CN109439618A - It is a kind of to activate preparation and its application for increasing ovarian follicle - Google Patents
It is a kind of to activate preparation and its application for increasing ovarian follicle Download PDFInfo
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- CN109439618A CN109439618A CN201811292953.4A CN201811292953A CN109439618A CN 109439618 A CN109439618 A CN 109439618A CN 201811292953 A CN201811292953 A CN 201811292953A CN 109439618 A CN109439618 A CN 109439618A
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Abstract
The present invention relates to ovary tissue Vitro Culture Techniques fields, disclose a kind of preparation and its application for activating and increasing ovarian follicle, said preparation is made of the agent of P13K/Akt/mTOR Pathway Activation and/or PTEN inhibitor SF1670, the P13K/Akt/mTOR Pathway Activation agent is 5-15 μM of statins, the SF1670 that the PTEN inhibitor is 20-50 μM.Ovarian follicle effect is increased by using the activation that said preparation can reach the prior art, facilitates the development and selection of clinically ovarian follicle Activation In Vitro agent, while the use of said preparation has also opened up a kind of new technical applications for statins.
Description
Technical field
The present invention relates to ovary tissue Vitro Culture Techniques fields, especially a kind of to activate the preparation for increasing ovarian follicle and its answer
With.
Background technique
In mammals, the growth of reproduction cell and maturation are the processes fine-tuned by hormone.These swash
Element includes hormone and growth factor of promoting sexual gland hormone and reproductive organs itself generation from hypothalamus etc..It is dynamic in female lactation
In object, ovarian follicle is the basic function unit for constituting ovary, by centrally located ovum and one or more layers peripheral granular cell
It constitutes.According to the different growth and development stages of egg mother cell in ovarian follicle, primordial follicle can be divided into, primary follicle, secondary follicle,
Chamber ovarian follicle, preovulatory follicle.Primordial follicle be in ovary be in quiescent condition ovarian follicle library, mammal before birth or birth
Afterwards, quantity is fixed, the primordial follicle about 400,000 or so in each ovary of people.In the growth and development of ovary
Cheng Zhong, the primordial follicle in ovary are ceaselessly activated, and pass through primary follicle and secondary follicle stage into growth period.Women goes out
When raw, since menarche, periodically ovulation gradually uses up these ovarian follicles, until when 1000-2000 remaining, it is original
Ovarian follicle is no longer developed, and women also enters menopause therewith.Since primordial follicle is after certain amount, just no longer develop, because
This does not represent yet and primordial follicle is not present in ovary even if can not see any ovarian follicle in ultrasound diagnosis.As long as and having original ovum
It can be waken up from dormant state using primordial follicle Activation In Vitro technology, enable it continue to develop and ovulate by the presence of bubble.
The current Activation In Vitro technology for increasing ovarian follicle mainly applies signal path activator extracorporeal treatment ovary tissue
To activate primordial follicle therein, it is of great significance for treatment premature ovarian failure patient.Current activation increases ovarian follicle
The main of preparation includes using PI3K signal path activator bpV and 740-YP and mTOR signal path activator phosphatidic acid and general
Naphthalene Luo Er's is applied in combination.However, the activator type as involved in above-mentioned patent is excessively complicated, and have extracorporeal treatment when
Between need 48 hours, in clinical application, patient has to receive two operations that therapeutic process could be completed, and therefore, how to find
New Activiation method shortens activationary time, completes this treatment by once operation, reduces patient's pain, easy to operate fast
Victory, by be the technology revolution leap.
Summary of the invention
The purpose of the present invention is to provide preparation and its applications that a kind of activation increases ovarian follicle, and said preparation is by P13K/Akt/
MTOR Pathway Activation agent statins and/or PTEN inhibitor SF1670 composition, increase the system of ovarian follicle by using the activation
Agent can reach prior art activation effect, while the use of said preparation has also been opened up a kind of new technology for statins and answered
Use field.
Technical solution: activation increases the preparation of ovarian follicle, by the agent of P13K/Akt/mTOR Pathway Activation and/or PTEN inhibitor
SF1670 composition, the P13K/Akt/mTOR Pathway Activation agent are 5-15 μM of statins, and the PTEN inhibitor is 20-
50 μM of SF1670.
Above-mentioned statins are one of Lovastatin, Pravastatin, Atorvastatin
Application of the above-mentioned preparation in preparation people's ovary cortex culture solution.
Above-mentioned people's ovary cortex culture solution, including following components: 1) P13K/Akt/mTOR Pathway Activation agent statins
And/or PTEN inhibitor SF1670；2) basic culture solution.
Above-mentioned people's ovary cortex culture solution, the basic culture solution are as follows: MEMa cell culture fluid, wherein adding Sodium Pyruvate
0.23mM, human serum albumins 10wt.%.
Above-mentioned people's ovary cortex culture solution, reagent set become: solution I: basic culture solution；Reagent I:P13K/Akt/mTOR
Pathway Activation agent is 5-15 μM of statins, according to the dosage packing in advance respectively of 30mL basic culture solution；Reagent II:
PTEN inhibitor is 20-50 μM of SF1670, according to the dosage packing in advance respectively of 30mL basic culture solution.
The application method of people's ovary cortex culture solution of non-diagnostic therapeutic purposes, steps are as follows:
1) it takes human ovarian tissue in 10mL-15mL cell culture fluid, separates ovary cortex and be cut into 1mm3Fritter；
2) it takes 1mL solution I to mix with reagent I and/or reagent II, is configured to the tissue culture medium of 10mL；
3) people's ovary cortex is incubated at the 24- orifice plate containing the small net that suspends, 400-3000 μ L group is added in small side off the net
Culture solution is knitted, is cultivated 2 hours, condition of culture is 37 DEG C, 5%CO2；
4) after 2 hours, tissue cultures are completed, and the tissue of culture can be used for subsequent operation.
The invention has the following advantages:
To sum up, it is formed containing P13K/Akt/mTOR Pathway Activation agent statins and/or PTEN inhibitor SF1670
People's ovary cortex culture solution culture mouse or people's ovary cortex (2 hours) activation primordial follicles and can promote in a short time
The development of ovarian follicle.The ovary cortex finished is cultivated, by surgical implantation art, parent can be remigrated back, continues normal hair
It educates, until reaching maturity, therefore the present invention provides a kind of primordial follicle activator being simple and efficient, so that the technology is more efficient
It is efficient and convenient to be applied to medicine supplementary reproduction field.
Detailed description of the invention
Fig. 1 is the development of ovary after the culture of mouse ovarian primordial follicle Activation In Vitro 2 hours.Left side (A) is control, right side
It (B) is the ovary after activation.The ImmunohistochemistryResults Results of AMH show the ovary (B) after activation after 48 hours of incubation, compared to
Control group ovary (A), has more ovarian follicles to enter the secondary follicle stage.
Fig. 2 is that after Mouse Ovary Tissues activate, mature ovum is taken out from processing group ovary, the embryo after carrying out ICSI
Fetal hair is educated.A carries out ICSI operation to mature oocyte；B, the embryo in 2 cell stages；C, the embryo in 4 cell stages
Tire；D, the embryo in 8 cell stages；E, the embryo in morula stage；F, the embryo in blastocyst stage.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.
Embodiment 1
Novel people's ovary cortex in vitro culture liquid, including following components:
Wherein, solution I preparation method is as follows:
Basic culture solution: MEMa (GIBCOTM Minimum Essential Medium(MEM)Alpha Medium(1X)
Liquid, Invitrogen) it is mother liquor, add human serum albumins 10wt.%, Sodium Pyruvate 0.23mM；
Wherein, reagent I preparation method is as follows:
P13K/Akt/mTOR Pathway Activation agent, containing 15 μM of Atorvastatin, according to the basic active cell culture of 30mL
The dosage of liquid dispenses in advance；
Above-mentioned activator is packaged into advance in parcel mounted box, is then packaged into larger packing box together with solution I
In, the ovary cortex cultural method according to described in specification is operated when use.
Culture solution application method is as follows:
1) it takes mouse ovarian in 10mL cell culture fluid, and is cleaned repeatedly more than three times with culture solution.
2) after taking 1mL solution I and reagent I, candidate agent I to be completely dissolved, the tissue culture medium of 10mL is configured to solution I.
3) by mouse ovarian be incubated at containing the small net of suspending (Millicell cell culture inserts,
Millipore 24- orifice plate) is added 500 μ L active cell culture solutions in small side off the net, cultivates 2 hours, condition of culture 37
DEG C, 5%CO2。
4) after 2 hours, activation is completed, by the transplanting of processed mouse ovarian under the kidney packing of Recipient mice, and
The bilateral ovaries of Recipient mice are extractd simultaneously.
5) second day after operation, Recipient mice are given follicle-stimulating hormone (FSH 1IU/ is only) and are injected, continuous 18 days.
6) in last day, human chorionic gonadotrophin (hCG) processing of Recipient mice 10IU is given.After 24 hours,
The ovary for collecting transplanting discharges the ovum reached maturity by needle-punching method.
7) by ovum with from the public mouse sperm of same strain carry out it is in vitro fertilization after, 2 cell stages that will develop
It transplants in pseudopregnant mouse, and is successfully established gestation.Its rate of fertilization of mature egg and production that ovary after last statistical disposition obtains
The difference for the mature egg that young number and normal adult ovary are obtained through superfecundation.
Experimental result is shown in Table 1, and table 1 is that the embryo of normal super ovulation and Activation In Vitro ovum 2 cells after in vitro fertilization sends out
Educate the comparison of survival rate after rate and embryo transfer.
Table 1
| MII | 2 cell stages | (%) | 2 cell stages | At son number | (%) | |
| Processing group ovum | 120 | 86 | 71.67 | 60 | 15 | 25.00 |
| Super ovulation | 81 | 69 | 85.18 | 60 | 22 | 36.67 |
Embodiment 2
Novel people's ovary cortex in vitro culture liquid, including following components:
Wherein, solution I preparation method is as follows:
Basic culture solution: MEMa (GIBCOTM Minimum Essential Medium(MEM)Alpha Medium(1X)
Liquid, Invitrogen) it is mother liquor, add human serum albumins 10wt.%, Sodium Pyruvate 0.23mM；
Wherein, reagent II preparation method is as follows:
PTEN inhibitor is dispensed containing 50 μM of SF1670 according to the dosage of the basic active cell culture solution of 30mL in advance；
Above-mentioned activator is packaged into advance in parcel mounted box, is then packaged into larger packing box together with solution I
In, the ovary cortex cultural method according to described in specification is operated when use.
Above-mentioned novel ovary cortex in vitro culture liquid is tested using the application method of embodiment 1, experimental result
2 are shown in Table, table 2 is normal super ovulation and the Activation In Vitro ovum embryo development rate of 2 cells and embryo transfer after in vitro fertilization
The comparison of survival rate afterwards.
Table 2
| MII | 2 cell stages | (%) | 2 cell stages | At son number | (%) | |
| Processing group ovum | 97 | 66 | 68.04 | 50 | 14 | 28.00 |
| Super ovulation | 110 | 102 | 92.73 | 50 | 33 | 33.00 |
Embodiment 3
Novel people's ovary cortex in vitro culture liquid, including following components:
Wherein, solution I preparation method is as follows:
Basic culture solution: MEMa (GIBCOTM Minimum Essential Medium(MEM)Alpha Medium(1X)
Liquid, Invitrogen) it is mother liquor, add human serum albumins 10wt.%, Sodium Pyruvate 0.23mM；
Wherein, reagent I preparation method is as follows:
P13K/Akt/mTOR Pathway Activation agent, containing 5 μM of Pravastatin, according to the basic active cell culture solution of 30mL
Dosage dispense in advance；
Wherein, reagent II preparation method is as follows:
PTEN inhibitor is dispensed containing 20 μM of SF1670 according to the dosage of the basic active cell culture solution of 30mL in advance；
Above-mentioned activator is packaged into advance in parcel mounted box, is then packaged into larger packing box together with solution I
In, the ovary cortex cultural method according to described in specification is operated when use.
Above-mentioned novel ovary cortex in vitro culture liquid is tested using the application method of embodiment 1,.Experimental result
It is shown in Table 3, after table is the embryo development rate and embryo transfer of normally super ovulation and Activation In Vitro ovum rear 2 cells in vitro fertilization
The comparison of survival rate.
Table 3
| MII | 2 cell stages | (%) | 2 cell stages | At son number | (%) | |
| Processing group ovum | 95 | 70 | 73.68 | 65 | 18 | 27.69 |
| Super ovulation | 83 | 75 | 90.36 | 65 | 23 | 35.38 |
From table 1, table 2, the result of table 3 can be seen that the mature egg of processing group after in vitro fertilization, 2 cell stages
Developmental rate be than normally surpass ovulation son 2 cell stages developmental rate it is low, but with the activation scheme in current existing patent
Comparison, the embryo development rate of 2 cell stages have been in higher level, although, the 2 cell stages development of all activated processing group
Rate is below normal super ovulation subgroup and illustrates this however, activation scheme involved in this patent obviously obtains more 2 cells
Activation scheme, which is formed by culture solution, can preferably promote follicular development and improve the quality of egg mother cell.
For above method test proves that effect is obvious, related effect test is as follows:
The development of ovary after I mouse ovarian primordial follicle Activation In Vitro culture 2 hours.
After mouse ovarian primordial follicle Activation In Vitro agent activates and cultivates 48 hours, by the fixed line flag point of going forward side by side of ovary
The immunohistochemistry detection of son.If Fig. 1, left side (A) are control, right side (B) is the ovary after activation.The ImmunohistochemistryResults Results of AMH
Ovary (B) after showing activation compared to control group ovary (A), has more ovarian follicles to enter secondary ovum after 48 hours of incubation
The bubble stage.
After the processing of II Mouse Ovary Tissues Activation In Vitro preparation, the ovarian follicle of normal development generation under Mouse Kidney envelope.
After the processing of Mouse Ovary Tissues Activation In Vitro preparation, through being developed under Mouse Kidney envelope, after hCG is handled 12 hours, receive
Collect ovary, puncture preovulatory follicle with Mechanical Method and collect mature egg mother cell, the embryonic development after carrying out carry out ICSI is commented
Valence body early embryo ectogenesis situation.By 2 cell stages after mature oocyte progress ICSI operation (A) as shown in Figure 2
(B), 4 cell stages (C), 8 cell stages (D), mulberry body (E) stage can normal development to be in blastocyst stage (F) embryo.
As it can be seen that the follicular development and function that the preparation activation mouse ovarian that application activating increases ovarian follicle generates are completely normal.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
In the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should be covered by the protection scope of the present invention.
Claims (7)
1. a kind of activate the preparation for increasing ovarian follicle, it is characterised in that inhibited by the agent of P13K/Akt/mTOR Pathway Activation and/or PTEN
Agent SF1670 composition, the P13K/Akt/mTOR Pathway Activation agent are 5-15 μM of statins, and the PTEN inhibitor is
20-50 μM of SF1670.
2. the preparation that a kind of activation as described in claim 1 increases ovarian follicle, it is characterised in that the statins are Lip river
Cut down one of statin, Pravastatin, Atorvastatin.
3. application of the preparation as claimed in claim 1 or 2 in preparation people's ovary cortex culture solution.
4. people's ovary cortex culture solution as claimed in claim 3, it is characterised in that including following components:
1) P13K/Akt/mTOR Pathway Activation agent statins and/or PTEN inhibitor SF1670；
2) basic culture solution.
5. people's ovary cortex culture solution as claimed in claim 4, it is characterised in that the basic culture solution are as follows: the training of MEMa cell
Nutrient solution, wherein addition Sodium Pyruvate 0.23mM, human serum albumins 10wt.%.
6. people's ovary cortex culture solution as claimed in claim 5, it is characterised in that reagent set becomes:
Solution I: basic culture solution；
Reagent I:P13K/Akt/mTOR Pathway Activation agent is 5-15 μM of statins, according to the agent of 30mL basic culture solution
Amount packing in advance respectively
Reagent II:PTEN inhibitor is 20-50 μM of SF1670, according to the dosage packing in advance respectively of 30mL basic culture solution.
7. the application method of people's ovary cortex culture solution such as the non-diagnostic therapeutic purposes of claim 6, it is characterised in that step is such as
Under:
1) it takes human ovarian tissue in 10mL-15mL cell culture fluid, separates ovary cortex and be cut into 1mm3Fritter；
2) it takes 1mL solution I to mix with reagent I and/or reagent II, is configured to the tissue culture medium of 10mL；
3) people's ovary cortex is incubated at the 24- orifice plate containing the small net that suspends, 400-3000 μ L tissue training is added in small side off the net
Nutrient solution is cultivated 2 hours, and condition of culture is 37 DEG C, 5%CO2；
4) after 2 hours, tissue cultures are completed, and the tissue of culture can be used for subsequent operation.
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| CN103387960A (en) * | 2013-07-22 | 2013-11-13 | 南京医科大学 | mTOR signaling pathway activator and applications thereof in in-vitro primordial follicle activation |
| CN104130972A (en) * | 2014-07-09 | 2014-11-05 | 南京医科大学 | Primordial follicle activator and applications thereof |
| CN108342355A (en) * | 2018-01-10 | 2018-07-31 | 南京艾维艾康生物技术有限公司 | Primordial follicle activator and its application in people's ovary cortex culture solution |
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2018
- 2018-11-01 CN CN201811292953.4A patent/CN109439618A/en active Pending
Patent Citations (4)
| 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 |
| CN103387960A (en) * | 2013-07-22 | 2013-11-13 | 南京医科大学 | mTOR signaling pathway activator and applications thereof in in-vitro primordial follicle activation |
| CN104130972A (en) * | 2014-07-09 | 2014-11-05 | 南京医科大学 | Primordial follicle activator and applications thereof |
| CN108342355A (en) * | 2018-01-10 | 2018-07-31 | 南京艾维艾康生物技术有限公司 | Primordial follicle activator and its application in people's ovary cortex culture solution |
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