CN108795849A - Application of the CDC42 signal paths in activating primordial follicle growth and development - Google Patents
Application of the CDC42 signal paths in activating primordial follicle growth and development Download PDFInfo
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Abstract
The present invention relates to biotechnologies, disclose application of the CDC42 signal paths in activating primordial follicle growth and development.The present invention confirms that quickly activation CDC42 can activate primordial follicle using CDC42 pathway inhibitors and over-express vector, and promote its development, it activates and develops for primordial follicle and provide a kind of completely new signal path, CDC42 activator and CDC42 over-express vectors can be applied in activating primordial follicle growth and development reagent and/or in vitro in non-treatment purpose activation primordial follicle growth.
Description
Technical field
The present invention relates to biotechnologies, more particularly to CDC42 signal paths are in activating primordial follicle growth and development
Application.
Background technology
Mammalian Ovary is the necessary reproductive organs of jenny reproduction procreation including female human, ovary
There are two functions for main tool：It supports Oocyte Development maturation and generates female sex hormone.From birth to puberty, child-bearing period, most
Climacteric and senescence phase are arrived afterwards, and ovary experienced a series of variations, these variations directly affect reproductive function, property feature and the heart
Reason.
And in female reproductive cell growth course, primordial follicle is the most basic functional unit of ovary again.Primordial follicle
By single suspend mode egg mother cell and be wrapped in surrounding one layer flat preceding granular cell and collectively form.The quantity of primordial follicle
I.e. primordial follicle library is decided in mammal period from prenatal to postnatal, and initial number is determined and is subject in early development stage
It is fixed, and primordial follicle library is non-renewable after adult.In ovary, the primordial follicle of suspend mode needs to be referred to as by one
The process of ovarian follicle activation is allowed for access subsequent development, and the stage of development of primary follicle, secondary follicle and antral follicles is then subjected to,
The ripe ovum of final discharge, after fertilization development are offspring individual.Thus, the quantity of primordial follicle determines female mammal
Using the quantity of reproduction resource in ovary, primordial follicle normally activates development then to determine the matter using reproduction resource
Amount.After primordial follicle activation occurs, the volume of egg mother cell increases by hundreds times, and the granular cell of surrounding is proliferated rapidly, by most
First simple squamous granular cell is developed to several layers of cubic thousands of a cells.Then when follicular development to antral follicles rank
Section, growth are developed to final ovulatory phases in turn by the stimulation of the follicle-stimulating hormone (FSH) FSH of pituitary.Finally preovulatory
Under the action of the peaks metakentrin LH, corpus luteum then occur for egg mother cell discharge, granular cell turn to the developments of fertilized eggs to prepare.
In the ovary of mammal, the primordial follicle in primordial follicle library is constantly activated and enters growth period, with people
For, women primordial follicle library is established initial stage, about 5,000,000 or so primordial follicle, in normal adult female's every month
About 1000 primordial follicles enter growth period, but the mature egg being discharged in life in women only have it is trivial hundreds of,
Utilization rate is very low.And when the primordial follicle in primordial follicle library is less than 1000, ovary will no longer be ovulated, i.e. women at this time
Into menopause.If the primordial follicle in ovary can be used, it will there is huge application value.And in some diseases
Under the conditions of reason, primordial follicle is unable to normal Activate and development, such as Turner syndrome and premature ovarian failure disease cause
Women of childbearing age is infertile, thus find it is a kind of it is effective adjust primordial follicle activation method have great importance.
It is existing research shows that PI3K signal paths and mTOR signal paths play in the activation of primordial follicle it is important
Role.Existing primordial follicle activator be PI3K signal paths activator, PTEN inhibitor and mTOR signal path activator,
But these reagents activation primordial follicle needs the time longer, and patient's Repeated Operation is caused to increase pain.It all needs to take out cortex block,
External freezen protective transplants back patient after patient restores a period of time after the cortex block of taking-up is carried out Activation In Vitro processing
In vivo, primordial follicle is developed step by step to take out ovum after mature egg, and supervention after carrying out in patient body is transplanted back after in vitro fertilization
It educates.Meanwhile currently used PI3K signal paths activator or mTORC1 signal path activator, it is commonly chemical synthesis point
Son, and since PI3K signal paths activator or mTORC1 signal paths with cancer there is direct relation, safety to need
Further assessment.Thus, it finds a kind of biological natural agent that can quickly activate primordial follicle and a kind of new Activiation method comes
Patient is avoided to have huge realistic meaning by the pain of Repeated Operation.It can not only be provided for infertile female patient more
Kind therapeutic scheme reduces the injury that operation is brought to patient；And it reduces that treated potential complication generates.
Invention content
In view of this, the purpose of the present invention is to provide CDC42 signal paths in activating primordial follicle growth and development
Using efficient activation primordial follicle growth and development by quickly activating or being overexpressed CDC42；
Another goal of the invention of the present invention is that provide CDC42 activator is activated with non-treatment diagnostic purpose in vitro
The application of primordial follicle growth and development, and answering in the reagent for preparing in vivo or in vitro activation primordial follicle growth and development
With；
Another object of the present invention is to provide a kind of specific in vivo or in vitro activation primordial follicle growth and development
Reagent；
Another object of the present invention, which is to provide, is overexpressed CDC42 carriers in vitro with the activation of non-treatment diagnostic purpose
The application of primordial follicle growth and development, and answering in the reagent for preparing in vivo or in vitro activation primordial follicle growth and development
With.
The present invention is small using CDC42 pathway inhibitors ML141 (Sigma companies) and ZCL278 (Selleck companies) processing
Mouse ovary simultaneously continues to cultivate, then ovary is fixed to and carried out histology, the results show that the place of CDC42 pathway inhibitors
Reason can cause the activation of primordial follicle to significantly inhibit, and CDC42 activity is remarkably decreased in ovary, and ovarian follicle count results show CDC42
Primordial follicle activity ratio is obviously lowered after activity suppression；
The above results indication CDC42 accesses may activate growth to have close ties with primordial follicle, therefore the present invention is built
Overexpression processing is carried out in CDC42 over-express vectors and transfection to mouse ovarian, it is aobvious that immune-blotting method shows that CDC42 is overexpressed
Work improves expression quantity of the CDC42 albumen in ovary, and ovarian follicle count results show original ovum after the processing of CDC42 overexpressions
Bubble activity ratio obviously raises, while histology shows to activate ovarian follicle significantly to increase after CDC42 overexpressions are handled.Above-mentioned two
There is apparent direct positive acting in activation primordial follicle growth in aspect the results show, CDC42 signal paths, you can
Efficient activation primordial follicle is grown by quickly activating or being overexpressed CDC42.
Based on the research conclusion of above-mentioned signal path, the present invention provides CDC42 activator to prepare activation primordial follicle
Application in growth agents and/or in vitro with the application of non-treatment purpose activation primordial follicle growth.
Wherein, proposed by the invention is in vitro in order into one with the application of non-treatment purpose activation primordial follicle growth
The step research subsequent growth and development process of ovarian follicle, simultaneously because studying in vitro, is unable to reach treatment used in basic research
Purpose.
It is due to the establishment that CDC42 signal paths act in activation primordial follicle growth, then all known in the art
CDC42 activator all can quickly activate CDC42 to go forward side by side the growth of one-step activation primordial follicle, and in the specific embodiment of the invention
In, the present invention is verified by CN02 (Cytoskeleton companies) and the specific activator of two kinds of EGF (Sigma companies)
Experiment.
Mouse Ovary Tissues are handled in vitro using CDC42 Pathway Activation agent, find ratio of the growing follicle than primordial follicle
Rate is noticeably greater than control group, and downstream marker molecule FOXO3a goes out core, Oocyte Activation after primordial follicle activation of oocytes
The phosphorylation of the downstreams critical path PI3K marker molecule FOXO3a and AKT obviously raise, and show that primordial follicle activity ratio is apparent
It is promoted.
And handle Mouse Ovary Tissues in vivo, it is found that ovary contains more growing follicles, the ratio shared by primary follicle
Example is apparently higher than control group, and reagent treatment and mode have no adverse effects to mouse in vivo, and with promotion female mice fertility
Effect.
According to above-mentioned technique effect, the present invention corresponds to the reagent for proposing a kind of activation primordial follicle growth, including CDC42
Activator, a concentration of 50-200ng/ml of the CDC42 activator, preferably 100ng/ml.
In the activation reagent handled in vitro, in addition to the CDC42 activator of core further include active cell culture solution and/or
Other published primordial follicles activate drug；In the specific embodiment of the invention, the active cell culture solution is DMEM/
F12 culture solutions, are preferably added ITS and antibiotic, and penicillin and streptomysin may be selected in antibiotic.More specifically, described
Reagent includes 100 times of ITS, 10Units/ml penicillin and 10 g/ml streptomysins and 50- using DMEM/F12 as mother liquor
The CDC42 activator of 200ng/ml；
In the activation reagent handled in vivo, in addition to the CDC42 activator of core further include Biological water colloidal sol and/or other
Primordial follicle activates drug, wherein Biological water colloidal sol to may be selected to be hyaluronic acid derivatives, more specifically the matrix hydrosol
Matrigel or uterine cavity cross-linked hyaluronic acid gel.
Based on the research conclusion of aforementioned signal access, another aspect of the present invention additionally provides CDC42 over-express vectors and is making
The standby application activated in primordial follicle growth agents and/or the application grown in vitro with non-treatment purpose activation primordial follicle.
Therein is still in vitro to further study the subsequent life of ovarian follicle with the application of non-treatment purpose activation primordial follicle growth
Long growth course, simultaneously because studying in vitro, is unable to reach therapeutic purposes used in basic research.
Preferably, the CDC42 over-express vectors can pass through genetic engineering skill based on over-express vector by commercially available
Art accesses CDC42 encoding genes, and the over-express vector is pSicoR or pLVX-IRES-ZsGreenl.
According to the application of CDC42 over-express vectors, the present invention also provides a kind of activation including CDC42 over-express vectors
The reagent of primordial follicle growth
Can explicitly it be seen according to experiment provided by the invention, CDC42 activator and CDC42 over-express vectors pass through
CDC42 signal paths, have activated primordial follicle, can cope with the low problem of ovarian follicle activation developmental potency.Therefore CDC42 activator
And/or CDC42 over-express vectors can be applicable in the drug of disease caused by preparing treatment ovarian follicle under-reserve, the ovarian follicle swashs
Lowly caused disease is premature ovarian failure or aging female ovary dysfunction to developmental potency living.
By above technical scheme it is found that the present invention is confirmed quickly using CDC42 pathway inhibitors and over-express vector
Activation CDC42 can activate primordial follicle, and promote its development, activate for primordial follicle and develop provide it is a kind of completely newly
CDC42 signal paths can apply CDC42 activator and CDC42 over-express vectors in activation primordial follicle growth and development examination
In agent and/or in vitro in non-treatment purpose activation primordial follicle growth.
Description of the drawings
Fig. 1, which is shown, inhibits CDC42 activity that primordial follicle is caused to activate the result being obstructed；Wherein, A is egg mother cell DDX4
With the immunofluorescence results of nucleus Hoechst, B is the immunoblot results of CDC42 expression, after C is inhibition CDC42 in ovary
Activate ovarian follicle quantity as a result, abscissa is processing group, control is control group, and ML141 and ZCL278 are to inhibit CDC42
Experimental group, ordinate be activation ovarian follicle quantity；
Fig. 2 show ovary and is overexpressed the result that CDC42 promotes primordial follicle activation；Wherein, A is exempting from for CDC42 expression
Epidemic disease Blot results, B are the accounting of activation ovarian follicle in ovary after being overexpressed CDC42 as a result, abscissa is to handle group, control
For control group, Cdc42-OE is the experimental group for being overexpressed CDC42, and ordinate is the accounting for activating ovarian follicle, and C is egg mother cell DDX4
With the immunofluorescence results of nucleus Hoechst；
Fig. 3 show immunofluorescence results of the newborn mice ovary after EGF is cultivated；
Fig. 4 show immunoblotting analysis result of the newborn mice ovary after EGF is cultivated；
Fig. 5 show hematoxylin coloration result of the newborn mice ovary after EGF cultures and the transplanting of kidney envelope；
Fig. 6 show ovarian follicle statistical result of the newborn mice ovary after EGF cultures and the transplanting of kidney envelope, and abscissa is place
Group is managed, control is control group, and EGF for 0.5h are the experimental group of in vitro culture, and left figure ordinate is growing follicle
Accounting, right figure ordinate are the result of ovarian follicle sum；
Fig. 7 show immunoblotting analysis result of the adult mice ovary after EGF is cultivated；
Fig. 8 show ovarian follicle statistical result of the adult mice ovary after EGF cultures and the transplanting of kidney envelope, and abscissa is place
Group is managed, control is control group, and EGF for 0.5h are the experimental group of in vitro culture, and ordinate is the accounting of growing follicle；
Fig. 9 show the result of injection matrix hydrosol retention time in ovary；
Figure 10 show the hematoxylin coloration result of the matrix hydrosol of the injection containing EGF in mouse ovarian；
It is horizontal that Figure 11 show the ovarian follicle statistical result of the matrix hydrosol of the injection containing EGF after a week, left figure in mouse ovarian
Coordinate is ovarian follicle rank, and primordial is primordial follicle, and primary is primary follicle, and secondary is secondary follicle,
Antral is antral follicles, and ordinate is the accounting of Follicles, and right figure abscissa is processing group, and control is control group,
The experimental group of the various concentration processing of 100ng/ml and 1000ng/ml, ordinate are the result of ovarian follicle sum；
It is horizontal that Figure 12 show the ovarian follicle statistical result of the matrix hydrosol of the injection containing EGF after two weeks, left figure in mouse ovarian
Coordinate is ovarian follicle rank, and primordial is primordial follicle, and primary is primary follicle, and secondary is secondary follicle,
Antral is antral follicles, and ordinate is the accounting of Follicles, and right figure abscissa is processing group, and control is control group,
The experimental group of the various concentration processing of 100ng/ml and 1000ng/ml, ordinate are the result of ovarian follicle sum；
Figure 13 show the fecundity testing result after the matrix hydrosol of the injection containing EGF in mouse ovarian, and left figure is place
The testing result of mouse fertility is managed, abscissa is the week old of mouse, and ordinate is average every female mice institute newborn's number, and A is indicated
EGF processing groups, B indicate control group；Right figure is processing the given birth to filial generation weight of mouse as a result, abscissa is the week old weighed, and indulges seat
It is designated as weight.
Specific implementation mode
The invention discloses application of the CDC42 signal paths in activating primordial follicle growth and development, people in the art
Member can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications
Apparent to those skilled in the art, they are considered as being included in the present invention.Activator of the present invention, mistake
Expression vector and its related application are described by embodiment, and related personnel can obviously not depart from the present invention
Hold, activator described herein, over-express vector and its related application be modified or suitably changed in spirit and scope and group
It closes, to realize and apply the technology of the present invention.
In the specific embodiment of the invention, it is related to contrast test, each test group is other than due difference, remaining experiment
Environment and raw material etc. are consistent.
Application of the CDC42 signal paths provided by the present invention in activating primordial follicle growth and development is done into one below
Walk explanation.
Embodiment 1：The reagent (external) of activation primordial follicle growth and development of the present invention
Solution I：Basic active cell culture solution；
Solution II：DMEM/F12 culture solutions are mother liquor, include ITS；
CDC42 Pathway Activation agent：EGF；
Reagent of the present invention can pack solution I, solution II and the agent of CDC42 Pathway Activations respectively, be packaged in together
In same packing box, wherein become activation primordial follicle reagent after solution I and CDC42 Pathway Activation agent mixing, solution II is in body
It is used as cleaning when outer activation primordial follicle to use；
Above-mentioned primordial follicle Activation In Vitro preparation method of reagent thereof, solution I：Basic active cell culture liquid making method is such as
Under：DMEM/F12 is mother liquor, includes 100 times of ITS, 10Units/ml streptomysin and 10 g/ml penicillin；Solution II is matched
Method processed is as follows：DMEM/F12 culture solutions are mother liquor, also containing 100 times of ITS, the agent of CDC42 Pathway Activations (EGF) working concentration
For 50-200ng/ml, when concrete application, uses 100ng/ml.
Reagent application method is as follows：
It takes mouse ovarian with 5ml solution IIs, is cleaned repeatedly with solution II more than three times.
It takes 1ml solution Is to be mixed with reagent, 10ml active cell culture solutions, a concentration of 50-200ng/ is configured to solution I
ml。
Mouse ovarian is incubated at the 24- orifice plates (millipore) containing cell culture cell, is added in culture hole
400L active cell culture solutions are cultivated 0.5 hour, and condition of culture is 37 DEG C, 5%CO2；
After 0.5h, activation is completed, and tissue can be used for subsequent operation.
Embodiment 2：The reagent (internal) of activation primordial follicle growth and development of the present invention
CDC42 Pathway Activation agent：EGF；
Water-soluable gel：Matrix hydrosol matrigel or uterine cavity cross-linked hyaluronic acid gel, the present embodiment use base
Matter hydrosol matrigel.
Reagent of the present invention can pack the agent of CDC42 Pathway Activations and water-soluable gel respectively, same being packaged in together
In one packing box, the agent of CDC42 Pathway Activations (EGF) a concentration of 50-200ng/ml when use is dissolved in the hydrosol, concrete application
Shi Caiyong 100ng/ml.
Reagent application method is as follows：
1) aseptically, reagent is sufficiently mixed in absorbable uterine cavity cross-linked hyaluronic acid gel；
2) gel for being mixed with reagent in the course of surgery, is applied to Ovarian surface by internal injection mode；
3) in vivo after processing, the development condition of ovarian follicle in periodic detection ovary, with natural conception after graaffian follicle or
Supplementary reproduction.
Embodiment 3：CDC42 signal paths are activated to primordial follicle and the influence of growth and development
1, CDC42 Inhibition tests
Experimental animal：Experiment animal used is newborn 6 days C57 mouse.
Inhibitor：CDC42 pathway inhibitors ML141 is purchased in Sigma companies, and ZCL278 is purchased in Selleck companies.
Processing 3 days is carried out to the newborn mice ovary of in vitro culture using two kinds of CDC42 inhibitor ML141 and ZCL278,
Followed by original follicular development situation in histotomy and reproduction cell specific immunofluorescence dyeing detection ovary, and use
CDC42 activity changes after immunoblotting mode detection process in ovary, and situation is activated by ovarian follicle counting statistics ovarian follicle.Knot
Fruit sees Fig. 1.
As shown in Figure 1, inhibit CDC42 activity that primordial follicle activation is caused to be obstructed.The result shows that utilizing two kinds of CDC42 in A
Inhibitor ML141 and ZCL278 handles the newborn mice ovary of in vitro culture, and the activation of primordial follicle can be caused notable
Inhibit；Ovary CDC42 activity is remarkably decreased after result illustrates inhibitor processing in B；Ovarian follicle count results show that CDC42 lives in C
Property inhibit after primordial follicle activity ratio obviously lower.
2, CDC42 is overexpressed experiment
Over-express vector is built
The primer sequence of the over-express vector of CDC42 is as follows：
Forward primer, 5 '-CTCGAGATGCAGACAATTAAGTGTGTTG-3 '；
Reverse primer, 5 '-GGATCCTAGCAGCACACACCTGCGGCTC-3 '.
CDS (code area) sequence in CDC42 gene mRNA sequences utilizes upper using the cDNA of embryonic-period mice ovary as template
Primer amplification target fragment is stated, the size of the target gene fragment of amplification is determined by agarose gel electrophoresis, passes through agarose
Gel recycles and measures the DNA concentration after recycling, selects different enzymes to do double digestion and to digestion according to the restriction enzyme site of selection
Product is purified.Digestion products after purification are connected to carrier, the product after connection are transformed into competent cell, root
The competent cell converted for requiring absorption different volumes according to the experiment, is added to the LB agar mediums containing corresponding antibiotic
On, it is incubated overnight 12-16h.
The LB liquid medium of 1mL is added into centrifuge tube, antibiotic is added into culture medium, chooses single bacterium colony and is put into training
It supports in base, 200rpm/min, 8-10h is shaken on 37 DEG C of shaking tables.It is expected by the way that whether bacterium solution PCR detections DNA fragmentation size meets
Size.The sequence of target gene and being consistent completely for design are determined by the method for sequencing later, qualified bacterium solution is added
To final concentration of 25%, -80 DEG C freeze 50% glycerite, and over-express vector is obtained by the method that no endogenous toxic material plasmid carries greatly
And transfected ovary is for activating ovarian follicle.
Over-express vector carries out processing 4 days by way of transfection to the newborn mice ovary of in vitro culture, utilizes group afterwards
Original follicular development situation in slice and reproduction cell specific immunofluorescence dyeing detection ovary is knitted, and uses immunoblotting side
CDC42 expression variations after formula detection process in ovary, and situation is activated by ovarian follicle counting statistics ovarian follicle.
By Fig. 2 results it is found that ovary, which is overexpressed CDC42, promotes primordial follicle activation.The result shows that passing through CDC42 mistakes in A
The transfection of expression vector carries out CDC42 overexpression processing to the newborn mice ovary of in vitro culture, and immune-blotting method shows
CDC42 overexpressions have been obviously improved expression quantity of the CDC42 albumen in ovary.Ovarian follicle count results show that CDC42 is overexpressed in B
Primordial follicle activity ratio obviously raises after processing；Activation ovarian follicle is notable after histology shows the processing of CDC42 overexpressions in C
Increase.
It can be proved that CDC42 signal paths are to activation primordial follicle, simultaneously enhancing development has result of both comprehensive
Positive correlation.
Embodiment 4：The agent of CDC42 Pathway Activations promotes the activation and development of mouse primordial follicle in vitro
Using the reagent and application method of embodiment 1 to the Whole ovary culture of newborn 6 days mouse (C57 mouse) and 35 days
Age mouse (C57 mouse) ovary is handled and is detected, and as a result sees Fig. 3-8；
1, the experiment of newborn 6 days mouse
After newborn 6 days mouse ovarians were handled 0.5 hour and are cultivated 12 hours with the CDC42 Pathway Activation agent EGF short time,
Ovary is fixed and carries out the Immunofluorescence test of activation of oocytes marker molecule.Such as Fig. 3, left side is control, and right side is sharp
Ovary after work.It is obviously improved the result shows that the FOXO3a of egg mother cell in primordial follicle can be caused to go out core ratio after EGF processing.
Meanwhile such as Fig. 4, Western blot results show that the phosphorylation of FOXO3, AKT obviously raise, and illustrate primordial follicle
Activation.
The new life ovarian transplantation in 6 days that activator is handled 0.5 hour takes out tissue to receptor mouse kidney envelope after 2 weeks, such as schemes
5, left side is control group, and right side is the ovary after activation.Then unite to the quantity and ratio of primordial follicle and growing follicle
Meter, such as Fig. 6, as a result, it has been found that the growing follicle of processing group than primordial follicle ratio be more than control group, and compare and processing
The ovarian follicle sum of group is without significant difference.Illustrate that the short time processing of CDC42 Pathway Activation agent EGF is original to newborn 6 days mouse
Ovarian follicle has activation.
2,35 day-old Mices are tested
35 days mouse ovarians of birth are cut into 4 pieces, handled 0.5 hour with CDC42 Pathway Activation agent EGF and cultivate 12 are small
When, such as Fig. 7, Western blot results show that the phosphorylation of FOXO3, AKT obviously raise, and illustrate that primordial follicle activates.It will swash
The birth ovarian transplantation in 35 days that agent living handles 0.5 hour takes out tissue, carrying out paraffin to it cuts to receptor mouse kidney envelope after 1 week
Piece and HE dyeing.Then the quantity and ratio of primordial follicle and growing follicle are counted, such as Fig. 8, as a result, it has been found that processing group
Growing follicle than primordial follicle ratio be more than control group.Illustrate the short time processing of CDC42 Pathway Activation agent EGF to birth
35 days mouse primordial follicles have activation.
Embodiment 5：The agent of CDC42 Pathway Activations promotes the activation and development of mouse primordial follicle in vivo
Reagent and 4 week old of application method pair using embodiment 2 or 6 week old C57 mouse, will be water-soluble by modus operandi
As a result gel microinjection is shown in Fig. 9-13 in ovarian bursa；
After 6 weeks mouse ovarian injection capsule matrix hydrosols, gel absorption situation is detected, such as Fig. 9 receives sample in different time points
Detection, experiment show that gel can be present in ovarian bursa the long period, are suitable ovary local administration pharmaceutical carriers.
The matrix hydrosol of CDC42 Pathway Activation agent EGF was contained to 4 weeks female mice ovary injection capsules, is being injected respectively
Afterwards 1 week and injection after collect ovary within 2 weeks, to ovary tissue carry out serial section, observe ovary in Follicles the case where, such as scheme
10, it is found that the processing group mouse ovarian containing CDC42 activator EGF contains more growing follicles.Count ovarian follicle in ovary
Quantity is calculated after growing follicle accounts for the ratio of total follicle and is found, after processing 1 week, such as Figure 11, shared by the primary follicle of processing group
Ratio be apparently higher than control group.After processing 2 weeks, such as Figure 12, difference is mainly reflected in secondary follicle.And to 6 weeks mouse eggs
After nest injection capsule contains the matrigel of EGF, mouse fertility and development subsequent scenario detection, such as Figure 13, experiment show that ovarian bursa is noted
It penetrates the matrigel containing EGF to have no adverse effects, and has the function of promoting female mice fertility.Illustrate CDC42 Pathway Activation agent
EGF has activation in such a way that water-soluble bases glue is carrier internal injection to the primordial follicle of 4 weeks mouse.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (12)
1.CDC42 activator prepare activate primordial follicle growth and development reagent in application and/or in vitro with non-treatment mesh
Activation primordial follicle growth and development application.
2. applying according to claim 1, which is characterized in that the CDC42 activator is CN02 or EGF.
3. a kind of reagent of activation primordial follicle growth and development, which is characterized in that including CDC42 activator.
4. reagent according to claim 3, which is characterized in that further include active cell culture solution and/or other primordial follicles
Activate drug.
5. reagent according to claim 4, which is characterized in that the active cell culture solution is DMEM/F12 culture solutions.
6. reagent according to claim 5, which is characterized in that the DMEM/F12 culture solutions contain ITS and antibiotic.
7. reagent according to claim 3, which is characterized in that further include Biological water colloidal sol and/or the activation of other primordial follicles
Drug.
8. reagent according to claim 3, which is characterized in that a concentration of 50-200ng/ml of the CDC42 activator.
9.CDC42 over-express vectors are in the application prepared in activating primordial follicle growth and development reagent and/or controlled in vitro with non-
Treat the application of purpose activation primordial follicle growth and development.
10. a kind of reagent of activation primordial follicle growth and development, which is characterized in that including CDC42 over-express vectors.
11.CDC42 activator and/or CDC42 the over-express vectors disease caused by preparing treatment ovarian follicle activation developmental potency lowly
Application in the drug of disease.
12. being applied according to described in claim 11, which is characterized in that the low disease of the ovarian follicle activation developmental potency is ovary
Early ageing or aging female ovary dysfunction.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810733153.5A CN108795849B (en) | 2018-07-05 | 2018-07-05 | Application of CDC42 signal path in activating primordial follicle growth and development |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690726A (en) * | 2020-07-01 | 2020-09-22 | 何元林 | Method for activating primordial follicles by BTC growth factor |
| CN114344331A (en) * | 2021-12-23 | 2022-04-15 | 华南理工大学 | Application of small molecule compound in preparation of medicine for activating primordial follicle |
| CN115068480A (en) * | 2022-08-09 | 2022-09-20 | 郑州大学第一附属医院 | Application of cyclin 42 small-molecule inhibitor in preparation of medicine for treating chronic kidney disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533642A (en) * | 2011-11-28 | 2012-07-04 | 李晶 | Kit for in-vitro primordial follicle activation |
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| CN102533642A (en) * | 2011-11-28 | 2012-07-04 | 李晶 | Kit for in-vitro primordial follicle activation |
Non-Patent Citations (3)
| Title |
|---|
| LEI GAO ET AL.: "Activation of Rho GTPase Cdc42 promotes adhesion and invasion in colorectal cancer cells", 《MED SCI MONIT BASIC RES》 * |
| RIFU XU ET AL.: "Inhibitory effect of SLIT2 on granulosa cell proliferation mediated by the CDC42-PAKs-ERK1/2 MAPK pathway in the prehierarchical follicles of the chicken ovary", 《SCIENTIFIC REPORTS》 * |
| 柳海珍等: "EGF在新生大鼠原始卵泡生长启动中的作用", 《中国科学（C辑）》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690726A (en) * | 2020-07-01 | 2020-09-22 | 何元林 | Method for activating primordial follicles by BTC growth factor |
| CN114344331A (en) * | 2021-12-23 | 2022-04-15 | 华南理工大学 | Application of small molecule compound in preparation of medicine for activating primordial follicle |
| CN115068480A (en) * | 2022-08-09 | 2022-09-20 | 郑州大学第一附属医院 | Application of cyclin 42 small-molecule inhibitor in preparation of medicine for treating chronic kidney disease |
| CN115068480B (en) * | 2022-08-09 | 2023-10-20 | 郑州大学第一附属医院 | Application of cyclin 42 small molecule inhibitor in preparation of medicines for treating chronic kidney disease |
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