94 (ShirotaM, SodaS, KatohC, AsaiS, the minimizing of SatoM, etal. (2003) primordial follicle quantity affects .Reproduction125:85-94 on follicular development to adolescence female rats);
9.TelferEE, GosdenRG, ByskovAG, SpearsN, AlbertiniD, etal. (2005) Onregeneratingtheovaryandgeneratingcontroversy.Cell122:8 21-822 (TelferEE, GosdenRG, ByskovAG, SpearsN, the dispute .Cell122:821-822 that AlbertiniD, etal. (2005) produce ovary regeneration);
10.JohnsonJ, BagleyJ, Skaznik-WikielM, LeeHJ, AdamsGB, etal. (2005) Oocytegenerationinadultmammalianovariesbyputativegermcel lsinbonemarrowandperipheralblood.Cell122:303-315 (JohnsonJ, BagleyJ, Skaznik-WikielM, LeeHJ, suppose there is sexual cell and develop into oocyte .Cell122:303-315 in AdamsGB, etal. (2005) Adult female mammal bone marrow and peripheral blood);
11.LeeHJ, SelesniemiK, NiikuraY, NiikuraT, KleinR, etal. (2007) Bonemarrowtransplantationgeneratesimmatureoocytesandresc ueslong-termfertilityinapreclinicalmousemodelofchemother apy-inducedprematureovarianfailure.JClinOncol25:3198-320 4 (LeeHJ, SelesniemiK, NiikuraY, NiikuraT, KleinR, etal. (2007) bone marrow transplantation produces .JClinOncol25:3198-3204 fertility of the clinical front mice that the treatment of immature oocyte causes because of long-term chemotherapeutic treatment premature ovarian failure),
12.SantiquetN, VallieresL, PothierF, SirardMA, RobertC, etal. (2012) Transplantedbonemarrowcellsdonotprovidenewoocytesbutresc uefertilityinfemalemicefollowingtreatmentwithchemotherap euticagents.CellReprogram14:123-129 (SantiquetN, VallieresL, PothierF, SirardMA, RobertC, do not generate new oocyte after the female mice bone marrow cell of etal. (2012) chemotherapeutic treatment but have therapeutic effect .CellReprogram14:123-129 to fertility),
13.MouXZ, LinJ, ChenJY, LiYF, WuXX, etal. (2013) Menstrualblood-derivedmesenchymalstemcellsdifferentiatei ntofunctionalhepatocyte-likecells.JZhejiangUnivSciB14:96 1-972 (MouXZ, LinJ, ChenJY, LiYF, the Derived from Mesenchymal Stem Cells functional hepatocyte-like cells .JZhejiangUnivSciB14:961-972 in WuXX, etal. (2013) menses source);
14.LaiD, WangF, ChenY, WangL, WangY, etal. (2013) Humanamnioticfluidstemcellshaveapotentialtorecoverovaria nfunctioninmicewithchemotherapy-inducedsterility.BMCDevB iol13:34 (LaiD, WangF, ChenY, WangL, WangY, etal. (2013) human amniotic fluid stem cell has the potential .BMCDevBiol13:34 recovering the infertile mouse ovarian function that chemotherapy causes);
15.MisrahiM, BeauI, MeduriG, BouvattierC, AtgerM, etal. (1998) Gonadotropinreceptorsandthecontrolofgonadalsteroidogenes is:physiologyandpathology.BaillieresClinEndocrinolMetab1 2:35-66 (MisrahiM, BeauI, MeduriG, BouvattierC, AtgerM, etal. (1998) gonadotropin receptor and gonadal steroids generate and control: physiology and pathology .BaillieresClinEndocrinolMetab12:35-66);
16.JohnsonJ, CanningJ, KanekoT, PruJK, TillyJL (2004) Germlinestemcellsandfollicularrenewalinthepostnatalmamma lianovary.Nature428:145-150 (JohnsonJ, CanningJ, KanekoT, PruJK, TillyJL (2004) postpartum breastfeeding animal ovary germ-line stem cell and follicle rebuild .Nature428:145-150);
17.ZouK, YuanZ, YangZ, LuoH, SunK, etal. (2009) Productionofoffspringfromagermlinestemcelllinederivedfro mneonatalovaries.NatCellBiol11:631-636 (ZouK, YuanZ, YangZ, LuoH, SunK, etal. (2009) derive from the filial generation .NatCellBiol11:631-636 of newborn ovarian germinal stem line);
18.WhiteYA, WoodsDC, TakaiY, IshiharaO, SekiH, etal. (2012) Oocyteformationbymitoticallyactivegermcellspurifiedfromo variesofreproductive-agewomen.NatMed18:413-421 (WhiteYA, WoodsDC, TakaiY, IshiharaO, the sexual cell that the splitting ability that SekiH, etal. (2012) origin comes from Women of childbearing age ovary is enlivened forms oocyte .NatMed18:413-421);
19.PatelAN, ParkE, KuzmanM, BenettiF, SilvaFJ, etal. (2008) Multipotentmenstrualbloodstromalstemcells:isolation, characterization, anddifferentiation.CellTransplant17:303-311 (PatelAN, ParkE, KuzmanM, BenettiF, SilvaFJ, etal. (2008) multipotency menses mescenchymal stem cell: be separated, phenotypic evaluation and differentiation .CellTransplant17:303-311);
20.HidaN, NishiyamaN, MiyoshiS, KiraS, SegawaK, etal. (2008) Novelcardiacprecursor-likecellsfromhumanmenstrualblood-d erivedmesenchymalcells.StemCells26:1695-1704 (HidaN, NishiyamaN, MiyoshiS, KiraS, the cardiomyocyte precursor like cell .StemCells26:1695-1704 that SegawaK, etal. (2008) people's menses Derived from Mesenchymal Stem Cells comes);
21.BorlonganCV, KanekoY, MakiM, YuSJ, AliM, etal. (2010) Menstrualbloodcellsdisplaystemcell-likephenotypicmarkers andexertneuroprotectionfollowingtransplantationinexperim entalstroke.StemCellsDev19:439-452 (BorlonganCV, KanekoY, MakiM, YuSJ, the menses cells show that AliM, etal. (2010) experiment is transplanted goes out the phenotype molecular marker of stem-like cell and has neuroprotective function .StemCellsDev19:439-452);
22.MurphyMP, WangH, PatelAN, KambhampatiS, AngleN, etal. (2008) Allogeneicendometrialregenerativecells:an " Offtheshelfsolution " forcriticallimbischemia? JTranslMed6:45 (MurphyMP, WangH, PatelAN, KambhampatiS, AngleN, etal. (2008) allogeneic endometrium regenerative cell: a kind of " ready-made solution " of limbs severe ischemia? JTranslMed6:45)
23.IchimTE, AlexandrescuDT, SolanoF, LaraF, CampionRdeN, etal. (2010) Mesenchymalstemcellsasanti-inflammatories:implicationsfo rtreatmentofDuchennemusculardystrophy.CellImmunol260:75-82 (IchimTE, AlexandrescuDT, SolanoF, LaraF, CampionRdeN, etal. (2010) mescenchymal stem cell is as antibiotic medicine: treatment Duchenne's muscular dystrophy .CellImmunol260:75-82);
24.RanganathSH, LevyO, InamdarMS, KarpJM (2012) Harnessingthemesenchymalstemcellsecretomeforthetreatment ofcardiovasculardisease.CellStemCell10:244-258 (RanganathSH, LevyO, InamdarMS, KarpJM (2012) utilize mescenchymal stem cell secretions Cardiovarscular .CellStemCell10:244-258);
25.SchachingerV, ErbsS, ElsasserA, HaberboschW, HambrechtR, etal. (2006) Intracoronarybonemarrow-derivedprogenitorcellsinacutemyo cardialinfarction.NEnglJMed355:1210-1221 (SchachingerV, ErbsS, ElsasserA, HaberboschW, the CFU-GM .NEnglJMed355:1210-1221 of derived from bone marrow in HambrechtR, etal. (2006) acute myocardial infarction arteria coronaria);
26.ParkMR, ChoiYJ, KwonDN, ParkC, BuiHT, etal. (2013) Intraovariantransplantationofprimordialfolliclesfailstor escuechemotherapyinjuredovaries.SciRep3:1384 (ParkMR, ChoiYJ, KwonDN, ParkC, injection primordial follicle treatment chemotherapy damaging ovary failure .SciRep3:1384 in BuiHT, etal. (2013) ovary);
27.OktemO, OktayK (2007) Quantitativeassessmentoftheimpactofchemotherapyonovarian folliclereserveandstromalfunction.Cancer110:2222-2229 (the follicle deposit that OktemO, OktayK (2007) qualitative assessment chemotherapy causes and interstitial function damage .Cancer110:2222-2229);
28.BucciLR, MeistrichML (1987) Effectsofbusulfanonmurinespermatogenesis:cytotoxicity, sterility, spermabnormalities, anddominantlethalmutations.MutatRes176:259-268 (BucciLR, MeistrichML (1987) busulfan is on the impact of mouse sperm: cytotoxicity, sterile, cacospermia and dominant lethal mutation .MutatRes176:259-268);
29.BrinsterRL, ZimmermannJW (1994) Spermatogenesisfollowingmalegerm-celltransplantation.Pro cNatlAcadSciUSA91:11298-11302 (sperm after the transplanting of BrinsterRL, ZimmermannJW (1994) male sex-cell produces .ProcNatlAcadSciUSA91:11298-11302);
30.BrinsterCJ, RyuBY, AvarbockMR, KaragencL, BrinsterRL, etal. (2003) Restorationoffertilitybygermcelltransplantationrequirese ffectiverecipientpreparation.BiolReprod69:412-420 (BrinsterCJ, RyuBY, AvarbockMR, KaragencL, BrinsterRL, etal. (2003) sexual cell transplanting recovers to require that receptor carries out and effectively prepares .BiolReprod69:412-420 fertility).
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of novelty teabag of people's endometrial stem cells----preparing the application in the medicine for the treatment of premature ovarian failure.
In order to solve the problems of the technologies described above, the invention provides the application of a kind of people's endometrial stem cells people endometrial stem cells in the medicine of preparation treatment premature ovarian failure.Application particularly in the cell drug and medical skill of preparation treatment premature ovarian failure.
By we find in research, people's endometrial stem cells is transplanted to chemotherapeutics and causes the recovery helping lend some impetus to its ovarian function in the Mice Body of premature ovarian failure, and the recovery (Fig. 2-3) of Mouse Weight and oestrous cycle can be promoted significantly.In addition, mating test result shows, though fail to reach normal group, all apparently higher than the matched group (Fig. 4) not carrying out cell transplantation at the pregnancy number of cell transplantation group small mouse and farrowing rate.
A large amount of animals and clinical experiment show, mescenchymal stem cell are injected in vivo by the method for pouring into or transplanting the functional rehabilitation contributing to tissue and organ.Such as, damaged tissues can be directly migrated to by the mescenchymal stem cell after intravenous injection or be promoted the reparation [24] of function of organization by biologically active secretory molecule.The mescenchymal stem cell of derived from bone marrow is injected in the Mice Body of coronary artery disease, although the 48th hour after cell transplantation fails donorcells to be detected, myocardial function [25] within 3 weeks, can be significantly improved in treatment.In experiment of the present invention, we also transplanted by bioluminescence imaging technology observation of cell after distribution situation in vivo.2 months after cell transplantation, donor GFP positive cell can migrate to ovary tissue and expresses the peculiar albumen of granular cell (FSHR), therefore, we think that people's endometrial stem cells can be divided into gonad granulocyte and then promote the recovery of ovarian function.
Research display, chemotherapeutics busulphan and cyclophosphamide all can cause the ovary of people and mice long-time, and irreversible damage.The conbined usage of busulphan and cyclophosphamide causes the increase of ovary apoptotic cell quantity and the quick depletion [26,27] of sexual cell.But, but seldom there is research to go to observe the impact of chemotherapeutics on Mouse Weight and hetero-organization organ thereof.Find in our study, the conbined usage of busulphan and cyclophosphamide causes female mice body weight obviously to decline and change (Fig. 2 and Fig. 3) of oestrous cycle.Simultaneously, the method that we are also dyeed by HE observes chemotherapeutics to the impact of other histoorgans, found that the Cardiac Fibroblasts generation edema of the 7th day after chemotherapy, glomerule is congested and atrophy is serious, and liver has in massive inflammatory cells infiltrated and spleen tissue and has a large amount of lymphopoiesis; And after chemotherapy the 14th day, the degree of injury of each histoorgan alleviates (Fig. 3) all to some extent.It is worth mentioning that, the impact performance of chemotherapeutics on ovary is the most serious and irreversible, mainly comprises the loss of ovum, the fibrosis of ovary tissue and infertile [Fig. 3, Fig. 4 C].Above result shows, ovarian function in chemotherapeutics major effect female mice.
Busulphan, as a kind of germline stem cell drug toxicity, is widely studied in the spermatogenesis of male mice.Study its on after the birth of male and female mice in follicle pond the impact of germline stem cell supply also by significant.In testis tissue, busulphan major effect germline stem cell and spermatogonium, and do not affect the sexual cell after mitosis, finally can cause spermatogenesis abnormal [28-30].Research finds, busulphan (30mg/kg) and cyclophosphamide (120mg/kg) single use and can cause the infertile and irreversible loss of germline stem cell of female mice.Recently, have research display can be separated from the ovary tissue of mice and people and obtain germline stem cell, this result shows that ovary has the ability producing new oocyte and follicle.In order to set forth busulphan and the impact of cyclophosphamide combined use on germline stem cell in mouse ovarian, we are by immunofluorescence and observe the distribution situation of germline stem cell to the method for positive cell technology and analyze the number change of germline stem cell in different experiments group.
Generally speaking, result of study of the present invention shows, the transplantation treatment of people's endometrial stem cells contributes to the recovery of mouse ovarian function, this in the future clinical middle chemotherapeutics cause the treatment of chain reaction in patients with premature ovarian to bring new hope.
The clinical treatment being found to be premature ovarian failure of menstrual blood Derived Stem Cells (people's endometrial stem cells) brings new hope, find that it has the potential to various kinds of cell differentiation after deliberation, there is the ability promoting that damaged tissue is repaired in the animal model being implanted into damage.It draws materials conveniently, and multiplication capacity is strong, and can obtain enough cell quantities for transplanting through cultivating, and research finds, in menses, the content of stem cell is about 30 times of derived from bone marrow.This kind of cell stem cell immunogenicity is low, without oncogenicity, all to can yet be regarded as the new seed cell of a regenerative medicine as autotransplantation or donor graft.Therefore, the stem cell (people's endometrial stem cells) in menses source has the ability promoting that ovarian function is repaired, that is, have the effect for the treatment of female infertility.
In order to treat premature ovarian failure, promoting the recovery of ovarian function, people's endometrial stem cells being carried out conventional transplanting; Transplanting mode can refer to the transplanting of current existing bone marrow stem cell.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 behaves the form of endometrial stem cells MP135, phenotype and multipotency analysis chart;
(A) the MP135 cell of In vitro culture has the form of Interstitial cell;
(B) GFP-transfected MP135 cell;
(C) chromosome karyotype analysis display, the 20th generation MP135 cell chromosome caryogram is normal;
(D) flow cytomery MP135 cell can express mesenchymal stem cells MSCs specific marker thing;
(E), in vitro under differentiation-inducing condition, flow cytomery MP135 can be divided into osteocyte, adipose cell and chondrocyte; Scalebars, 200 μm (A, B).
Fig. 2 endometrial stem cells of behaving promotes the recovery situation figure of infertile Mouse Weight and oestrous cycle;
(A) in whole experimentation, the body weight of untreated fish group mice obviously declines, and the body weight of cell transplantation group mice recovers (P < 0.01) gradually from the 4th cycle, but with Normal group no significant difference (P > 0.05);
(B) different experiments small mouse proestrus, rutting period, metoestrus and diestrus account for the percentage analysis of oestrous cycle. from after cell transplantation the 4th week, the oestrous cycle of cell transplantation group small mouse is tending towards Normal group mice gradually, and the mice of untreated fish group then mainly maintains diestrus.
In figure, DE represents diestrus, and ME represents metoestrus, and E represents rutting period, and PE represents proestrus.
Fig. 3 is that people's endometrial stem cells transplants latter 2nd, 4,8 weeks, different experiments group small mouse oestrous cycle vaginal exfoliated cell smear observed result.
As shown in the figure: in the vaginal smear result of the mice of cell transplantation group, can be observed typical superficial cell (black arrow) and fern cell (white arrow), unprocessed control animals is then without significant change.
Fig. 4 is the recovery situation of infertile mice fertility after people's endometrial stem cells is transplanted, and carries out evaluation map by the cycle of the continuous copulation of assessment different experiments group small mouse to its fertility;
A figure is the farrowing situation of Normal group, untreated fish group and people's endometrial stem cells transplantation group small mouse; Arrow is the stillborn fetus in untreated fish group;
B figure is the average farrowing amount (mean+/-standard error) of pregnant mouse. every treated animal n=10.**P<0.001;
C figure is after mating test terminates, ovarian sections haematoxylin and eosin stains (H & E) result, non-treatment group ovary shows as fibrosis formation, without follicle structure, people's endometrial stem cells transplantation group ovary and normal group similar, have Follicles to exist.Scalebars,100um。
Fig. 5 is the tracer analysis of people's endometrial stem cells in infertile Mice Body:
A figure is the people endometrial stem cells distribution situation Mice Body in of tail vein injection with GFP labelling of bioluminescence imaging technology observation; Observed result is presented at the 6-12 hour after cell transplantation, and first GFP positive cell enters in pelvic tissue; Migrated in thymic tissue gradually at the 24th hour; And the 48th hour after the transfer, GFP fluorescence signal dies down gradually; After cell transplantation the 7th day, only a small amount of GFP positive signal can be detected in pelvic tissue;
B figure is the expression on the bean vesicular ovarian follicles of human specific nuclear antigen (Hu) in the ovary tissue of mice, finds the positive cell that can be observed GFP and Hu double labelling in the ovary tissue of 2 months after cell transplantation;
C figure is after cell transplantation 2 months, the antral follicle count in ovary tissue can be observed the positive cell .Scalebars:100 μm (B, C) of GFP and FSHR double labelling, 10 μm (B, Cinsets).
Fig. 6 is that people's endometrial stem cells transplants the multiplication capacity situation improving infertile mouse ovarian germline stem cell.
A figure is in different experiments group, and the two target positive cell of BrdU and MVH mainly expresses .Scalebars:50 μm in the epidermal tissue of mouse ovarian; Insets, 10 μm;
B figure is the count results analysis display of different experiments group small mouse ovarian germinal stem cell, result shows that chemotherapy decreases germline stem cell quantity, and people's endometrial stem cells transplants the reparation (mean value ± standard error, every treated animal n=5) facilitating germline stem cell.
Detailed description of the invention
Embodiment 1, people's endometrial stem cells are identified
Cell derived and cultivation
People's endometrial stem cells system MP135 derives from the menstrual blood of 40 years old Chinese women, and MP135 the 3rd generation cell is provided by Zhejiang University professor Xiang Chunsheng, and the use of human cell obtains the approval of Ethics Committee of attached international peace maternity & child care center of Shanghai Communications University.
MP135 cell grows [13] in the stem cell media containing endometrium/menstruation, is positioned over containing 5%CO
2in 37 DEG C of incubators.Cell is through trypsinization, and Secondary Culture, carries out subsequent experimental when cell confluency reaches 80-90%.
Flow cytometry
Flow cytometer is utilized to analyze MP135 cell surface specific marker thing CD29, CD90, CD34, CD45, CD117, HLA-DR and OCT4.Specific as follows:
(concentration is made to be 1 × 10 by resuspended for MP135 cell
6cells/ml) in the PBS containing 2% bovine serum albumin, add the mouse anti human CD90 of PE labelling, CD29 and CD34 antibody respectively, FITC labelling mouse anti human CD45, OCT4 antibody, use FC500 flow cytometer and CoulterCXP software to analyze.
People's endometrial stem cells differentiation-inducing
By the Function Identification test kit induction people endometrial stem cells differentiation of human marrow mesenchymal stem cell.Osteocyte and chondrocyte are respectively by rabbit anti-human osteocalcin polyclonal antibody and rabbit anti-human collagen protein II antigen Anti-TNF-α body tag.The oil red O stain of frozen section is lipid dyeing.
Result is as follows:
People's endometrial stem cells (MP135) grows rapidly in vitro, and morphological category is similar to fibroblast-like cells (Figure 1A-B).Chromosome karyotype analysis result shows, the cell in the 20th generation of going down to posterity is diploid cell, does not have the generation (Fig. 1 C) of chromosomal aberration.We adopt flow cytometry technique to detect the phenotype of the 5th generation MP135 cell, and result shows, and the cells show of 99.1% goes out the CD29 positive, and 99.4% cells show CD90 is positive, but does not express CD34 and CD45.People's endometrial stem cells does not express HLA-DR antigen.Meanwhile, 18.9% cellular expression OCT4,10.9% cellular expression CD117 (Fig. 1 E).In addition, people's endometrial stem cells also has multi-lineage potential, can be induced to differentiate into the various kinds of cell such as adipose cell, chondroblast and osteocyte.Above result of study shows that people's endometrial stem cells has the characteristic of mescenchymal stem cell.
Remarks illustrate: Fig. 1 is endometrial stem cells morphology, phenotype and versatility.(A) and the endometrial stem cells of (B) In vitro culture there is the feature of mescenchymal stem cell; (C) chromosome analysis still keeps normal karyotype after display endometrial stem cells subculture in vitro separately 20 generation; (D) flow cytometry endometrial stem cells expression mescenchymal stem cell shows mark; (E) endometrial stem cells differentiation-inducing fat, cartilage and osteocyte in vitro.
Embodiment 2, people's endometrial stem cells transplant the recovery that can promote Mouse Weight and oestrous cycle
Zoopery
1. choose C57BL/6 wild females mice in 6 week age (60), set up premature ovarian failure animal model by lumbar injection busulfan (30mg/kg) and cyclophosphamide (120mg/kg);
2. choose age-matched female mice lumbar injection DMSO equally as experiment contrast group (10);
3., latter 1 week of chemotherapeutics injection, make Animal Anesthesia by lumbar injection pentobarbital sodium (45mg/kg);
4. by (2 × 10
6cells) people's endometrial stem cells injects in Mice Body through caudal vein;
5. matched group gives the cell training liquid of Isodose according to the same manner;
6., after people's endometrial stem cells transplants 1 week, vaginal smear method observes the oestrous cycle change of mice, body weight change;
7. people's endometrial stem cells transplant after the 4th and Animal Anesthesia was put to death in 8 weeks, collect ovary tissue preparation section, carry out h and E dyeing.
8. the mice choosing equal number in cell processed group and untreated fish group respectively after chemotherapy carries out the mating test of 1 month by a definite date, and carries out record to every nest yield number.
Remarks illustrate: all zooperies are all carried out in the health care of Shanghai Institutional Animal with under using committee's approval.That is, in order to whether further analyst's endometrial stem cells can promote the recovery of ovarian function effectively, the animal model of the method inducing ovarian senilism that we adopt above-mentioned chemotherapeutics busulfan commissural arch phosphamide to use; This kind of method can destroy most sexual cell in ovary.
Specific as follows:
People's endometrial stem cells is transplanted, and body weight is assessed, vaginal smear and mating test
Latter 1 week of above-mentioned chemotherapeutics injection, make Animal Anesthesia by lumbar injection pentobarbital sodium (45mg/kg), volume is about the cell suspension (2 × 10 that 6 μ L contain people's endometrial stem cells
6cells) inject in Mice Body (20) through caudal vein, and matched group gives cell training liquid (20) of Isodose according to the same manner.
After people's endometrial stem cells transplants 1 week, every morning 9:00 observes Normal group by vaginal smear method, the change of the oestrous cycle of cellular transplantation therapy group and non-treatment group mice.And in whole process, record the change of Mouse Weight.
When people's endometrial stem cells grows into 85% density, the slow virus infection object cell (Figure 1A-B) of green fluorescent protein (GFP) labelling will be carried.
After chemotherapeutics uses the 7th day, by the mode of tail vein injection by the people's endometrial stem cells (about 2 × 10 with GFP labelling
6cells) inject in Mice Body, the death caused because of transplanting does not occur in the mice transplanted.
The 4th week after cell transplantation starts, and the recovery of cell transplantation group small mouse body weight is apparently higher than matched group (P<0.01, Fig. 2 A).In whole experimentation, the method for vaginal smear is adopted to observe the change of Mouse Oestrous Cycle (mainly comprising diestrus, proestrus, rutting period and metoestrus).Result shows, start to change in the oestrous cycle of cell transplantation vaginal smear display after 1 week, in about the 4th week cell transplantation group, the change of female mice oestrous cycle is tending towards the change of Normal group gradually, and the change of the oestrous cycle of chemotherapy group mice and fail picture Normal group, its main manifestations is more grow or shorter rutting period, diestrus (Fig. 2 B, Fig. 3) is mainly maintained in whole experimentation.Above result shows that the transplantation treatment of people's endometrial stem cells can promote the recovery of Mouse Weight and ovarian function significantly.
Remarks illustrate:
Fig. 2 be endometrial stem cells transplant infertile mice can make Mouse Weight increase and recover the estrus cycle.(A) curve above is normal mouse, and centre is treatment group mice, is untreated infertile mice below.(B) different experiments group is in different treatment time diestruss, metoestrus, rutting period, the time interval ratio of proestrus.Endometrial stem cells is transplanted infertile mice and is recovered normal after 4 weeks.
Fig. 3 is that after vaginal exfoliated cell smear and cervical mucus crystalization method display endometrial stem cells are transplanted, infertile mice recovers the estrus cycle.
As described in Figure 2:
A is the recovery that people's endometrial stem cells promotes infertile Mouse Weight and oestrous cycle;
In whole experimentation, the body weight of untreated fish group mice obviously declines; And the body weight of cell transplantation group mice recovers (P < 0.01) gradually from the 4th cycle, but with Normal group no significant difference (P > 0.05);
B is different experiments small mouse proestrus, and in rutting period, metoestrus and diestrus account for the percentage analysis of oestrous cycle;
From after cell transplantation the 4th week, the oestrous cycle of cell transplantation group small mouse was tending towards Normal group mice gradually, and the mice of untreated fish group then mainly maintains diestrus.
As shown in Figure 3:
In the vaginal smear result of the mice of cell transplantation group, can be observed typical superficial cell (black arrow) and fern cell (white arrow), unprocessed control animals is then without significant change.
Embodiment 3, people's endometrial stem cells transplant the fertility can recovering mice
In order to study the impact of people's endometrial stem cells transplanting on mice fertility, within the 8th week after cell transplantation, start female mice in different groups and Normal male mice to match the mating test carrying out 3 months by a definite date.The pregnancy number of different group small mouse and farrowing quantity are carried out statistical analysis.Result shows, and the use of chemotherapeutics obviously reduces the fertility of mice (comprising pregnant frequency and average farrowing amount).All have three successfully gestation generations the mice of cell transplantation group and Normal group, but only there is twice gestation in the animal in chemotherapy group, comprises stillborn fetus (Fig. 4 A).Although the average matched group (3.47versus0.3 that uses apparently higher than chemotherapeutics of the farrowing amount of every female mice in cell transplantation group, P<0.001, Fig. 4 B), but its farrowing amount does not reach the farrowing quantity (3.47versus7.4 of Normal group, P<0.001, Fig. 4 B).
After completing animal mating experiment, the ovarian sections that we choose middle tangent plane carries out histologic analysis, found that in chemotherapeutics processed group, ovarian size is significantly less than matched group and cell transplantation group (Fig. 4 C).In addition, in the ovary of cell transplantation group, have the follicle being in growth different times in a large number similar to Normal group, and the ovary of chemotherapy group only there is a large amount of Interstitial cell not have follicle appearance (Fig. 4 C).Above result shows that the transplanting of people's endometrial stem cells can promote the recovery of mice fertility.
Remarks illustrate: Fig. 4 is the infertility of endometrial stem cells transplantation treatment mice.(A) newborn mice, the left side is from normal mouse copulation, and the middle mice not having to transplant, the right side is the mice that endometrial stem cells is transplanted.(B) every pregnant Mus litter size, the above is normal mouse, and centre is the mice that endometrial stem cells is transplanted, and does not transplant the mice of endometrial stem cells below.(C) ovary sectional drawing, the left side is normal mouse, and centre is the mice not having endometrial stem cells to transplant, and the mice of endometrial stem cells is transplanted on the right side.
Fig. 4 endometrial stem cells of behaving transplants the recovery contributing to infertile mice fertility; By the cycle of the continuous copulation of assessment different experiments group small mouse, its fertility is evaluated; (A) picture is shown as Normal group, the farrowing situation of untreated fish group and people's endometrial stem cells transplantation group small mouse. and arrow is the stillborn fetus in untreated fish group; (B) the average farrowing amount (mean+/-standard error) of pregnant mouse. every treated animal n=10.**P<0.001; (C) after mating test terminates, ovarian sections haematoxylin and eosin stains (H & E) result, non-treatment group ovary shows as fibrosis formation, without follicle structure; People's endometrial stem cells transplantation group ovary and normal group similar, have Follicles to exist.Scalebars,100um。
Embodiment 4, people's endometrial stem cells are divided into granular cell and promote ovarian function reparation
Since people's endometrial stem cells can promote the recovery of ovarian function; So we suppose that people's endometrial stem cells may be play a significant role by moving and being incorporated in the ovary tissue of damage.In order to prove this guess, we adopt the method for living imaging to observe from 6 after cell transplantation hour by 14 days, the Expression and distribution situation of GFP fluorescence signal in Mice Body.Result shows, and first the 6-12 hour after cell transplantation, GFP positive cell enters in pelvic tissue, moves in thymic tissue at 24 hour cells.48 hours GFP signals after the transfer die down gradually, and within after the transfer the 7th day, only a small amount of in pelvic tissue signal can be detected, and in other two groups of matched groups not fluorescence signal be detected (Fig. 5 A).
Although from the 7th after cell transplantation day, bioluminescence imaging technology can not detect the positive signal of GFP, we adopt the distribution of people's endometrial stem cells in the method for immunofluorescence further spike receptor ovary tissue.Immunofluorescence results shows, 2 weeks after cell transplantation, the GFP positive cell partly in the interstitial of ovary tissue can be observed arrive.In order to confirm in receptor ovary, whether GFP positive cell is the people's endometrial stem cells coming from donor; After cell transplantation 2 months, We conducted GFP and human specific nuclear antigen (Hu) immunofluorescence double staining, in the interstitial of ovary, can be observed GFP
+-Hu
+double-labeled cell (Figure5B).
As everyone knows, gonad granulocyte plays vital effect for the growth of women's ovary and the maturation of follicle.In order to analyze the characteristic implanting cell further, we select a kind of glycoprotein hormone receptor, and namely Human Fallicle-Stimulating Hormone's receptor (FSHR) identifies gonad granulocyte [15].Immunofluorescence results shows, and 2 months after cell transplantation, in ovary tissue, can be observed FSHR
+-GFP
+two target cell (Figure5C).Above result shows, people's endometrial stem cells that a part is implanted in the damage ovary of induced by chemotherapeutic agents can be divided into gonad granulocyte and play a role.
Specifically as described in Figure 5:
Fig. 5. the tracer analysis of people's endometrial stem cells in infertile Mice Body (A) observes the people endometrial stem cells distribution situation Mice Body in of tail vein injection with GFP labelling by bioluminescence imaging technology. the 6-12 hour after cell transplantation, first GFP positive cell enters in pelvic tissue, migrated in thymic tissue gradually at the 24th hour, and the 48th hour after the transfer, GFP fluorescence signal dies down gradually, after cell transplantation the 7th day, only can detect on the bean vesicular ovarian follicles that a small amount of GFP positive signal (B) human specific nuclear antigen (Hu) is mainly expressed in the ovary tissue of mice in pelvic tissue, and can be observed the positive cell of GFP and Hu double labelling in the ovary tissue of 2 months after cell transplantation. (C) after cell transplantation 2 months, antral follicle count in ovary tissue can be observed the positive cell .Scalebars:100 μm (B of GFP and FSHR double labelling, C), 10 μm of (B, Cinsets).
Embodiment 5, people's endometrial stem cells are transplanted and are improved mouse propagation stem cell updating ability
In order to identify and confirm the existence of germline stem cell in ovary tissue (GSCs), we adopt the method for generally acknowledged BrdU and MVH immunofluorescence double staining to observe expression and the quantity situation [16-18] of entovarial germline stem cell.Each treated animal is 1 hr collections ovary tissue after giving BrdU lumbar injection, and immunofluorescence double staining is carried out in preparation section.
Immunofluorescence results shows, and in ovary tissue, the cell of the MVH positive only expresses in sexual cell (SupplementFig.1).In addition, all can be observed BrdU in the Ovarian surface epithelium in different experiments group
+– MVH
+double-labeled cell (Figure6A).In Normal group, 78 germline stem cells (GSCs) are about had in the average each ovary of female mice in 8 week age, and dropping to 56 gradually to the quantity of the 15th week germline stem cell, this result is pointed out, and in ovary, the storage level of germline stem cell reduces gradually under normal circumstances.But in chemotherapy group, after chemotherapeutics uses, the quantity of germline stem cell of 2 weeks declines 35.8% (28/78), and to the 8th week ovary early in germline stem cell almost do not observe.By comparison, in cell transplantation group, the quantity of germline stem cell slightly increases, and as after cell transplantation the 8th week, in cell transplantation group, the quantity of germline stem cell was about 78.3% (39/49.8) (Figure5B).Above result shows, people's endometrial stem cells transplants the exhaustion contributing to reducing germline stem cell in the ovary that causes of chemotherapeutics.
Specifically as shown in Figure 6:
Fig. 6 endometrial stem cells of behaving is transplanted and is improved the multiplication capacity of infertile mouse ovarian germline stem cell. and (A), in different experiments group, BrdU and MVH pair of target positive cell mainly expresses .Scalebars:50 μm in the epidermal tissue of mouse ovarian; Insets, 10 μm. the count results analysis display of (B) different experiments group small mouse ovarian germinal stem cell, chemotherapy decreases germline stem cell quantity, and people's endometrial stem cells transplants the reparation (mean value ± standard error, every treated animal n=5) facilitating germline stem cell.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (2)
1. the application of people's endometrial stem cells in the medicine of preparation treatment premature ovarian failure.
2. the application of people's endometrial stem cells according to claim 1 in the medicine of preparation treatment premature ovarian failure, is characterized in that: be the application in the cell drug and/or medical skill of preparation treatment premature ovarian failure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510079968.2A CN105055450A (en) | 2015-02-13 | 2015-02-13 | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510079968.2A CN105055450A (en) | 2015-02-13 | 2015-02-13 | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105055450A true CN105055450A (en) | 2015-11-18 |
Family
ID=54485132
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510079968.2A Withdrawn CN105055450A (en) | 2015-02-13 | 2015-02-13 | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105055450A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106913584A (en) * | 2017-04-27 | 2017-07-04 | 广州资生生物科技有限公司 | A kind of Endometrial stem cell preparation and its application |
| CN107080754A (en) * | 2017-04-27 | 2017-08-22 | 广州资生生物科技有限公司 | A kind of Endometrial stem cell preparation and its application |
| CN110123841A (en) * | 2018-02-09 | 2019-08-16 | 上海市第六人民医院 | Load purposes of the human pluripotent stem cells excretion body in preparation treatment genital system diseases drug of resveratrol |
| CN111107858A (en) * | 2017-06-19 | 2020-05-05 | 美商生命科学公司 | Treatment of sexual dysfunction and improvement of quality of sexual life |
| CN113633754A (en) * | 2021-09-28 | 2021-11-12 | 北京远胜达生物科技发展有限公司 | Application of ovarian stem cells in preparation of ovarian anti-aging drugs |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374760A (en) * | 2012-04-19 | 2013-10-30 | 孙勇 | Construction of human endometrium (menstrual blood) stem cell bank |
-
2015
- 2015-02-13 CN CN201510079968.2A patent/CN105055450A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374760A (en) * | 2012-04-19 | 2013-10-30 | 孙勇 | Construction of human endometrium (menstrual blood) stem cell bank |
Non-Patent Citations (3)
| Title |
|---|
| TE LIU ET AL.: "transplantation of human menstrual blood stem cells to treat premature ovatian failure in mouse model", 《STEM CELLS AND DEVELOPMENT》 * |
| 丁晓静: "子宫内膜肝细胞的研究进展", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 经济参考报: "子宫内膜再生细胞治疗卵巢早衰", 《经济参考报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106913584A (en) * | 2017-04-27 | 2017-07-04 | 广州资生生物科技有限公司 | A kind of Endometrial stem cell preparation and its application |
| CN107080754A (en) * | 2017-04-27 | 2017-08-22 | 广州资生生物科技有限公司 | A kind of Endometrial stem cell preparation and its application |
| CN111107858A (en) * | 2017-06-19 | 2020-05-05 | 美商生命科学公司 | Treatment of sexual dysfunction and improvement of quality of sexual life |
| CN110123841A (en) * | 2018-02-09 | 2019-08-16 | 上海市第六人民医院 | Load purposes of the human pluripotent stem cells excretion body in preparation treatment genital system diseases drug of resveratrol |
| CN113633754A (en) * | 2021-09-28 | 2021-11-12 | 北京远胜达生物科技发展有限公司 | Application of ovarian stem cells in preparation of ovarian anti-aging drugs |
| CN113633754B (en) * | 2021-09-28 | 2022-08-30 | 上海揽微赛尔生物科技有限公司 | Application of ovarian stem cells in preparation of ovarian anti-aging drugs |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3517118B1 (en) | Sperm activator and uses thereof | |
| Abd-Allah et al. | Mechanistic action of mesenchymal stem cell injection in the treatment of chemically induced ovarian failure in rabbits | |
| Okazaki et al. | Seminal plasma damages sperm during cryopreservation, but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality | |
| Forcada et al. | Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep | |
| JP4749331B2 (en) | Cell differentiation of adipose-derived progenitor cells | |
| Aubard | Ovarian tissue xenografting | |
| CN105055450A (en) | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment | |
| JPH06508527A (en) | Hormone-secreting cells maintained in long-term culture | |
| JP6241819B2 (en) | Sperm activation method and use thereof | |
| KR20080005563A (en) | N-acetylcysteine amide(nac amide) for treatment of oxidative stress associated with infertility | |
| WO2014053418A9 (en) | Method for obtaining mesenchymal stem cells and use thereof | |
| CN109481466A (en) | Use the method and cell preparation of placenta mesenchyma stem cell treatment premature ovarian failure | |
| Rodriguez-Dorta et al. | Effect of coculture with oviduct epithelial cells on viability after transfer of vitrified in vitro produced goat embryos | |
| CN109652366A (en) | For treating the placenta mesenchyma stem cell preparation of premature ovarian failure | |
| WO2001034775A1 (en) | Mesenchymal stem cells and/or progenitor cells, their isolation and use | |
| Trasorras et al. | Production, preservation, and transfer of South American camelid embryos | |
| von Schönfeldt et al. | Advanced follicle development in xenografted prepubertal ovarian tissue: the common marmoset as a nonhuman primate model for ovarian tissue transplantation | |
| EP3680322A2 (en) | Stem cell-derived sertoli-like cell, preparation method therefor, and use thereof | |
| Kurotaki et al. | Practical reproductive techniques for the common marmoset | |
| CN101287826A (en) | Compositions and methods for culturing embryos and oocytes | |
| CN110732017B (en) | Use of SP in preparing medicine for treating male sterility | |
| Van Saen et al. | Bone marrow stem cells transplanted to the testis of sterile mice do not differentiate into spermatogonial stem cells and have no protective effect on fertility | |
| Tahmasbpour Marzouni et al. | Stem cells and organs-on-chips: new promising technologies for human infertility treatment | |
| JP7257012B1 (en) | Egg maturation promoter and use thereof | |
| CN112057471B (en) | Pharmaceutical application of mesenchymal stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20151118 |