CN102771471A - Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof - Google Patents
Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof Download PDFInfo
- Publication number
- CN102771471A CN102771471A CN2012101935712A CN201210193571A CN102771471A CN 102771471 A CN102771471 A CN 102771471A CN 2012101935712 A CN2012101935712 A CN 2012101935712A CN 201210193571 A CN201210193571 A CN 201210193571A CN 102771471 A CN102771471 A CN 102771471A
- Authority
- CN
- China
- Prior art keywords
- ovary
- ovarian
- cryopreservation
- protection liquid
- big
- 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.)
- Pending
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to an ovarian large cortex piece vitrified cryopreservation protection liquid and a cryopreservation method thereof. Although the vitrified cryopreservation method is widely applied to experimental animals, a universal cryopreservation method and a standard cryopreservation liquid preparation scheme are absent at present, so that the sized transplantation subjected to vitrified cryopreservation is at a small-size stage, an ovarian small cortex piece has limited follicles, and the service life of the transplanted follicles is influenced. The invention provides vitrified cryopreservation protection liquid suitable for an ovarian large cortex piece, and gonadotropin is added on the basis of the common vitrified liquid. According to the vitrified cryopreservation protection liquid suitable for the ovarian large cortex piece, especially the ovaries of experimental animals, improved variety livestock and wild endangered species in sudden death are conveniently cryopreserved at the most proper osmotic equilibrium time of performing vitrified cryopreservation on the ovarian large cortex pieces of different sizes; and moreover, according to the method, the survival rate of the follicles can be improved, the blood reperfusion time of the transplant is shortened, and the survival rate of the cryopreserved ovarian transplantation in vivo is improved.
Description
Technical field
The present invention relates to the technical field of the ultralow temperature preservation of cell, tissue and organ, be specifically related to a kind of ovary big cortex slice glassivation frozen solution and freezing method.
Background technology
Glass freezing is the new technology that the supper-fast ultralow temperature that at first put forward by Rall and Fahy in 1985 is preserved; Not only method of operating is easy; And it is frozen effective; Can better preserve the biologically active of lived cell, tissue and organ, have economy, easy and advantage efficiently, be one of cryobiology field research direction with fastest developing speed in recent years.
Cancer patient's ovary is carried out frozen before carcinosis radiotherapy and chemotherapy, after the cancer control, in frozen ovarian transplantation ex vivo, recover the endocrine function and the fecundity of patient's ovary to a certain extent.In recent years many scholars have reported that in succession mouse, rat, sheep, monkey and people's ovary tissue glass freezing success is (referring to document: Kagabu S Umezu M.Transplantation of Cryopreserved mouse; Chinese hamster, rabbit, Japanese monkey and rat ovaries into rat recipients [J] .Exp Anim; 2000; 49 (1): 17-21.Alaghbari AM, Jr Menino AR.Survival of oocytes recovered from vitrified sheep ovarian tissues [J] .Annimal Reprod Science, 2002; 71 (1-2): 101-110.Lee KA; Lee SH, Yoon SJ, et al.Resumption of the human primordial follicle growth in xenografts after vitrification of the ovarian tissue [J] .Fertil Steril; 2000,74 (3): S214.).Though vitrifying freeze process has been widely used in the laboratory animal, go back the freezing liquid allocation plan of general freezing method of neither one and standard at present, cause the volume transplanting of glass frozen preservation to be paced up and down in the small size stage.(1mm * 2mm * 2mm) contained ovarian follicle is limited but owing to ovary cuticle matter sheet; Because holding time physiological reproductive endocrine state, the consumption of ovarian follicle lacks (2～3 years) after transplanting in the ex vivo; Simultaneously, can destroy the minimizing that too much cause ovarian follicle quantity because of ovarian follicle during cutting.Big cortex sheet (80mm
3More than) transplant because of containing the primordial follicle enormous amount, with the functional lifetime that obviously prolongs graft, at present; Largest cortical piece area is 15mm * 5mm, and thickness is the 1-2mm size, and all adopts the protectant freezing preservation method at a slow speed of low concentration (referring to document: J Donnez; MM Dolmans, D Demylle, P Jadoul; C Pirard, J Squifflet, B Martinez-Madrid; And A vanLangendonckt.Livebirth after orthotopic transplantation of cryopreserved ovarian tissue..2004 .Lancet, Oct 2004; 364 (9443): 1405-10; Dror Meirow; Jacob Levron, Talia Eldar-Geva et al.Pregnancy after Transplantation of Cryopreserved Ovarian Tissue in a Patient with Ovarian Failure after Chemotherapy.N Engl J Med2005; 353; 318-321.), the slow freezing method is that to adopt the frozen cooling appearance to be undertaken by the program that is provided with freezing, and the refrigerating process time is long, and it is many to consume liquid nitrogen, and needs special instrument, and common laboratory is difficult for enforcement.
The glass freezing of the big cortex sheet of ovary is focus and the difficult point that recent research person pays close attention to; Concrete operation method faces following challenge: piece of tissue too senior general prolongs the time of penetration of cortex sheet in vetrifying solution; Though can provide time enough to make protectant get into middle section; But fringe region just will be exposed to the overlong time in the high concentration protectant, can produce the cytotoxicity damage.If time of penetration is shorter, then cause the cryoprotector at cortex sheet center can not reach valid density.Therefore how to reduce to greatest extent under the Cytotoxic condition, guaranteeing that the protectant infiltration of middle section is to guarantee the freezing successful key of the big cortex slice glassivation of ovary.
Still there are not the ovary of being applicable to big cortex slice glassivation frozen solution and freezing method thereof at present.
Summary of the invention
The object of the present invention is to provide a kind of glass freezing protection liquid that is applicable to the big cortex sheet of ovary, another object of the present invention provides a kind of vitrification method that is applicable to the big cortex sheet of ovary.
Technical problem to be solved by this invention mainly is that the frozen infiltration and the time of appearing owing to protection liquid of big cortex sheet is long, and the toxicity time of the cryoprotective agent that stands is also long, possibly face bigger toxic damages.Simultaneously, big cortex sheet does not have vascular anastomosis to be transplanted and will face the hypoxic-ischemic damage owing to blood flow can not in time arrive, and can lose basis---the primordial follicle that egg mother cell and female sex hormone produce.Therefore, solve above technical problem, at first must have a kind of apoptosis that produces in the frozen process of can resisting to promote freezing back to transplant the glass freezing protection liquid that ovary is replied blood flow fast again.
The invention provides a kind of glass frozen preservation protection liquid that is applicable to the big cortex sheet of ovary, this glass frozen preservation protection liquid is that gonadotropin is joined in the frozen use liquid as anti-apoptosis agent,
Used glass freezing protection liquid is: EG5.5/30 liquid, this protection liquid is made up of 5.5mol/L ethylene glycol (EG)+30% ficoll+0.5mol/L sucrose, has added gonadotropin (gonadotropins) on this basis.
Described gonadotropin can be HMG (HMG), lutropin (LH) or short follicle-stimulating hormone (FSH).Preferred short follicle-stimulating hormone (FSH).
Add 0.2IU/mL～0.6IU/mL FSH, LH or HMG (HMG) among glass freezing protection liquid: the vetrifying solution EG5.5/30 (5.5mol/L EG+30% ficoll+0.5mol/L sucrose vetrifying solution) of the big cortex sheet of adult mice ovary organ, concentration is 0.3IU/mLFSH best.
Adding the follicle-stimulating hormone (FSH) (FSH) of 5 μ g/mL～20 μ g/mL among glass freezing protection liquid: the vetrifying solution EG5.5/30 (5.5mol/LEG+30% ficoll+0.5mol/L sucrose vetrifying solution) of the big cortex sheet of sheep ovary, is the FSH of 10 μ g/mL best.
The glass freezing protection liquid that is applicable to the big cortex sheet of ovary provided by the invention not only can be resisted the apoptosis that ovary tissue produces in frozen, and promote transplanting afterwards blood flow shift to an earlier date 12-24h at infusion time.Utilize the glass frozen preservation of this glass freezing protection liquid to adult mice ovary organ, the big cortex sheet of sheep; Frozen effective; Can better preserve the biologically active of lived cell, tissue and organ; And the big cortex sheet of frozen ovary is transplanted in the ex vivo, and endocrine function, reproductive function isoreactivity are not affected.
Use glass freezing protection liquid of the present invention, the big cortex sheet of frozen ovary, the volume of the big cortex sheet of ovary is 80mm
3More than, generally at 80mm
3-200mm
3Between, thickness≤2mm.
The present invention also provides a kind of vitrification method that is applicable to the big cortex sheet of ovary, and this vitrification method relates generally to the big cortex sheet of ovary volume glass frozen preservation osmotic equilibrium Best Times control technology.
Further; The present invention finds out the rule according to the freezing tissue area size of the big cortex sheet of ovary osmotic equilibrium time of confirming it in glass freezing protection liquid, and the grasp of the glass freezing time of convenient big cortex sheet ovary tissue has also guaranteed frozen effect.
The inventor by volume is divided into sample (40mm with the sheep ovary cortex sample at 2～6 monthly ages
3=5mm * 4mm * 2mm), middle appearance (80mm
3=10mm * 4mm * 2mm), full-page proof (160mm
3=10mm * 8mm * 2mm), adopt two step osmotic equilibrium methods to carry out glass frozen preservation, overall process is carried out under room temperature (22～24 ℃).
1. preparatory osmotic equilibrium: 1.5mol/L ethylene glycol (EG)+20% calf serum (ICS), sample: 15min, middle appearance: 20min, full-page proof: 25min;
2. vitrifying osmotic equilibrium: EG 5.5/30 liquid (5.5mol/L EG+30% ficoll+0.5mol/L sucrose vetrifying solution), sample: 4min～8min, middle appearance: 10min～13min, full-page proof: 17min～20min;
3. directly drop into liquid nitrogen.
After thawing; Judgement to the glass frozen preservation result: to the ovary after thawing carry out ordinary optical microscope, and the relevant immunohistochemistry of apoptosis detect three kinds of different volumes the ovary cortex piece in glass freezing liquid after the osmotic equilibrium that suits, carry out glass frozen preservation.The infiltration of all vetrifying solutions is not good enough, glass frozen preservation thaw the coming off of back histology performance follicle cell, arrangement disorder; Slight crack, egg mother cell distortion etc. appear in the egg mother cell endochylema.This is that the EG of infiltration in follicle cell and the egg mother cell does not reach vitrifying concentration, and making freezingly has ice crystal to form inside and outside the cell when thawing, and causes the mechanical injuries of cell and makes a large amount of ovarian follicle structural deteriorations.Time of penetration is long, then shows as the cell nucleus that a large amount of pyknosis engrains appears in primordial follicle, dehydration is described excessively, and shrinkage is dead.The osmotic equilibrium 7min group that the small size group is best, middle volume are the 11min group, and big volume is the 19min group.Identical with observed result to normal ovarian follicle to apoptosis result's judgement, the ovarian follicle of apoptosis is minimum in the best frozen ovary tissue of osmotic equilibrium time, and no difference of science of statistics between fresh ovarian group.
The present invention sums up under the controlled condition of 22～24 ℃ of scopes and thickness≤2mm from experimental result, suitable osmotic equilibrium time T (min) and maximum area S (mm in the vetrifying solution
2) there is such relation: T=1/5 S+3.
The present invention also provides a kind of vitrification method that is applicable to the big cortex sheet of ovary, and this method is specially:
Glass freezing protection liquid used during the vitrifying osmotic equilibrium is: added gonadotropin on the basis of vetrifying solution EG5.5/30 (5.5mol/L EG+30% ficoll+0.5mol/L sucrose vetrifying solution) commonly used;
Osmotic equilibrium time T (min) and the big cortex sheet of ovary area S (mm
2) relation be T=1/5 S+3; The osmotic equilibrium temperature is 22～24 ℃;
The big cortex sheet of described ovary volume is 80mm
3More than, thickness≤2mm.
Use the EG5.5/30 glass freezing of above-mentioned interpolation gonadotropin to protect on the basis of liquid; Find out rule according to the freezing tissue area size of the big cortex sheet of ovary osmotic equilibrium time of confirming it in glass freezing protection liquid; In conjunction with the osmotic equilibrium formula, can make things convenient for, the small animal ovary organ and the big ovary cortex tissue of glass frozen preservation different volumes effectively.
Glass freezing protection liquid of the present invention and vitrification method are specially adapted to the big cortex sheet of ovary; The optimum osmotic equilibrium time with the glass frozen preservation ovary is provided, has made things convenient for wild endangered species ovary frozen of laboratory animal, the big domestic animal of breeding, die by visitation of God; Through use adding the EG5.5/30 vetrifying solution of gonadotropin, can improve the ovarian follicle survival, shorten the time that the blood flow of graft pours into again, improve the survival rate of frozen big cortex sheet.
Description of drawings
Fig. 1: to the big cortex sheet of the ovary after thawing ordinary optical microscope figure
A: sheep ovary 40mm
3Cortex sheet (sample) thaws in different glass and deposits osmotic equilibrium performance of the morphology after frozen the thawing under the time, and 7min-8min organizes best;
B: sheep ovary 80mm
3Cortex sheet (middle appearance) thaws in different glass and deposits osmotic equilibrium performance of the morphology after frozen the thawing under the time, and 11min-12min organizes best;
C: sheep ovary 160mm
3Cortex sheet (full-page proof) thaws in different glass and deposits osmotic equilibrium performance of the morphology after frozen the thawing under the time, and 18min-19min organizes best.
Fig. 2: the histology picture (* 400) of respectively organizing ovarian follicle behind the glass frozen preservation
A wherein: the fresh control group (fresh control group, FCG); B: non-intervention glass frozen preservation group (vitrification control group, VCG); C:FSH intervention lower-glass group (FSH treatment vitrification group, FSH-VG);
" → " expression primordial follicle among the figure, " ★ " representes primary follicle, " ▲ " expression secondary follicle, Bar is 50 μ m.
Fig. 3: 0.3IU/FSH intervene lower-glass thaw deposit thaw after, the caspase-3 SABC result of ovary activation:
A wherein: the fresh control group, B: non-intervention vitrifying group, C:FSH intervenes vitrifying group down, shows that the caspase-3 expression of activation is lower than non-intervention group; Bar is 50 μ m.
Fig. 4: 0.3IU/FSH intervene lower-glass thaw deposit thaw after, the ovary slit connects PROTEIN C X43 SABC result (Cx43 albumen mainly is expressed on the follicular cell film)
A wherein: the fresh control group, B: non-intervention vitrifying group, C:FSH intervenes vitrifying group down, shows that the CX43 expression is superior to non-intervention group; Bar is 50 μ m.
" → " expression primordial follicle among the figure, " ★ " representes primary follicle, " ▲ " expression secondary follicle.
Fig. 5: FSH intervention lower-glass thaws and deposits the back ovary Cx43 albumen western Western blotting result of thawing.
FCG is the fresh control group, and VG is not for there being the vitrifying of intervention group, and FSH-VG is the vitrifying group under FSH intervenes.
Embodiment
Below in conjunction with specific embodiment and accompanying drawing, further set forth the present invention.Should understand these embodiment and only be used to explain the present invention, and be not used in restriction range of application of the present invention.
Embodiment 1: the big cortex slice glassivation of ovary freezing method
(1) glass freezing
It is thick that the big cortex sheet of the sheep ovary sample at 2～6 monthly ages is fixed as 2mm, by volume is divided into:
Sample (40mm
3=5mm * 4mm * 2mm),
Middle appearance (80mm
3=10mm * 4mm * 2mm),
Full-page proof (160mm
3=10mm * 8mm * 2mm),
Adopt two step osmotic equilibrium methods to carry out glass frozen preservation, overall process is carried out under room temperature (22～24 ℃):
The first step is preparatory osmotic equilibrium: 1.5mol/L ethylene glycol (EG)+20% calf serum (ICS), sample: 15min, middle appearance: 20min, full-page proof: 25min;
Second step was that (the concrete prescription of EG 5.5/30 liquid is vitrifying osmotic equilibrium: EG 5.5/30 liquid: EG+30% ficoll+0.5mol/L sucrose); Sample: 5min～8min, middle appearance: (the vitrifying osmotic equilibrium time of every kind of cortex sheet was a group with one minute to 10min～13min, full-page proof: 17min～20min, and promptly sample cortex sheet time of penetration is grouped into 5min, 6min, 7min, 8min group; The time of penetration of middle appearance cortex sheet is grouped into 10min, 11min, 12min, 13min; The time of penetration of full-page proof cortex sheet is grouped into 17min, 18min, 19min, 20min.）；
Directly drop into liquid nitrogen then.
(2) thaw
After the big cortex sheet of ovary tissue goes out liquid nitrogen, in 37 ℃ of water-baths, thaw, the steps in sequence of specifically thawing is A to B to C to D:
Table 1: the big cortex sheet of ovary tissue goes out the step of thawing behind the liquid nitrogen
(3) glass frozen preservation result's judgement:
To the big cortex sheet of the ovary after thawing carry out ordinary optical microscope, and the relevant immunohistochemistry of apoptosis detect three kinds of different volumes the big cortex sheet of ovary in glass freezing liquid after the osmotic equilibrium that suits, carry out glass frozen preservation.The infiltration of all vetrifying solutions is not good enough, glass frozen preservation thaw the coming off of back histology performance follicle cell, arrangement disorder; Slight crack, egg mother cell distortion etc. appear in the egg mother cell endochylema.This is that the EG of infiltration in follicle cell and the egg mother cell does not reach vitrifying concentration, and making freezingly has ice crystal to form inside and outside the cell when thawing, and causes the mechanical injuries of cell and makes a large amount of ovarian follicle structural deteriorations.Time of penetration is long, then shows as the cell nucleus that a large amount of pyknosis engrains appears in primordial follicle, dehydration is described excessively, and shrinkage is dead.The osmotic equilibrium 7min group that sample is best, what middle appearance was best is that 11min organizes, what full-page proof was best is the 19min group.Identical with observed result to normal ovarian follicle to apoptosis result's judgement, the ovarian follicle of transferring in the best frozen ovary tissue of osmotic equilibrium time is minimum, sets up no difference of science of statistics with fresh ovary.
From experimental result we to sum up in 22～24 ℃ of scopes and thickness be under the controlled condition of 2mm, suitable osmotic equilibrium time T (min) and maximum area S (mm in the vetrifying solution
2) there is such relation: T=1/5 S+3.
With 10mm * 4mm * 2mm cortex piece is example, and maximum area is 10mm * 4mm=40mm
2, T=(40+15)/5 is T=11.
Table 2: different volumes ovary cortex slice glassivation liquid time of penetration during thickness≤2mm
| S（mm 2） | T＝1/5?S+3（min） |
| 4 | 4 |
| 10 | 5 |
| 20 | 7 |
| 40 | 11 |
| 80 | 19 |
The present invention uses T=1/5 S+3 formula and can calculate easily when thickness is 1～2mm, and area one regularly adopts the osmotic equilibrium time of glass frozen preservation ovary, makes things convenient for wild endangered species ovary frozen of laboratory animal, the big domestic animal of breeding, die by visitation of God; Through use adding the EG5.5/30 vetrifying solution of gonadotropin, can improve the ovarian follicle survival, shorten the time that the blood flow of graft pours into again, improve the survival rate of frozen big cortex sheet.
Embodiment 2: the big cortex slice glassivation of sheep ovary is freezing
The present invention adds suitable gonadotropin in 5.5mol/L EG+30% ficoll+0.5mol/L sucrose vetrifying solution.Wherein sheep is added to 10 μ g/mL FSH.Through comparing with not adding the gonadotropin group, no matter be culture in vitro, transplant in the body after still freezing, all show as and add the follicular development of gonadotropin group, endothelial growth factors increases, and the survival follicle number is many, significant difference.
1.1 material
1.1.1 sheep ovary
2-6 monthly age sheep after the sheep ovary is butchered from Islamic beef and mutton slaughter house, Na Jia family, Yongning county, Yinchuan City, Ningxia discards ovary, sends the laboratory in butchering back 2 hours back in inherent 4 ℃ of aseptic culture fluids.
1.1.2 laboratory animal
8～12 age in week the BALB/CA male nude mouse by Beijing dimension tonneau China company credit number is provided: SCXK (capital) 2006-008, body weight 25 ± 1.8g.On the laminar-flow rack of raising in the Experimental Animal Center clean room, guaranteed aseptic feeding environment thereby have tight purification facility in the clean room, temperature maintenance is about 25 ℃, and the air velocity in the clean room is 20cm
3/ s, relative air humidity are 55%.
1.2 divide into groups
Fresh group of (fresh culture group; FCG): the base fluid that contains 10% calf serum is fresh+and FSH organizes (fresh culture group+FSH; FCFG): add 10 μ g/mL FSH vitrifying group (vitrificated culture group in the culture fluid; VCG): contain that the base fluid vitrifying of 10% calf serum+(vitrificated culture group+FSH, VCFG): culture fluid adds 10 μ g/mLFSH to FSH group
1.3 cultural method
Fresh and frozen ovary is all from same sheep.Ovary tissue after fresh and the freeze thawing recovery is cut into about 1mm * 1mm * 1mm fritter with aseptic eye scissors, puts into the blake bottle that is added with the 6mL culture fluid at random, every bottle 6 block organization.CO
237 ℃ of cultivations in the incubator are changed culture fluid once behind every 48h, cultivate altogether 6 days.
1.4 the big cortex sheet of sheep ovary tissue xenogenesis heterotopic transplantation:
Under the gnotobasis, with 0.3% yellow Jackets intraperitoneal injection of anesthesia nude mice (0.1mL/10g).With fresh or thaw after the sheep ovary cortex of 5mm * 4mm * 2mm size implant the neck hypodermis,, and skin suture after dripping several culture fluids in the graft place.
1.5 morphological observation
Ovary tissue is fixed conventional dehydration of alcohol, FFPE, serial section, the thick 5 μ m of sheet, HE dyeing back om observation respectively at cultivating 0 day, 1 day, 2 days, 6 days in 4% paraformaldehyde.
FD is measured: select that the oocyte nuclei person of existence measures in the non-apoptosis ovarian follicle.Because most ovarian follicles is also non-circular, so the length average of ovarian follicle two vertical radial lines is this FD size.Selecting the basilar memebrane inboard of primordial follicle or primary follicle during measurement is measurement point.
1.6 immunohistochemistry detects the expression of cultivating VEGF in the ovary tissue
The ovary tissue of cultivating 24h and 48h is carried out the immunohistochemical staining of VEGF.One anti-is the anti-mouse of rabbit, sheep VEGF polyclonal antibody, and two anti-ly are single mark goat anti-rabbit igg.Adopt SABC Evison method, negative control replaces one to resist with PBS.The strict by specification of operating procedure carries out.
1.7 transplanting the back vigor judges
Histological observation: during 6 weeks after the sheep ovary tissue is transplanted; Nude mice is put to death in the cervical vertebra dislocation, and graft row 4% paraformaldehyde of getting survival immediately is liquid-solid fixed, conventional dehydration of alcohol; FFPE, serial section; The thick 6 μ m of sheet, HE dyeing, light microscopic is observed the ovarian follicular growth situation in the back survival ovary of transplanting down.Because of sheep primordial follicle average diameter 20～30 μ m; Variation for sheep ovary tissue ovarian follicle quantity after freeze thawing is transplanted of the research that quantizes and statistical unit volume; So in order to following method counting: the thick 6 μ m of sheet, continuous 4 sections are a primordial follicle, read first section after; Read one again after 3 sections at interval, by that analogy.The ovarian follicle counting is carried out in every section under low-power field, and calculates ovarian follicle sum in all sections.Ask its mean value, unit is ovarian follicle number/graft.
2. result
2.1FSH influence to the follicular development of culture in vitro ovary tissue
Add in the ovary of FSH in the fresh and freeze thawing group culture fluid, FD comparatively fresh not culture group enlarges markedly, and difference has statistical significance, and P < 0.05.Concrete outcome is seen table 3:
Table 3:FSH is to cultivating the influence of FD in 6 days ovary tissues
*vs?FG，P<0.05
2.2FSH influence to culture in vitro ovary tissue vegf expression
The expression of cultivating 24h and 48h VEGF is adds FSH group height, and the FCFG group is the highest, and FCG and VCG are that freezing front and back vegf expression difference does not have statistical significance, P in the non-FSH intervention group>0.05, concrete outcome is seen table 4:
Table 4:FSH is to cultivating the influence of VEGF positive expression in the ovary tissue
*Vs FCFG organizes P＜0.05,
▲Vs FCFG, VCFG organize P＜0.05
2.3FSH the follicle number purpose after the heterotopic transplantation of sheep ovary tissue xenogenesis is influenced
The result shows that fresh transplantation group follicle number is maximum, and freeze thawing transplantation group follicle number significantly is lower than fresh group, and the more non-intervention group of FSH intervention group follicle number is high in the freeze thawing group.Concrete outcome is seen:
Table 5: transplant and respectively organize graft ovarian follicle survival 6 weeks
*：vs?FTG，P＜0.05?
■vs?FTG，P＜0.01?
▲vs?VTFG，P＜0.05
Embodiment 3: the different period FSH of mouse ovarian glass frozen preservation intervene the correlative study that reduces follicle apoptosis
The present invention adds suitable gonadotropin in 5.5mol/L EG+30% ficoll+0.5mol/L sucrose vetrifying solution.Wherein mouse is added 0.3IU/mL FSH.FSH not only has anti-apoptotic effect as the survival factors of ovarian follicle, also participates in the growth and the maturation of ovarian follicle.The growth of ovarian follicle be unable to do without the cell communication between the connection of slit, and Cx43 can strengthen this intercellular signal alternating current function.For this reason, this research is intervened with FSH in the different links of glass frozen preservation, the expression of the caspase-3 (active caspase-3) through detecting Cx43 and activation, and observation FSH is to the protection effect of ovary glass frozen preservation.
1.1 material
Laboratory animal: to clean a level C56BL/6J female mice 4 ages in week is research object; Add injection recombinant human follicle-stimulating stimulin (fruit receive sweet smell) (Merck Xue Lannuo company) 0.3IU/ml forever in the liquid at glass frozen preservation, get anoestrum mouse bilateral ovaries, complete ovary random packet; Every group 6 pieces
(control group, FCG): after extracting ovary tissue, it is directly fixing or directly extract albumen not apply any intervention for A, fresh control group.
B, glass frozen preservation control group (vitrification control group, VCG): no FSH.
C, 0.3IU/mL FSH intervene glass frozen preservation group (FSH-VG) glass frozen preservation and thaw routine with before similar.
1.2FSH intervene the effect of influence through Cx43, the observation of active caspase-3 immunohistochemistry and CX43 protein quantification analysis and judgement are intervened to frozen result.
2. result
2.1FSH intervene influence to follicle number
The ovary tissue morphosis of intervening through FSH keeps better, and normal follicle number is compared significant difference (P < 0.05) with non-intervention group; But normal follicle number there was no significant difference (seeing Fig. 2, table 6) between the FSH intervention group.
Table 6: the follicle number/HPF after ovary is frozen
★ compares with other each groups respectively, and the result is P, and < 0.05, difference has statistical significance.
2.2FSH intervene influence to each group ovary active caspas-3 and Cx43 expression
Immunohistochemistry result is visible, and glass frozen preservation is omnidistance, and to add FSH group active caspas-3 minimum, and the Cx43 expressing quantity is the highest, and and other each groups between compare in twos, < 0.05, difference has statistical significance (see Fig. 3,4, table 7) to be P.The gonadotropin intervention can suppress the expression of active caspas-3, thereby suppresses the granular cell apoptosis due to frozen, makes more ovarian follicle be able to post-thaw survival.
Table 7: FSH intervenes the influence of organizing Cx43 to express to mouse ovarian in the glass frozen preservation
★ compares with other each groups respectively, and P < 0.05.
2.3FSH intervene the western-blot result that each group ovary Cx43 is expressed
The Cx43 of each vitrifying group expressed and all is higher than fresh group and non-intervention vitrifying group under FSH intervened, and the Cx43 that different links are added in intervention group expression is also variant, and is the highest with the omnidistance interpolation of glass frozen preservation FSH group Cx43 expressing quantity.Compare P < 0.05 between each group.Concrete outcome is seen Fig. 5.
Claims (7)
1. a glass freezing protection liquid that is applicable to the big cortex sheet of ovary adds gonadotropin in the vetrifying solution of 5.5mol/L ethylene glycol+30% ficoll+0.5mol/L sucrose, and the big cortex sheet of described ovary volume is 80mm
3More than, thickness≤2mm.
2. a kind of glass freezing protection liquid that is applicable to the big cortex sheet of ovary according to claim 1 is characterized in that described gonadotropin is HMG, lutropin or short follicle-stimulating hormone.
3. a kind of glass freezing protection liquid that is applicable to the big cortex sheet of ovary according to claim 1 and 2; It is characterized in that the big cortex sheet of described ovary is the big cortex sheet of adult mice ovary, described gonadotropin is HMG, lutropin or the short follicle-stimulating hormone of 0.2IU/mL～0.6IU/mL.
4. a kind of glass freezing protection liquid that is applicable to the big cortex sheet of ovary according to claim 3 is characterized in that described gonadotropin is the short follicle-stimulating hormone of 0.3IU/mL.
5. a kind of glass freezing protection liquid that is applicable to the big cortex sheet of ovary according to claim 1 and 2; It is characterized in that the big cortex sheet of described ovary is the big cortex sheet of sheep ovary, described gonadotropin is the short follicle-stimulating hormone of 5 μ g/mL～20 μ g/mL.
6. a kind of glass freezing protection liquid that is applicable to the big cortex sheet of ovary according to claim 5 is characterized in that gonadotropin is the short follicle-stimulating hormone of 10 μ g/mL.
7. vitrification method that is applicable to the big cortex sheet of ovary is characterized in that this method is:
Glass freezing protection liquid such as claim 1 to 6 used during the vitrifying osmotic equilibrium are arbitrary said;
The osmotic equilibrium temperature is 22～24 ℃; Osmotic equilibrium time T (min) and the big cortex sheet of ovary area S (mm
2) relation be T=1/5 S+3;
The big cortex sheet of described ovary volume is 80mm
3More than, thickness≤2mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101935712A CN102771471A (en) | 2012-08-22 | 2012-08-22 | Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101935712A CN102771471A (en) | 2012-08-22 | 2012-08-22 | Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102771471A true CN102771471A (en) | 2012-11-14 |
Family
ID=47116788
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101935712A Pending CN102771471A (en) | 2012-08-22 | 2012-08-22 | Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102771471A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444700A (en) * | 2013-09-04 | 2013-12-18 | 重庆市畜牧科学院 | Simple in-vitro ovary storage method |
| CN104222071A (en) * | 2014-09-22 | 2014-12-24 | 四川大学华西第二医院 | Ovarian tissue cryopreservation method |
| CN106153622A (en) * | 2015-04-22 | 2016-11-23 | 北京大学深圳医院 | Glass freezing finder and application thereof |
| CN106172376A (en) * | 2016-08-30 | 2016-12-07 | 宁夏医科大学 | Ovary method for glass frozen preservation under lutropin intervention |
| CN106386784A (en) * | 2016-08-30 | 2017-02-15 | 宁夏医科大学 | Method for improving ovary vitrification cryopreservation effect with sphingosine-1-phosphate |
| CN108641999A (en) * | 2018-03-26 | 2018-10-12 | 阮祥燕 | A kind of ovary tissue freeze and method for resuscitation |
| CN109644991A (en) * | 2019-01-31 | 2019-04-19 | 力盟低温医学(深圳)有限公司 | In conjunction with freezing sample processing method of the laser technology under without permeability protective agent |
| CN110352949A (en) * | 2018-03-26 | 2019-10-22 | 首都医科大学附属北京妇产医院 | A kind of ovary tissue freezes protection and resuscitation fluid |
| CN117898229A (en) * | 2024-03-19 | 2024-04-19 | 三亚热带水产研究院 | Artificial fertilization method for Chlamys nobilis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986780A (en) * | 2006-12-14 | 2007-06-27 | 宁夏医学院 | New-type of vetrifying solution |
-
2012
- 2012-08-22 CN CN2012101935712A patent/CN102771471A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986780A (en) * | 2006-12-14 | 2007-06-27 | 宁夏医学院 | New-type of vetrifying solution |
Non-Patent Citations (8)
| Title |
|---|
| B.IMTHURN ET. AL.: "Gonadotrophin administration can benefit ovarian tissue grafted to the body wall: implications for human ovarian grafting", 《MOLECULAR AN CELLULAR ENDOCRINOLOGY》 * |
| 党玲: "绵羊卵巢组织大皮质超速玻璃化冻存研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 孙静等: "小鼠卵巢自体及异体移植前的尿促性素体外干预对卵泡存活的影响", 《器官移植》 * |
| 庞龙等: "小鼠卵巢组织的超速冻存法研究", 《中国实验动物学报》 * |
| 李梅等: "卵巢组织冷冻保存及其应用的研究进展", 《现代妇产科进展》 * |
| 覃颖等: "卵巢组织玻璃化冷冻保存和移植的研究进展", 《中国组织工程研究》 * |
| 鞠晓红等: "大鼠卵巢组织的冷冻移植", 《中国组织工程研究与临床康复》 * |
| 马黔红等: "长效促性腺激素释放激素激动剂用于体外受精-胚胎移植中降调节后延迟卵巢启动时间的探讨", 《实用妇产科杂志》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103444700A (en) * | 2013-09-04 | 2013-12-18 | 重庆市畜牧科学院 | Simple in-vitro ovary storage method |
| CN104222071A (en) * | 2014-09-22 | 2014-12-24 | 四川大学华西第二医院 | Ovarian tissue cryopreservation method |
| CN106153622A (en) * | 2015-04-22 | 2016-11-23 | 北京大学深圳医院 | Glass freezing finder and application thereof |
| CN106172376A (en) * | 2016-08-30 | 2016-12-07 | 宁夏医科大学 | Ovary method for glass frozen preservation under lutropin intervention |
| CN106386784A (en) * | 2016-08-30 | 2017-02-15 | 宁夏医科大学 | Method for improving ovary vitrification cryopreservation effect with sphingosine-1-phosphate |
| CN108641999A (en) * | 2018-03-26 | 2018-10-12 | 阮祥燕 | A kind of ovary tissue freeze and method for resuscitation |
| CN110352949A (en) * | 2018-03-26 | 2019-10-22 | 首都医科大学附属北京妇产医院 | A kind of ovary tissue freezes protection and resuscitation fluid |
| CN110352949B (en) * | 2018-03-26 | 2021-07-23 | 首都医科大学附属北京妇产医院 | Cryopreservation protection and resuscitation solution for ovarian tissues |
| CN113207869A (en) * | 2018-03-26 | 2021-08-06 | 首都医科大学附属北京妇产医院 | Cryopreservation protective solution for ovarian tissues |
| CN113207869B (en) * | 2018-03-26 | 2022-06-07 | 首都医科大学附属北京妇产医院 | Cryopreservation protective solution for ovarian tissues |
| CN109644991A (en) * | 2019-01-31 | 2019-04-19 | 力盟低温医学(深圳)有限公司 | In conjunction with freezing sample processing method of the laser technology under without permeability protective agent |
| CN117898229A (en) * | 2024-03-19 | 2024-04-19 | 三亚热带水产研究院 | Artificial fertilization method for Chlamys nobilis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102771471A (en) | Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof | |
| Sansone et al. | Storage of buffalo (Bubalus bubalis) semen | |
| Memon et al. | Effect of antioxidants on post thaw microscopic, oxidative stress parameter and fertility of Boer goat spermatozoa in Tris egg yolk glycerol extender | |
| Hu et al. | RETRACTED: The cryoprotective effect of trehalose supplementation on boar spermatozoa quality | |
| Morrell | Artificial insemination: current and future trends | |
| Memon et al. | Effect of butylated hydroxytoluene on cryopreservation of Boer goat semen in Tris egg yolk extender | |
| Renard | Cooling and freezing tolarances in embryos of the Pacific oyster, Crassostrea gigas: methanol and sucrose effects | |
| Guignot et al. | Improved vitrification method allowing direct transfer of goat embryos | |
| Meirow et al. | Ovarian tissue cryopreservation and transplantation: a realistic, effective technology for fertility preservation | |
| Zomborszky et al. | Sperm collection from shot red deer stags (Cervus elaphus) and the utilisation of sperm frozen and subsequently thawed | |
| Gook et al. | The effects of cryopreservation regimens on the morphology of human ovarian tissue | |
| Garg et al. | Effect of different cryoprotectant concentrations for ultrarapid freezing of immature goat follicular oocytes on their subsequent maturation and fertilization in vitro | |
| Yavaş et al. | Cryopreservation of scaly carp (Cyprinus carpio) sperm: effect of different cryoprotectant concentrations on post-thaw motility, fertilization and hatching success of embryos | |
| Herranz-Jusdado et al. | Eel sperm cryopreservation: an overview | |
| Van den Hurk et al. | Development of fresh and cryopreserved early-stage ovarian follicles, with special attention to ruminants | |
| JP2001247401A (en) | Cooling preservation liquid for tissue | |
| Luvoni et al. | Effects of slow and ultrarapid freezing on morphology and resumption of meiosis in immature cat oocytes | |
| Colombo et al. | Artificial fertilization and induction of triploidy and meiogynogenesis in the European sea bass, Dicentrarchus labrax L. | |
| Vatanparast et al. | Evaluation of sheep ovarian tissue cryopreservation with slow freezing or vitrification after chick embryo chorioallantoic membrane transplantation | |
| Šćekić et al. | A novel strategy for conservation of European eel (Anguilla anguilla) genetic resources: Cryopreservation of ovarian stem cells | |
| Mohammed | Application of advanced reproductive biotechnologies for buffalo improvement with focusing on Egyptian buffaloes | |
| Olurode et al. | Effects of storage temperature and extension media on motility of caprine spermatozoa | |
| Munyai | Cryopreservation of South African indigenous ram semen | |
| FERNANDES et al. | Oocyte recovery and in vitro embryo production in cows treated with a single dose offollicle-stimulating hormone | |
| Letsoalo | Characterisation and Cryopreservation of Semen from Indigenous Namaqua Afrikaner Sheep Breed, in Comparison with Dorper and Dohne Merino Breeds |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20121114 |