CN110129402A - A kind of research method that Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis - Google Patents
A kind of research method that Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis Download PDFInfo
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Abstract
The invention discloses the research methods that a kind of Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis, 12 days mouse ovarians after taking-up is raw, preantral follicle is obtained by different separation methods, ovarian follicular growth and In vitro maturation are carried out by the way of droplet.Experimental setup blank control group and processing group containing Y27632.Ideal vitro culture system can be obtained using of the invention.By the study show that, which can reduce the apoptosis of granular cell in Ovarian Follicles really, this for ovarian follicle continue development provide reliable guarantee.
Description
Technical field
The invention belongs to field of biotechnology, specifically, being related to a kind of Rho-ROCK signal pathway inhibitor Y27632
The research method that mouse eggs bubble ectogenesis is influenced.
Background technique
Ovary is the organ of female mammal reproductive endocrine function, and the performance of function depends on Basic Structure And Functions
Unit-ovarian follicle.The growth course of one complete ovarian follicle mainly undergoes the package and proliferation, thecacells of granular cell
Occur, the processes such as the maturation of egg mother cell and sex hormone regulation, primordial follicle, just is roughly divided into according to the number of plies of granular cell
Grade ovarian follicle, secondary follicle, tertiary follicle and graaffian follicle, former three form antrum folliculi, therefore also known as preantral follicle not yet,
The two is then referred to as antral follicles afterwards.In humans and animals one complete life cycle, most of ovarian follicle rank before chamber
Duan Douhui, which degenerates, to be latched.How to develop and use and avoid the waste of reproduction resource so that the in-vitro separation of ovarian follicle, culture even it is cold
Freeze the hot spot all become in Developmental Biology.
Rho/Rho correlation coiled-coil protein kinases (ROCK) signal path is that cytoskeleton and cell fate determine key
One of signal path can participate in proliferation, migration, differentiation and the apoptosis of cell by the adjusting of cell micro-environment.The signal is logical
Road inhibitor Y27632 sends out embryo by inhibiting the apoptosis of embryonic stem cell with the ATP competitive binding catalytic site time limit
During life, the differentiation of endoderm cell can be inhibited by TGF-β and SMAD signal path, and in improving, ectoderm cell
Differentiation capability.The signal paths play certain adjusting to the development of preantral follicle by the proliferation to granular cell and make
With.
Summary of the invention
It is an object of the invention to propose a kind of Rho-ROCK signal pathway inhibitor Y27632 to mouse eggs foam outside
The research method of development impact.By adding Y27632 in more mature preantral follicle culture system in vitro, it is right to inquire into its
The proliferation of granular cell and the influence of Oocyte Development.New approaches are provided to influence the molecular mechanism of follicular development.
Its technical solution is as follows:
A kind of research method that Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis,
The following steps are included:
The separation of step 1, preantral follicle: obtaining mouse ovarian under stereoscope, in superclean bench, uses separating liquid
After cleaning 3 times, mechanical phonograph recorder separation: by ovarian metastasis into the glass surface ware containing 2mL separating liquid, with dissecting needle stereoscopic aobvious
Blunt separation ovarian follicle under micro mirror guarantees that the time of one ovary ovarian follicle in-vitro separation does not exceed 30min.Three are cleaned with culture solution
After secondary, detection form is complete, ovarian follicle of the diameter at 100~130 μm.Ovary: being directly put into the recessed ware of digestive juice glass by enzyme process,
After 37 DEG C of culture 1h, remove the tissue block in digestive juice, abandons supernatant after being added in follicle culture liquid low-speed centrifugal 5min, then use
After culture solution cleaning twice, the detection complete preantral follicle of form is cultivated.Enzyme process-Mechanical Method: 20min is handled with digestive juice
Afterwards, then use above-mentioned Mechanical Method separation method.
The culture of step 2, preantral follicle: selecting diameter is the culture dish of 60mm, and 12h prepares the ovarian follicle training that 30 μ L rise in advance
Nutrient solution drop, and 2mL mineral oil is being covered above.The preantral follicle that separation obtains is put into drop with self-control glass rim suction pipe
(1 piece /) in 37 DEG C of incubator cultures, every 2d half, which is measured, changes liquid, and collects culture solution supernatant.
Step 3, the extraction of ovarian follicle RNA, cDNA reverse transcription and quantitative fluorescent PCR: collect 5 culture before and culture after 2d,
The mouse ovarian follicle of 4d, 6d and 8d after cleaning 3 times in PBS, are operated in strict accordance with the specification of micro RNA extracts kit, are used
Micro-spectrophotometer detects standard compliant RNA sample and can carry out according to reverse transcription reagent box and PCR kit for fluorescence quantitative
The detection of related gene.Result input Graphpad prism software is analyzed and charted.
Step 4, follicular activity detection: for the survival rate for assessing and determining ovarian follicle, choose by different number of days developmental
Ovarian follicle with fluorescent reagent Calcein AM and Eethidium homodimer (EthD-1) under dark condition into dyeing,
15min is placed in 37 DEG C of incubators, then in the activity of fluorescence microscopy microscopic observation ovarian follicle.Wherein EthD-1 can enter and occur
In the cell of cell membrane breakage or apoptosis, bright red fluorescence is issued.Feux rouges is all issued according to granular cell and egg mother cell
Ratio judge the existing state of ovarian follicle.
The measurement of step 5, ovarian follicle and egg mother cell: after changing liquid, the developmental state of ovarian follicle is observed simultaneously under inverted microscope
It takes pictures, the long axis and minor axis diameter of ovarian follicle and egg mother cell is measured with ImageJ software, and average value is female as ovarian follicle and ovum
The diameter of cell, while recording the survival condition of ovarian follicle.When the egg mother cell and/or granular cell color for observing ovarian follicle become
Secretly, when not having refractivity under the microscope, then it is judged as that death has occurred for ovarian follicle.
Step 6, statistical method
As a result it is analyzed using 21.0 statistical software of SPSS, measurement data is indicated with mean ± standard deviation (x ± s).Respectively
Compared between group using one-way analysis of variance.P≤0.05 is that difference is statistically significant.
Further, in step 1, the ingredient of the separating liquid are as follows: L-15 culture medium+10%FBS+1% blueness/streptomysin.
Further, in step 1, the ingredient of the digestive juice are as follows: the clostridiopetidase A+0.1g/mL DNase of 1mg/mL.
Further, the ingredient of the ovarian follicular growth culture solution are as follows: α-MEM culture medium+3%BSA+1%ITS+1% blueness/chain
Mycin, 0.5IU/mL FSH+0.23mM PNa+50g/ml Vc.
Further, the ingredient of oocyte maturation liquid are as follows: ovarian follicular growth culture solution+1.5IU/mL HCG+10ng/mL EGF
The invention has the benefit that
Result of study of the invention is shown, can obtain ideal vitro culture system using of the invention.Pass through
The study show that the inhibitor can reduce the apoptosis of granular cell in Ovarian Follicles really, this is ovarian follicle after supervention
It educates and provides reliable guarantee.
Detailed description of the invention
Fig. 1 is the form under different separation methods after follicle culture, wherein Figure 1A: the ovarian follicle of enzyme digestion culture 5 days；
Figure 1B: the ovarian follicle of enzyme combination Mechanical Method culture 6 days；Fig. 1 C: the ovarian follicle of enzyme process culture 6 days, egg mother cell occur excessive；Fig. 1 D:
First polar body is discharged in the ovarian follicle of Mechanical Method culture 8 days, egg mother cell.Arrow show oolemma in figure, and five-pointed star show
One polar body, scale size are 50 microns；
Fig. 2 is influence of the Y27632 to granular cell and Oocyte Development, wherein Fig. 2A: real time fluorescent quantitative detection
The expression of granular cell (Amh, Lhr, Fshr and Foxl2) and oocyte gene (Bmp15).* P < 0.05, * * P <
0.001 compared with the control group；Fig. 2 B: the distribution of spindle microtubule (α-tublin) after Immunofluorescence test oocyte maturation；
Fig. 3 A: the viability examination after follicle culture 6 days；
Fig. 3 B: the expression of apoptosis-related genes Bad and apoptosis suppressor Xiap in fluorogenic quantitative detection ovarian follicle；* P <
0.05, * * P < 0.001 is compared with the control group.
Specific embodiment
Technical solution of the present invention is described in more detail with reference to the accompanying drawings and detailed description.
In such a way that Mechanical Method, enzyme (clostridiopetidase A) digestion method and the two combine, preantral follicle is separated,
It finds through statistics, is obtained by Mechanical Method (after obtaining ovary, one ovary guarantees to separate ovarian follicle within half an hour)
Egg mother cell quantity is most, ova nuda minimum number (table 1).
The ovarian follicle and oocyte number (x ± s) that the different separation methods of table 1 obtain
Table1 The count ofsingle follicle and denuded oocyte at different
method
As shown in Figure 1, after follicle culture, either enzyme process or enzyme-Mechanical Method can all cause membrana follicularis different degrees of
Breakage makes granular cell that excessive (Figure 1A and Figure 1B) occur, causes ovarian follicle to be unable to normal development, egg mother cell discharges in advance.With
Mechanical Method is compared, and the oolemma of this egg mother cell is also relatively thin (Fig. 1 C), it is difficult to be developed coelosis, but by Mechanical Method, can be obtained
First polar body can be discharged, mention after HORMONE TREATMENT by obtaining basal lamina of follicles and the more complete ovarian follicle of granular cell, late stage of culture
Show that ovarian follicle completes initial meiosis (Fig. 1 D).
By 30 microlitres of drop culture of the single ovarian follicle obtained using Mechanical Method, addition Rock inhibitor Y- is found
After 27632, to the apparent growth of growth diameter (table 2 and table 3) nothing of ovarian follicle and egg mother cell or inhibiting effect.
The diameter (x ± s) of 2 different disposal group ovarian follicle of table
Table2 The diameter offollicle in different treatment.
The diameter (x ± s) of 3 different disposal group egg mother cell of table
Table3 The diameter ofoocyte in different treatment.
The method for further using real time fluorescent quantitative, examines the expression of granular cell and egg mother cell related gene
It surveys.Wherein, granular cell expressing gene Amh, Lhr, the increase of Fshr, Foxl2 with cultivated days, of inhibited dose of processing
Granulocyte expression quantity is higher with respect to untreated fish group, and egg mother cell specific gene Bmp15 does not have significant change, shows inhibitor
There is certain influence to the development of granular cell.After culture 8 days, after processing group ovarian follicle passes through mature liquid culture, first polar body is discharged,
It completes initial meiosis (Fig. 2).
In order to be observed in Ovarian Follicles in real time with the presence or absence of apoptosis phenomenon, the ovum of random selection culture different number of days
Bubble, has carried out cell viability detection.Experimental result discovery, in the ovarian follicle of culture the 6th day, red fluorescence sun in control group ovarian follicle
Property granular cell number more than addition Y27632 group, being developed the color by DAPI shows, granular cell DNA fragment is equally more than processing group.
Further through fluorescence quantitative PCR detection apoptosis-related genes Bad and apoptosis suppressor Xiap, follicle culture later period, control group
The obvious up-regulation of Bad expression, shows that ovarian follicle has started apoptosis in ovarian follicle.Meanwhile apoptosis suppressor Xiap is the 4th of follicular development
Its obvious up-regulation shows that Y27632 has apparent inhibiting effect to the apoptosis of ovarian follicle, but this inhibiting effect is then inadequate at the 8th day
Obviously (Fig. 3 A, Fig. 3 B).
Ovarian follicle is the basic function unit that egg mother cell occurs and develops, and establishes perfect preantral follicle in-vitro separation and training
Feeding system be not only research Growth of Oocytes, maturation, the effective means of granulosa cell proliferation, and be also reproduction resource it is cold
Freeze deposit --- the unique one external approach for obtaining mature oocyte after ovarian follicle freezing, to female cancer patients fertility
It saves significant.
Influencing the ectogenetic primary factor of ovarian follicle is the suitable preantral follicle separation method of selection, it can be common that enzymatic hydrolysis
The method that method, mechanical phonograph recorder separation and the two combine.Wherein, enzyme process mostly uses clostridiopetidase A, trypsase, dispase and release enzyme etc.
Structure between vitellophag and cell or between cell and extracellular matrix, to achieve the purpose that the single ovarian follicle of separation.Though
Right part result of study shows that the ovarian follicle quantity obtained by enzyme process is more compared with Mechanical Method, but to thecacells, ovarian follicle base
All there is certain influence in the granular cell of film or even egg mother cell outer layer, and these be all influence the development of ovarian follicle later period must
Want factor.In our experimental result, enzyme process is equally also demonstrated to ovarian follicle, the integrality of even egg mother cell has centainly
Destruction.Mechanical Method mainly uses 25-30 diameter syringe needle, divides under Stereo microscope ovary tissue
The method for solving and removing single ovarian follicle, this method are higher to the precision and operating time requirement of operator, and general control exists
Each ovary is removed in 30 minutes and is finished.In our current research, by a large amount of operation practice, can guarantee to remove in 20 minutes
One complete ovary tissue, selection wherein form excellent about 25 pieces of single ovarian follicle or so for cultivating, this is to guarantee ovarian follicle
Continue the basic guarantee of development.
Suitable follicle culture technology is the key factor of external follicular development.Currently, according to culture extracellular matrix architecture
Follicle culture system can be divided into 3D stereoscopic culture and 2D plane cultivating system by feature, the former farthest remains maintenance ovum
The stereochemical structure of growth is steeped, convenient for balanced, autocrine and the paracrine path between guarantee cell in gap between cell and cell
Normal pass, in order to maintain this structure, frequently with the methods of sodium alginate, fibrin, the cell transwell.And it adopts
Microdrop culture with simulation 3D is easy to operate with its, and the advantages such as brief extracorporeal treatment time can also be used to do preliminary
Follicle culture.This research the results show that ideal vitro culture system can also be obtained using this method.
Rho/ROCK signal pathway inhibitor Y27632 is in the maintenance for promoting multipotential stem cell self-renewing and inhibits apoptosis
Aspect has numerous research, but the research in terms of reproduction cell is very few.Correlative study shows by inhibiting the signal path,
But the relevant albumen of apoptosis reduces 20%-30%.By the study show that, which can reduce follicular development really
The apoptosis of granular cell in journey, this provides reliable guarantee for the development that continues of ovarian follicle.
As a result by Mechanical Method, enzyme-Mechanical Method, every mouse of enzyme process respectively can get 167.25 ± 67.25,135.33 ±
74.11 and 52.33 ± 38.89 pieces of single ovarian follicles.And ovum can all be destroyed using the separation method containing enzyme by morphological observation
Steep integrality.Y27632 is added to the balanced growth diameter (25.9 ± 3.69) of ovarian follicle and the balanced growth diameter of egg mother cell
(1.87 ± 1.29) are compared with the control group without significant difference.It detects and finds through fluorescent quantitative PCR technique, not with ovarian follicle
Disconnected development, granular cell express related gene Amh, Lhr, Fshr, Foxl2, are in compared with the control group significant difference, and ovum is female
Cell-specific genes Bmp15 is then without significant change.It is found moreover, being dyed through micro-pipe, the addition of Y27632 has no effect on ovum mother
Cell completes initial meiosis.It is learnt by the result of ovarian follicle viability examination, the apoptosis of granulosa cell phenomenon of processing group is few
In control group, apoptogene Bad is significantly lower than control group, and can cause the significant up-regulation of apoptosis suppressor Xiap.Conclusion machine
Tool method can obtain more single ovarian follicle, and the addition of Y27632 can improve the expression of the gene of granular cell, and to egg mother cell
It influences little.The apoptosis of granular cell can be reduced simultaneously, to maintain the normal development of ovarian follicle.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Claims (4)
1. the research method that a kind of Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis, feature
It is, comprising the following steps:
The separation of step 1, preantral follicle: obtaining mouse ovarian under stereoscope, in superclean bench, is cleaned with separating liquid
After 3 times, mechanical phonograph recorder separation: by ovarian metastasis into the glass surface ware containing 2mL separating liquid, with dissecting needle in stereomicroscope
Lower blunt separation ovarian follicle guarantees that the time of one ovary ovarian follicle in-vitro separation does not exceed 30min；It is cleaned three times with culture solution
Afterwards, detection form is complete, ovarian follicle of the diameter at 100~130 μm；Ovary: being directly put into the recessed ware of digestive juice glass by enzyme process, in
After 37 DEG C of culture 1h, remove the tissue block in digestive juice, abandon supernatant after being added in follicle culture liquid low-speed centrifugal 5min, then with training
After nutrient solution cleaning twice, the detection complete preantral follicle of form is cultivated；Enzyme process-Mechanical Method: 20min is handled with digestive juice
Afterwards, then use above-mentioned Mechanical Method separation method；
The culture of step 2, preantral follicle: selecting diameter is the culture dish of 60mm, and 12h prepares the follicle culture liquid that 30 μ L rise in advance
Drop, and 2mL mineral oil is being covered above；The preantral follicle that separation obtains is put into drop in 37 with self-control glass rim suction pipe
DEG C incubator culture, every 2d half, which is measured, changes liquid, and collects culture solution supernatant；
Step 3, the extraction of ovarian follicle RNA, cDNA reverse transcription and quantitative fluorescent PCR: 5 are collected and cultivates 2d, 4d, 6d after preceding and culture
With the mouse ovarian follicle of 8d, after being cleaned 3 times in PBS, operated in strict accordance with the specification of micro RNA extracts kit, use is micro
Spectrophotometer detects standard compliant RNA sample and carries out dependency basis according to reverse transcription reagent box and PCR kit for fluorescence quantitative
The detection of cause；Result input Graphpad prism software is analyzed and charted；
Step 4, follicular activity detection: for the survival rate for assessing and determining ovarian follicle, the developmental ovarian follicle chosen by different number of days
15min is placed in 37 DEG C of incubators, then into dyeing under dark condition with fluorescent reagent Calcein AM and EthD-1
In the activity of fluorescence microscopy microscopic observation ovarian follicle；Wherein EthD-1 enters in the cell that cell membrane breakage or apoptosis has occurred, hair
Bright red fluorescence out；The ratio that feux rouges is all issued according to granular cell and egg mother cell judges the existing state of ovarian follicle；
The measurement of step 5, ovarian follicle and egg mother cell: after changing liquid, observing the developmental state of ovarian follicle and taken pictures under inverted microscope,
With the long axis and minor axis diameter of ImageJ software measurement ovarian follicle and egg mother cell, and average value is as ovarian follicle and egg mother cell
Diameter, while recording the survival condition of ovarian follicle；When the egg mother cell and/or granular cell color of observing ovarian follicle are dimmed, aobvious
When not having refractivity under micro mirror, then it is judged as that death has occurred for ovarian follicle；
Step 6, statistical method
As a result it is analyzed using SPSS21.0 statistical software, measurement data is indicated with mean ± standard deviation (x ± s)；Between each group
Compared using one-way analysis of variance；P≤0.05 is that difference is statistically significant.
2. Rho-ROCK signal pathway inhibitor Y27632 according to claim 1 influences mouse eggs bubble ectogenesis
Research method, which is characterized in that in step 1, the ingredient of the separating liquid are as follows: L-15 culture medium+10%FBS+1% blueness/strepto-
Element.
3. Rho-ROCK signal pathway inhibitor Y27632 according to claim 1 influences mouse eggs bubble ectogenesis
Research method, which is characterized in that in step 1, the ingredient of the digestive juice are as follows: the clostridiopetidase A+0.1g/mL DNase of 1mg/mL.
4. Rho-ROCK signal pathway inhibitor Y27632 according to claim 1 influences mouse eggs bubble ectogenesis
Research method, which is characterized in that the ingredient of the follicle culture liquid are as follows: α-MEM culture medium+3%BSA+1%ITS+1% blueness/chain
Mycin, 0.5IU/mL FSH+0.23mM PNa+50g/ml Vc.
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| 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 |
| CN112980773A (en) * | 2021-03-15 | 2021-06-18 | 中山大学附属第六医院 | Method for culturing mouse secondary follicle in vitro |
| CN112980773B (en) * | 2021-03-15 | 2023-11-17 | 中山大学附属第六医院 | Method for in vitro culture of mouse secondary follicles |
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