CN102363766A - Method for promoting in vitro maturation of immature oocytes by using activin A (ActA) - Google Patents
Method for promoting in vitro maturation of immature oocytes by using activin A (ActA) Download PDFInfo
- Publication number
- CN102363766A CN102363766A CN2011103417554A CN201110341755A CN102363766A CN 102363766 A CN102363766 A CN 102363766A CN 2011103417554 A CN2011103417554 A CN 2011103417554A CN 201110341755 A CN201110341755 A CN 201110341755A CN 102363766 A CN102363766 A CN 102363766A
- Authority
- CN
- China
- Prior art keywords
- ovocyte
- acta
- maturation
- days
- drop
- 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
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for promoting in vitro maturation of immature oocytes by using activin A (ActA). The method comprises the following steps of: collecting preantral granular cells; placing the preantral granular cells into growth liquid drops; cultivating in a culture box overnight to prepare a granular cell layer; placing the collected oocytes of 55 to 65 mu m on the granular cell layer; and adding the ActA at the concentration of 100 ng/ml into a culture medium and cocultivating to realize in vitro maturation of the immature oocytes. Under the cocultivation condition of ova nuda and the granular cells, the ActA can promote the oocytes to enter meiosis, so that the maturation quantity of the oocytes can be increased. On the third day to the fourth day of cultivation, the apoptosis ratio of the oocytes is 14.3 +/- 1.7 percent; 7 days after cocultivation, the diameter change of the ova nuda is from 61.79 +/- 3.2 to 67.27 +/- 3.5 mu m; and the glutathione (GSH) expression quantity of the oocyte cytoplasm is 11.33 +/- 1.38 (mu m). The method is easy to operate; an ectogenesis and development system of small follicles is established; development of the oocyte resource is facilitated; and the method is applied to biological and medical research.
Description
Technical field:
The present invention relates to the method that a kind of ActA (Activin A, activator A) promotes the prematurity oocyte in vitro maturation, particularly relate to and a kind ofly when the vitro culture ovocyte, in substratum, add ActA, promote the sophisticated method of immature egg parent cell.The field of biomedicine technology that belongs to reproductive medicine.
Background technology:
The a large amount of ovarian follicles that contain each different developmental phases in the Mammalian Ovary.A maximum part is a primordial follicle.Yet, in female period, have only the sub-fraction ovarian follicle to produce and have ovulability and sophisticated ovocyte with reproductivity.The last locking of remaining folliculus ovarii (＞99.9%).In order to obtain extra female gamete resource, be devoted to develop the ovocyte technology with developmental potency of growing fully and have very big meaning in the production of ovarian follicle early development stage.Research to the transition mechanisms of primary follicle is very important and has clinical meaning to the adjusting primordial follicle in the ectogenesis research of early stage ovarian follicle; And can be applied to auxiliary procreation technology vitro culture primordial follicle and produce mature oocyte, in order to produce the wild kind of valuable domestic animal and extinction in imminent danger.
The foundation of ovarian follicle vitro culture technology can be applicable to fields such as biology and medical science, can be used for remedying ovocyte in the preservation process owing to damage the forfeiture shortcoming (environmental factors, radiotherapy, chemotherapy) of the fertility that causes; Preserve their Fertility for cancered young woman when the cancer therapy and bring new hope; And, create huge social and economic worth for therapeutic cloning and ovary Infertility patient provide the fine ovocyte.
Summary of the invention:
The object of the present invention is to provide a kind of ActA to promote the method for prematurity oocyte in vitro maturation; This method is through when the common cultivation of ovocyte and granulosa cell; In substratum, add the maturation in vitro that ActA promotes the immature egg parent cell, good technique means is provided for exploitation ovocyte resource is applied to biomedicine.
In order to realize the foregoing invention purpose, a kind of ActA of the present invention promotes the method for prematurity oocyte in vitro maturation, and granulosa cell is put into the growth drop of getting ready in advance before the collecting chamber, and incubated overnight in incubator is made granular cell layer; Ovocyte between 55～65 μ m that collect is put on the granular cell layer of cultivating behind the 24h, and the ActA that in substratum, adds concentration and be 100ng/ml cultivates altogether, realizes the maturation in vitro of immature egg parent cell.
The inventive method comprises following concrete operations step:
The collection of granulosa cell and cultivation before the first step, the chamber:
With 12～14 days the mouse in birth back, behind the taking-up ovary, be digested to ova nuda and granulosa cell with digestive ferment, the collecting granules cell is put into the growth drop of getting ready and was cultivated 24 hours, and culture condition is each drop 100 μ l, 37 ℃, 5%CO
2, saturated humidity incubator in, process granular cell layer;
Said digestive ferment is to contain 0.25% pancreatin, the digestive ferment of 0.2% collagenase IV and 0.02%DNAse-I (DNA digestive ferment I is available from sigma company).
The preparation of said growth media: α-MEM (Mi Libo high glucose medium, HyClone company); BSA (bovine serum albumin, Roche company) 3mg/ml; Lipid acid (Fetuin) 1mg/ml; 1%ITS-mix (Regular Insulin Transferrins,iron complexes Sodium Selenite mixture, HyClone company); FSH (follitropin, HyClone company) 10mIU/ml; EGF (Urogastron, HyClone company) 5ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate.
The collection and the cultivation of second step, 12～14dpp ovocyte:
After mechanical process was separated 12～14 days mouse ovarians in birth back, stereoscope thorn down was broken and with digestive ferment digestion, and mouthful suction pipe takes out ovocyte, chooses the ovocyte of diameter between 55～65 μ m then, in grown cultures liquid, cultivates, and culture condition is 37 ℃, 5%CO
2, saturated humidity incubator in, every half liquid that changed at a distance from 2 days; Put on the granular cell layer of cultivating behind the 24h with 20 ovocytes of each drop and to cultivate, in substratum, adding concentration is the ActA of 100ng/ml;
Said digestive ferment is to contain 0.25% pancreatin, the digestive ferment of 0.2% collagenase IV and 0.02%DNAse-I.
The preparation of ovocyte grown cultures liquid: α-MEM; 10%FCS (foetal calf serum); 1%ITS-mix; FSH 10mIU/ml; EGF 5ng/ml; ActA 100ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate.
The maturation of the 3rd step, ovocyte is cultivated:
Ovocyte was cultivated after 7 days, chose the ovocyte that does not have apoptosis, with 20 of each drops, put into the cultivation drop (6cm petridish, each drop 100 μ l, 37 ℃, the 5%CO that get ready in advance
2, saturated humidity incubator in overnight cultures), at 37 ℃, 5%CO
2, cultivate 18h in the incubator of saturated humidity, the ratio of statistics oocyte maturation.
The preparation of maturation culture solution: α-MEM; BSA 3mg/ml; Fetuin 1mg/ml; 1%ITS-mix; FSH 100mIU/ml; EGF 1ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate.
The present invention is the method that a kind of ActA promotes the prematurity oocyte in vitro maturation; This method is put into the growth drop to granulosa cell before the chamber of collecting earlier and is cultivated; Process granular cell layer; The ovocyte of diameter between 55～65 μ m of collecting put on the granular cell layer again and cultivated, in substratum, adding concentration is the ActA of 100ng/ml, realizes the maturation in vitro of immature egg parent cell.Ova nuda and granulosa cell are total under the culture condition, and ActA can promote ovocyte to get into reduction division, improves the ripe quantity of ovocyte.When cultivating 3～4 days, the apoptosis generation ratio of ovocyte is 14.3 ± 1.7%; After cultivating 7 days altogether, the vary in diameter of ova nuda is 61.79 ± 3.2～67.27 ± 3.52 μ m; After cultivating 7 days altogether, ovocyte kytoplasm GSH (gsh) expression amount is 11.33 ± 1.38 (μ M).This method is simple to operate, has set up the external generation and the growth system of little ovarian follicle, helps developing the ovocyte resource and is used for biology and medical research.
Description of drawings:
Fig. 1 is 12～14dpp Oocyte in Vitro incubation growth microgram.
Fig. 2 is different culture group ovocyte vary in diameter situation maps.
Fig. 3 is different culture group ovocyte apoptosis situation maps.
Fig. 4 is the detection figure of ovocyte GSH.
Fig. 5 induces back GVBD (germinal vesicle breaks) and the ovocyte microgram of MII phase (MII) for vitro culture 12～14dpp oocyte maturation.
The scale map that Fig. 6 takes place for GVBD.
The scale map that Fig. 7 takes place for MII.
Embodiment:
Below in conjunction with specific embodiment the inventive method is done further elaboration.
Embodiment 1,
The first step: the collection of granulosa cell and cultivation before the chamber
1, behind 12～14 days the mouse ovarian in taking-up birth back, with containing 0.25% pancreatin, the digestive ferment of 0.2% collagenase IV and 0.02%DNAse-I (DNA digestive ferment I) is digested to ova nuda and granulosa cell; Add stop buffer (containing 10% FCS and 90% DMEM) and end digestion, granulosa cell is put into the 1.5ml centrifuge tube, the centrifugal 5min of 1500rpm; Wash twice with growth media, put into growth drop (6cm petridish, each drop 100 μ l of getting ready in advance; 37 ℃, 5%CO
2, saturated humidity incubator in overnight cultures) cultivate 24h, process granular cell layer.
2, the preparation of growth media: α-MEM; BSA 3mg/ml; Fetuin 1mg/ml; 1%ITS-mix; FSH (Follicle-stimulating Hormone, FSH) 10mIU/ml; EGF (epidermal growth factor, EGF) 5ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate.
Second step: the collection of 12～14dpp ovocyte and cultivation:
1, chooses 12～14 days the mouse in birth back; After mechanical process was separated the taking-up ovary, stereoscope stung broken down and uses 0.25% pancreatin, 0.2% collagenase IV and 0.02%DNAse-I digestion; The mouth suction pipe takes out ovocyte, chooses the ovocyte of diameter between 55～65 μ m then;
2, the ovocyte of collecting is divided into two portions: first part puts into 20 ovocytes of each drop on the granular cell layer of cultivating behind the 24h ,+/-ActA cultivates; Second section is put into the cultivation drop of getting ready in advance ,+/-ActA cultivates, and culture condition is 37 ℃, 5%CO
2, saturated humidity incubator in.Every half liquid that changed at a distance from 2 days.
3, the ovocyte growth media of control group and experimental group is following:
The preparation of control group ovocyte nutrient solution: α-MEM; 10%FCS; 1%ITS-mix; FSH 10mIU/ml; EGF 5ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate.
The preparation of experimental group ovocyte nutrient solution: α-MEM; 10%FCS; 1%ITS-mix; FSH 10mIU/ml; EGF 5ng/ml; ActA 100ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate.
The 3rd step: the maturation of ovocyte is cultivated:
1, after the ovocyte of cultivation and single culture is cultivated 7d altogether, chooses the ovocyte that does not have apoptosis,, put the people and shift to an earlier date (6cm petridish, 37 ℃ of each drop 100 μ l, 5%CO in the ready-made cultivation drop with 20 of each drops
2, saturated humidity incubator in overnight cultures), at 37 ℃, 5%CO
2, cultivate 18h in the incubator of saturated humidity, the ratio of statistics oocyte maturation.
2, the preparation of maturation culture solution: α-MEM; BSA 3mg/ml; Fetuin 1mg/ml; 1%ITS-mix; FSH 100mIU/ml; EGF 1ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate.
In the present embodiment, granulosa cell is taken from ovarian follicle before 12～14 days mouse ovarian chambeies, carry and cultivate granular cell layer previous day, employing be that drop is cultivated.Granulosa cell is cultivated and is added the ovocyte of 20 diameters at 55～65 μ m after one day.Among Fig. 11 and 2 expressions derive between 12～14dpp mouse, 60～70 μ m ovocyte separately in grown cultures liquid (/+ActA) cultivated 0 day, 4 days and 7 days, and carry out maturation and induce 16～18 hours upgrowth situation; Mouse denuded oocyte and 12～14 day mouse granulosa cell of 3 and 4 expressions between 60～70 μ m among Fig. 1 (/+ActA) cultivated altogether 0 day, 4 days and 7 days, and carry out maturation and induce 16～18 hours upgrowth situation.Ovocyte single culture test (DOs) is as among Fig. 11 and 2.Shown in Figure 3, it is little to the influence of ovocyte growth in the process of cultivating, whether to add ActA, 22.3 ± 2.2% and 24.3 ± 2.7% (/+apoptosis (P＞0.05) taken place in ActA) ovocyte.Be total to culture experiment (DOs+PAGCs); Shown among Fig. 13 and 4, the ovocyte upgrowth situation that is grown on the granulosa cell is fine, when cultivating 3～4 days; Granulosa cell can cover ovocyte; Under this situation, the apoptosis generation ratio of ovocyte is very low, be 25.5 ± 2.1% and 14.3 ± 1.7% respectively (/+ActA).Therefore, we can obtain, and do not have under the condition of granulosa cell existence, and ActA can not exert an influence to the growth of ovocyte.
For further research ActA is to the influence of growing of ovocyte, we detect through two aspects, and the one, the diameter of ovocyte, the 2nd, the expression amount of GSH.Fig. 2 explains in the process of Oocyte in Vitro cultivation, does not add and adds the ActA test group and cultivate diameter variation situation after 7 days.The vary in diameter of ova nuda is respectively 63.27 ± 3.12～61.03 ± 2.87 μ m and 63.42 ± 3.72～61.36 ± 4.04 μ m; The vary in diameter of cultivating altogether is respectively 61.54 ± 4.03～66.37 ± 3.05 μ m and 61.79 ± 3.2～67.27 ± 3.52 μ m.In the process of above data declaration ovocyte single culture; Two groups of ovocyte diameters all diminish; With the ovocyte that granulosa cell is cultivated altogether, the test group diameter that adds ActA increases 5.48 ± 1.2 μ m, does not add the ActA test group and increases 4.83 ± 0.9 μ m (P＜0.05).Fig. 4 represent 14 and the 21 days oocyte of mouse in birth back, ovocyte single culture (/+ActA) 7 days and ovocyte and granulosa cell cultivate altogether (/+ActA) expression amount of the ovocyte kytoplasm GSH (gsh) after 7 days; Be respectively 6.95 ± 0.97; 14.28 ± 0.87; 8.07 ± 0.47,7.95 ± 0.55,9.8 ± 1.32 and 11.33 ± 1.38 (μ M).* represent significant difference P＜0.05, * * represents extremely significantly P＜0.01 of difference.In ovocyte and the granulosa cell co-culture experiments, add ActA, the GSH expression amount significantly raises, but is lower than interior 21 days level of body.
In order to detect ActA to the sophisticated influence of the nucleus of ovocyte, we carry out maturation to the ovocyte of vitro culture and induce, and detect the ratio of oocyte maturation.Fig. 5 is in the synoptic diagram of GVBD phase and MII phase for ovocyte.In the ova nuda culturing process, do not add and add ActA and cultivate after 7 days, ratio takes place GVBD is respectively 16.1 ± 2.2% and 15 ± 1.7% (shown in Figure 6), and MII generation ratio is 3.7 ± 2.5% and 4.9 ± 1.6% (shown in Figure 7) respectively.Not adding and adds ActA with 12～14 days mouse ovarian granulosa cells in birth back, to cultivate the ratio that GVBD takes place ovocyte after 7 days altogether be respectively 20.5 ± 3.1% and 24.5 ± 4.5% (P＜0.05), ratio difference 20.5 ± 4.1% and 27.6 ± 3.3% (P＜0.05) of MII.This data declaration oocyte of mouse can spontaneously recover reduction division; But do not have under the condition of granulosa cell existence; GVBD takes place in ovocyte and the ratio of MII phase does not have difference; And ova nuda and granulosa cell are total under the culture condition, and ActA can promote ovocyte to get into reduction division, improves the ripe quantity of ovocyte.
Above test-results shows do not have under the condition of granulosa cell existence, and ActA can't exert an influence to the g and D of ovocyte, and it is to act on granulosa cell that ActA acts on the ovocyte approach, and then acts on ovocyte.
The present invention has tested and has implemented repeatedly, and the result of test is successful, has realized the purpose of invention.
Claims (2)
1. an ActA promotes the method for prematurity oocyte in vitro maturation; It is characterized in that operating: the collection of granulosa cell and cultivation before the first step, the chamber:, behind the taking-up ovary, be digested to ova nuda and granulosa cell with digestive ferment with 12～14 days the mouse in birth back according to following steps; The collecting granules cell is put into the growth drop of getting ready and was cultivated 24 hours; Culture condition is each drop 100 μ l, 37 ℃, and 5%CO
2, saturated humidity incubator in, process granular cell layer; The collection and the cultivation of second step, 12～14dpp ovocyte: after mechanical process is separated afterwards 12～14 days mouse ovarians of birth; Stereoscope stings broken down and digests with digestive ferment; The mouth suction pipe takes out ovocyte, chooses the ovocyte of diameter between 55～65 μ m then, in grown cultures liquid, cultivates; Culture condition is 37 ℃, 5%CO
2, saturated humidity incubator in, every half liquid that changed at a distance from 2 days; Put on the granular cell layer of cultivating behind the 24h with 20 ovocytes of each drop and to cultivate, in substratum, adding concentration is the ActA of 100ng/ml; The maturation of the 3rd step, ovocyte is cultivated: ovocyte was cultivated after 7 days, chose the ovocyte that does not have apoptosis, with 20 of each drops, put into the cultivation drop of getting ready in advance, 6cm petridish, each drop 100 μ l, 37 ℃, 5%CO
2, saturated humidity incubator in overnight cultures, at 37 ℃, 5%CO
2, cultivate 18h in the incubator of saturated humidity, the ratio of statistics oocyte maturation; Wherein, the preparation of growth media in the first step: Mi Libo high glucose medium; Bovine serum albumin 3mg/ml; Lipid acid 1mg/ml; 1% Regular Insulin Transferrins,iron complexes Sodium Selenite mixture; Follitropin 10mIU/ml; Urogastron 5ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate; The preparation of ovocyte grown cultures liquid in second step: Mi Libo high glucose medium; 10% foetal calf serum; 1% Regular Insulin Transferrins,iron complexes Sodium Selenite mixture; Follitropin 10mIU/ml; Urogastron 5ng/ml; ActA 100ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate; The preparation of maturation culture solution in the 3rd step: Mi Libo high glucose medium; Bovine serum albumin 3mg/ml; Lipid acid 1mg/ml; 1% Regular Insulin Transferrins,iron complexes Sodium Selenite mixture; Follitropin 100mIU/ml; Urogastron 1ng/ml; 1% Sodium.alpha.-ketopropionate; 1% penicillium mould and Streptomycin sulphate.
2. a kind of ActA according to claim 1 promotes the method for prematurity oocyte in vitro maturation, it is characterized in that said digestive ferment contains 0.25% pancreatin, 0.2% collagenase IV and 0.02%DNA digestive ferment I.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103417554A CN102363766A (en) | 2011-11-02 | 2011-11-02 | Method for promoting in vitro maturation of immature oocytes by using activin A (ActA) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103417554A CN102363766A (en) | 2011-11-02 | 2011-11-02 | Method for promoting in vitro maturation of immature oocytes by using activin A (ActA) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102363766A true CN102363766A (en) | 2012-02-29 |
Family
ID=45690360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103417554A Pending CN102363766A (en) | 2011-11-02 | 2011-11-02 | Method for promoting in vitro maturation of immature oocytes by using activin A (ActA) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102363766A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074296A (en) * | 2013-01-21 | 2013-05-01 | 安徽农业大学 | In vitro maturation method and in vitro maturation culture solution for mouse naked ovum |
| CN103525759A (en) * | 2013-10-06 | 2014-01-22 | 吉林大学 | Application of cordycepin in in-vitro maturation culture of oocytes of small pig follicles |
| CN110129402A (en) * | 2019-04-15 | 2019-08-16 | 宁夏医科大学 | A kind of research method that Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis |
| CN112386743A (en) * | 2020-11-18 | 2021-02-23 | 华中科技大学同济医学院附属同济医院 | Artificial ovary stent constructed by 3D biological printing technology, artificial ovary and construction method and application thereof |
| CN113005075A (en) * | 2021-03-10 | 2021-06-22 | 宁夏医科大学 | Application of ROCK inhibitor in promotion of up-regulation of oocyte gene Bmp15 and Gdf9 expression level |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989004366A1 (en) * | 1987-11-03 | 1989-05-18 | Grob Howard S | Method for in vitro maturation of oocytes |
-
2011
- 2011-11-02 CN CN2011103417554A patent/CN102363766A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989004366A1 (en) * | 1987-11-03 | 1989-05-18 | Grob Howard S | Method for in vitro maturation of oocytes |
Non-Patent Citations (5)
| Title |
|---|
| HIDEMI YOKOTA ET AL: "Paradoxical action of activin A on folliculogenesis in immature and adult mice", 《ENDOCRINOLOGY》 * |
| ZHANBIAO LI ET AL: "A co-culture system with preantral follicular granulosa cells in vitro induces meiotic maturation of immature oocytes", 《HISTOCHEM CELL BIOL》 * |
| 朱辉 等: "激活素A对离体培养大鼠卵泡发育的影响", 《基础医学与临床》 * |
| 李占彪 等: "颗粒细胞或卵丘细胞提高小鼠未成熟卵母细胞恢复减数分裂能力的研究", 《青岛农业大学学报》 * |
| 陈颖 等: "激活素_抑制素_卵泡抑素系统对小鼠卵母细胞体外成熟及早期胚胎发育潜能的影响机制", 《生殖与避孕》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074296A (en) * | 2013-01-21 | 2013-05-01 | 安徽农业大学 | In vitro maturation method and in vitro maturation culture solution for mouse naked ovum |
| CN103074296B (en) * | 2013-01-21 | 2014-06-11 | 安徽农业大学 | In vitro maturation method and in vitro maturation culture solution for mouse naked ovum |
| CN103525759A (en) * | 2013-10-06 | 2014-01-22 | 吉林大学 | Application of cordycepin in in-vitro maturation culture of oocytes of small pig follicles |
| CN110129402A (en) * | 2019-04-15 | 2019-08-16 | 宁夏医科大学 | A kind of research method that Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis |
| CN110129402B (en) * | 2019-04-15 | 2023-08-01 | 宁夏医科大学 | Method for researching influence of Rho-ROCK signal channel inhibitor Y27632 on mouse follicle in-vitro development |
| CN112386743A (en) * | 2020-11-18 | 2021-02-23 | 华中科技大学同济医学院附属同济医院 | Artificial ovary stent constructed by 3D biological printing technology, artificial ovary and construction method and application thereof |
| CN113005075A (en) * | 2021-03-10 | 2021-06-22 | 宁夏医科大学 | Application of ROCK inhibitor in promotion of up-regulation of oocyte gene Bmp15 and Gdf9 expression level |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101491228B (en) | Sea no-nucleus pearl incubation method | |
| CN103422176B (en) | Construction method of human amniotic mesenchymal stem cell bank | |
| CN102363766A (en) | Method for promoting in vitro maturation of immature oocytes by using activin A (ActA) | |
| CN102344906B (en) | Hair follicle stem cell separation culture method | |
| CN102311938A (en) | Serum-free medium for culturing hepatic cells | |
| CN1962855A (en) | Method for construction of stem cell in-vitro multiplication and differentiation microenvironment | |
| CN102178946B (en) | Application of baby hamster kidney(BHK)-21 cell serum-free suspension culture technology in foot-and-mouth disease vaccine production | |
| Ye et al. | Dynamic changes in meiotic progression and improvement of developmental competence of pig oocytes in vitro by follicle-stimulating hormone and cycloheximide | |
| CN101372682B (en) | Construction method of Epinephelus fuscoguttatus fin cell line | |
| CN104419659A (en) | Cultivation and mass-production method of adipose-derived stem cells and stem cell secretion of adipose-derived stem cells | |
| CN103074297B (en) | Livestock spermatogonial stem cell medium | |
| CN105018418B (en) | Human oocytes In-vitro maturation liquid containing Endothelin-1 and application | |
| CN102363765B (en) | Technological method for promoting ectogenesis of premeiotic female germ cells by using activin A (ActA) | |
| CN103451149A (en) | Transgenic hamster and preparation method thereof | |
| CN101096653A (en) | Method for expanding uterus intima cell externally on a large scale | |
| CN102776260A (en) | Method for effectively expressing recombinant human coagulation factor VIII | |
| CN102796698A (en) | Method for inducing differentiation of placenta-derived mesenchymal stem cells into islet-like cells | |
| CN104894056A (en) | Method for constructing spleen tissue cell line of acipenser dabryanus | |
| JPH03502045A (en) | How to mature oocytes in vitro | |
| CN114369573B (en) | Method for constructing in-situ primary nasopharyngeal carcinoma animal model | |
| CN105316282A (en) | Acipenser dabryanus spermatogonium culture solution and application thereof | |
| CN104195100A (en) | In-vitro culture method of mammary gland epithelial cells | |
| CN107034176A (en) | A kind of nutrient solution and its cultural method for improving in vitro culture porcine oocytes developmental potentiality | |
| CN101886059A (en) | Culture solution used for embryo vitro production and method for bovine embryo vitro production | |
| CN102676449A (en) | Ghrelin-containing sheep embryo in-vitro culture solution and culture method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120229 |