CN104130972A - Primordial follicle activator and applications thereof - Google Patents
Primordial follicle activator and applications thereof Download PDFInfo
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Abstract
The invention discloses a primordial follicle activator and applications thereof. The activator comprises an mTOR signal path activator I and II, wherein the mTOR signal path activator I is 50 to 200 [mu]M of phosphatidic acid, and the mTOR signal path activator II is 20 to 50 [mu]M of propranolol; and a PI3K signal path activator composed of a PI3K signal path activator III and IV; wherein the PI3K signal path activator III is 30 to 100 [mu]M of bpV, and the PI3K signal path activator IV is 250 to 500 [mu]g/mL of 740Y-P. The invention relates to an in-vitro activated kit, which co-uses mTOR and PI3K signal path activators to activate primordial follicle cells in ovarian tissues of mouse and human and promote the growth of ovarian follicle. The kit can be widely used to assist the treatment on diseases caused by deficient ovarian follicle storage such as premature ovarian failure in the fertility field.
Description
Technical field
The present invention relates to a kind of primordial follicle activator and application thereof, particularly a kind of mTOR and the application of PI3K signal path activator in kit for in-vitro primordial follicle activation.
Background technology
In Mammals, the growth of sexual cell and maturation are processes that is subject to the meticulous adjusting of hormone.These hormones comprise from the hormone of hypothalamic gonad-stimulating hormone and reproductive organ self generation and somatomedin etc.In female mammal body, ovarian follicle is the basic function unit of an ovary, by being positioned at central ovum and peripheral one or more layers granulosa cell forms.Different growth and development stages according to ovocyte in ovarian follicle, can be divided into primordial follicle, primary follicle, secondary follicle, chamber ovarian follicle, preovulatory follicle.Primordial follicle is the ovarian follicle storehouse in quiescent condition in ovary, Mammals in utero or birth after, its quantity is fixed, nearly 400,000 left and right of primordial follicle in each ovary of people.In the growth and development process of ovary, the primordial follicle in ovary is ceaselessly activated, and enters vegetative period through primary follicle and secondary follicle stage.In the secondary follicle stage, the in the situation that of there is no gonad-stimulating hormone (FSH) secretion before puberty, these ovarian follicles will be dead and follicle atresia occurs, and after puberty, under the stimulation of the FSH of hypothalamus secretion, just have one or several secondary follicle and periodically further grow and grow up until preovulatory follicle.In this process, the volume of ovocyte can increase hundred times, and around granulosa cell quantity also can increase sharply, and by an initial individual layer 6-9 cell, is increased to several layers of thousands of cell.Except the gonad-stimulating hormone of hypothalamus secretion, the growth of ovarian follicle is also subject to the inner hormone producing of ovary and the impact of somatomedin, such as the growing of the other side that just can interact by the form of paracrine at ovulum parent cell and granulosa cell around.Finally under total use at ovulation prohormone peak, oocyte maturation in preovulatory follicle and ovulating, there is luteinization in residual granulosa cell and theca cell, and the growth that secreting hormone is zygote is prepared.
In mammiferous ovary, in primordial follicle storehouse, not stopping has primordial follicle to be activated and enters vegetative period, and this process is called as initial recruitment (initial recruiment).Initial recruitment is not subject to the adjusting of FSH, but is subject to the impact of some somatomedins of ovary generation.Many somatomedins are all found to have participated in this process as PDGF, SDF, EGF etc.In people's every month, the primordial follicle of nearly 1000 left and right enters vegetative period, and primordial follicle in primordial follicle storehouse is while being not enough to 1000, and ovary will no longer be ovulated, and now women also will enter climacteric.The age that normal female enters climacteric all about 50 years old, and under some pathological conditions, causes Premature Ovarian Failure because under-reserve in primordial follicle storehouse in ovary makes women enter in advance the impasse phase (before 40 years old).Premature Ovarian Failure is a kind of very common infertility, and the sickness rate in reproduction age women is probably 1%.At present, the Mechanism Study activating about primordial follicle is not also clear especially, except above-mentioned some somatomedins of mentioning, scientists is by the research of knock out mice, and discovery signals path PTEN-PI3K-FOXO3 and mTOR-p70S6K-rpS6 play an important role in the activation of primordial follicle.Therefore how to find an effective way to regulate the activation of primordial follicle to have very important significance.
Summary of the invention
The technical problem solving: the invention provides a kind of primordial follicle activator and application thereof, another object of the present invention is to provide a kind of kit for in-vitro primordial follicle activation.
Technical scheme: primordial follicle activator, by mTOR signal path activator and PI3K signal path activator, formed, described mTOR signal path activator comprises mTOR signal path activator I and II, mTOR signal path activator I is 50-200 μ M phosphatidic acid, and mTOR signal path activator II is 20-50 μ M Proprasylyte; Described PI3K signal path activator comprises PI3K signal path activator III and IV, and PI3K signal path activator III is the bpV of 30-100 μ M, and PI3K signal path activator IV is the 740Y-P of 250-500 μ g/mL.
Above-mentioned phosphatidic acid is preferably 200 μ M, and Proprasylyte is preferably 50 μ M, and described bpV is preferably 30 μ M, and 740Y-P is preferably 250 μ g/mL.
Above-mentioned primordial follicle activator activates primordial follicle and promotes the application in follicular development medicine in preparation.
Kit for in-vitro primordial follicle activation, comprises following component: 1) mTOR signal path activator I and II; 2) PI3K signal path activator III and IV; 3) basic activating cells nutrient solution; 4) L-15 nutrient solution.
A kit for in-vitro primordial follicle activation, mTOR signal path activator I is phosphatidic acid, its concentration is: 50-200 μ M; MTOR signal path activator II is Proprasylyte, and its concentration is: 20-50 μ M; PI3K signal path activator III is bpV, and its concentration is 30-100 μ M; PI3K signal path activator IV is 740Y-P, and its concentration is 250-500 μ g/mL; Basic activating cells nutrient solution formula: MEMa is mother liquor, includes Sodium.alpha.-ketopropionate 0.23mM, human serum albumin 10wt.%, L-AA 50 μ g/mL, Streptomycin sulphate 50mg/L and penicillin 75mg/L.
A kit for in-vitro primordial follicle activation, reagent set becomes:
Solution I: basic activating cells nutrient solution;
Solution II: L-15 nutrient solution is mother liquor, includes human serum albumin 10wt.%;
Reagent I:mTOR signal path activator I and II, the Proprasylyte of the phosphatidic acid that contains 50-200 μ M and 20-50 μ M, according to the packing in advance respectively of the dosage of the basic activating cells nutrient solution of 10mL;
Reagent II:PI3K signal path activator III and IV, contain 30-100 μ M bpV and 250-500 μ g/mL 740Y-P, according to the packing in advance respectively of the dosage of the basic activating cells nutrient solution of 10mL.
Another kind of kit for in-vitro primordial follicle activation, reagent set becomes:
Solution I: basic activating cells nutrient solution;
Solution II: L-15 nutrient solution is mother liquor, includes human serum albumin 10wt.%;
Reagent I: the mTOR signal path activator I and the II that mix in advance, the Proprasylyte of the phosphatidic acid that contains 50-200 μ M and 20-50 μ M, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL;
Reagent II: the PI3K signal path activator III and the IV that mix in advance, the 740Y-P of the PA that contains 30-100 μ M and 250-500 μ g/mL, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL.
Above-mentioned kit for in-vitro primordial follicle activation, packs solution I, reagent I, reagent II, solution II respectively, is more together packaged in same packing box.
The preparation method of above-mentioned kit for in-vitro primordial follicle activation, solution I: basic activating cells nutrient solution compound method is as follows: MEMa is mother liquor, contains Sodium.alpha.-ketopropionate 0.23mM, human serum albumin 10wt.%, L-AA 50 μ g/mL, Streptomycin sulphate 50mg/L and penicillin 75mg/L; Described reagent I contains mTOR signal path activator and comprises: (1) mTOR signal path activator I, the phosphatidic acid of 50-200 μ M, (2) mTOR signal path activator II, the Proprasylyte of 20-50 μ M; Described reagent II contains PI3K signal path activator and comprises: (1) PI3K signal path activator I, the PA of 30-100 μ M, (2) PI3K signal path activator II, the 740Y-P of 250-500 μ g/mL; The compound method of solution II is as follows: L-15 nutrient solution is mother liquor, also contains human serum albumin 10wt.%.
The application method of above-mentioned non-diagnoses and treatment object kit for in-vitro primordial follicle activation, step is as follows: 1) get ovary cortex in 5mL solution II, ovary cortex is cut into 1mm
3fritter, by solution II, repeatedly clean; 2) get 1mL solution I and mix with reagent I, after candidate agent I dissolves completely, by solution I, be mixed with the activating cells nutrient solution of 10mL; 3) ovary cortex is incubated to the 24-orifice plate that contains the little net that suspends, little side off the net, adds 400 μ L activating cells nutrient solutions, cultivate 24 hours, culture condition is 37 ℃, 5%CO
2; 4) after 24h, get 1mL solution I and mix with reagent II, after candidate agent II dissolves completely, by solution I, be mixed with the activating cells nutrient solution of 10mL; 5) nutrient solution in culture plate is blotted, add the activation nutrient solution of new configuration, continue to cultivate 24 hours, culture condition is 37 ℃, 5%CO
2; 6) after 24 hours, activation process completes, and tissue can be used for subsequent operations.
Beneficial effect: to sum up, the kit for in-vitro primordial follicle activation that application mTOR and PI3K signal path activator form is processed mouse or people's ovary cortex, can activate the primordial follicle in tissue and promote the growth of ovarian follicle.Activate in vitro complete ovary cortex, through Srgery grafting art, can remigrate back parent, continue normal growth, until reach maturity, therefore the invention provides a kind of mTOR signal path activator, provide a kind of test kit for vitro culture operation, so that this technology is applied to larger medical science supplementary reproduction field simultaneously.
Accompanying drawing explanation
To be mouse ovarian process after 24h with mTOR signal path activator Figure 1A, collects that the method for ovary tissue by western-blot detects downstream marker molecule S6K1 after mTOR signal path activates and the phosphorylation of rps6 changes.P-S6K1, the S6K1 that p-rps6 is phosphorylation and rps6.
Figure 1B is that newborn mice ovary application mTOR signal path activator was processed after 24 hours, the expression of phosphorylation rps6 in newborn mice ovary.Arrow, is present in the positive signal in primordial follicle ovocyte.Scale=100 μ m.
Fig. 1 C, for the ovary cortex after activating is implanted into acceptor Mouse Kidney tunicle after lower 14 days, can promote the growth schematic diagram of ovary.C is the untreated ovary of contrast; PA, PRO, the ovary that mTOR activator PA and PRO process.Scale=1mm.
Fig. 1 D is ovary serial section ovarian follicle count results schematic diagram.Original, primordial follicle; Elementary, primary follicle; Secondary, secondary follicle; There is chamber, have in early days chamber ovarian follicle; There is greatly chamber, have greatly chamber ovarian follicle.*, P<0.05, significant difference; *, P<0.01, difference is extremely remarkable.
Fig. 2 Activation In Vitro is processed the quality that does not affect ovocyte.After ovarian transplantation 18 days, acceptor mouse disposable celiac injection hCG ovulation rate, collected ovary, by the ovulate method recovery of ova of ovarian follicle of acupuncture.Wherein a part of ovum carries out evaluation fetal development situation in vitro fertilization, and a part of ovum and zygote are fixed and immunofluorescence analysis.A: the MII mature egg of collection; B:MII ovum tubulin immunofluorescence result, in the visual field, strip is the spindle body of tubulin mark, strip center is Hoechst33258 dyeing, labeled cell core; C:MII ovum 5MeC immunofluorescence result, edge, the visual field is 5MeC dyeing, the methylation level of mark ovocyte, center is nucleus; D: after 10h in vitro fertilization, the zygote that female-male pronucleus forms.What depart from center, the visual field is 5MeC dyeing, the female pronucleus of mark in methylation state, and center, the visual field is Hoechst33258 dyeing, labeled cell core, the female-male pronucleus in visible zygote, male pronucleus is demethylation state, and 5MeC dyes negative.E:2 cell stage; F: blastaea; G: 2 cells are carried out to the healthy offspring that produces after embryo transfer.
Fig. 3 is that PI3K signal path activator individual curing can activate the primordial follicle schematic diagram in newborn mice ovary.A) mouse ovarian primordial follicle Activation In Vitro is cultivated the growth of ovary after 48 hours.Mouse ovarian activates and cultivates after 48 hours with PI3K activator, and fixedly the go forward side by side immunohistochemical methods of line flag molecule of ovary is detected.Left side (a, c, e) is contrast, and right side (b, d, f) is the ovary after activating.A, the ImmunohistochemistryResults Results that b is FOXO3, in figure, arrow indication is the primordial follicle after activating, the dyeing of FOXO3 is transferred to cytoplasm by nucleus (seeing contrast).C, the coloration result that d is BrdU shows, cells a large amount of in the ovary tissue after activation are in vegetative state.E, the ImmunohistochemistryResults Results that f is AMH shows, the ovary after activation, after cultivating 48 hours, compared to control group ovary, has more ovarian follicle to enter the secondary follicle stage.B) ovary of cultivating is carried out transplanting under Mouse Kidney tunicle the growing state schematic diagram of latter 14 days ovaries.At every pictures, be control group ovary at the middle and upper levels, lower floor is the ovary after activating, and represents different treatment group.
Fig. 4 is the growth schematic diagram that mTOR and PI3K signal path activator synergy promote newborn mice ovary.One side ovary of newborn mice is processed through mTOR and the PI3K activator of various combination, after opposite side ovary application mTOR or PI3K activator individual curing, transplants under the kidney tunicle of the left and right sides of same acceptor mouse and grows 14 days.After 14 days, the ovary of transplanting is taken out, observe the developmental state of ovary.
Fig. 5 A is that people's ovary tissue is processed the also developmental state of vitro culture ovarian follicle after 6 days through mTOR or PI3K signal path activator.A and b, the primordial follicle before cultivating in ovary cortex, the lower picture of the high power lens that b is a (20X); C and d, the ovarian follicle in transforming to primary follicle in control group ovary cortex; E and f, mTOR signal path activator is processed the secondary follicle that in ovary cortex, cluster exists, and e is the picture of low power lens (10X), and f is the picture in the different visuals field under high power lens (20X); G, PI3K signal path activator is processed representational secondary follicle in rear ovary cortex; H, representational secondary follicle in ovary cortex after mTOR and PI3K activator combined utilization.Scale=100 μ m.
Fig. 5 B, for cultivating after 6 days, cultivates the cell proliferation schematic diagram of ovarian follicle.A and b, the ovary tissue of control group; C and d, ovary cortex after mTOR and PI3K activator combined utilization; B and d, for high power lens (20X) and from the different visuals field.Brown mark is the nucleus in proliferation period of BrdU mark.
Fig. 5 C is the statistical analysis schematic diagram of ovarian follicle count results.Control group, space management; Process 1, mTOR signal path activator treatment group; Process 2, PI3K signal path activator treatment group; Process 3, mTOR activator and PI3K activator combined utilization treatment group; Original, primordial follicle; Elementary, primary follicle; Secondary, secondary follicle; Degenerate, the ovarian follicle of degeneration.*, P<0.05, * *, P<0.01, difference is extremely remarkable.
Fig. 5 A～C shows that mTOR and PI3K signal path activator synergy promote activation and the growth of people's primordial follicle.People's ovary cortex is applied respectively mTOR, PI3K activator separately and combination treatment, and after processing, ovary cortex continues to be cultured to the 6th day, collects ovary cortex and analyzes.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done and replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Embodiment 1
Kit for in-vitro primordial follicle activation, comprises following component:
Wherein, solution I compound method is as follows:
Basic activating cells nutrient solution: MEMa (GIBCO
tMminimum Essential Medium (MEM) Alpha Medium (1X) liquid, Invitrogen) be mother liquor, also contain human serum albumin 10wt.%, Sodium.alpha.-ketopropionate 0.23mM, L-AA (vitamins C) 50 μ g/mL, Streptomycin sulphate 50mg/L and penicillin 75mg/L;
Wherein, reagent I compound method is as follows:
MTOR signal path activator I and II, the propranolol of the PA that contains 200 μ M and 50 μ M, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL;
Wherein, reagent II compound method is as follows:
PI3K signal path activator III and IV, the 740Y-P of the bpV that contains 30-100 μ M and 250-500 μ g/mL, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL;
Wherein, under the compound method of solution II enters:
The mixed solution of L-15 (GIBCO, Invitrogen) and human serum albumin, wherein human serum albumin accounts for solution II system 10wt.%;
By these three kinds of solution difference splendid attires, be more together packaged in same packing box, during use, according to the primordial follicle Activation In Vitro method of describing in specification sheets, operate.
Test kit using method is as follows:
1) get mouse ovarian in 5mL solution II, by solution II, repeatedly clean more than three times.
2) get 1mL solution I and mix with reagent I, after candidate agent I dissolves completely, by solution I, be configured to the activating cells nutrient solution of 10mL.
3) mouse ovarian is incubated to the 24-orifice plate that contains the little net (Millicell cell culture inserts, Millipore) that suspends, little side off the net, adds 400 μ L activating cells nutrient solutions, cultivate 24 hours, culture condition is 37 ℃, 5%CO
2.
4) after 24h, get 1mL solution I and mix with reagent II, after candidate agent II dissolves completely, by solution I, be mixed with the activating cells nutrient solution of 10mL;
5) nutrient solution in culture plate is blotted, add the activation nutrient solution of new configuration, continue to cultivate 24 hours, culture condition is 37 ℃, 5%CO
2;
6) after 24 hours, activation process completes, and the mouse ovarian of processing is transplanted under the kidney packing of acceptor mouse, and the bilateral ovaries of acceptor mouse is extractd simultaneously.
7) second day after operation, acceptor mouse gives follitropin (FSH1IU/ only) injection, continuous 18 days.
8) in the end one day, the human chorionic gonadotrophin (hCG) that gives acceptor mouse 10IU was processed.After 24 hours, collect the ovary of transplanting, by needle-punching method, discharge fully-developed ovum.
9) ovum and the public mouse sperm that comes from same strain are carried out in vitro fertilization after, by growth to 2 cell stages transplant in false pregnancy mouse, and successfully set up gestation.Finally add up the difference of the mature egg that its rate of fertilization of mature egg that the ovary after Activation In Vitro test kit is processed obtains and litter size and normal adult ovary obtain through superovulation.
Experimental result is in Table 1, table 1 be normal super ovulation in vitro fertilization with Activation In Vitro ovum after the comparison of surviving rate after the embryo development rate of 2 cells and embryo transfer.
From the results shown in Table 1, the mature egg for the treatment of group is after in vitro fertilization, and the developmental rate of 2-cell stage is 64.3%, and the sub 2-cell stage developmental rate of super ovulation is 86.7%, therefore,, at 2-cell stage developmental rate, the ovum for the treatment of group is lower than super ovulation.By 2 cell stages, after embryo transfer, aspect litter size, these two kinds of ovums are but almost as broad as long.Therefore, application kit for in-vitro primordial follicle activation is processed after ovary, can't affect fertilization and the embryo development procedure of ovocyte.
Table 1
| MII | 2 cell stages | (％) | 2 cell stages | Survive young number | (％) | |
| Treatment group ovum | 129 | 83 | 64.3 | 66 | 33 | 50 |
| Super ovulation | 45 | 39 | 86.7 | 39 | 19 | 49 |
Embodiment 2
MTOR signal path activator, comprise mTOR signal path activator I and II, mTOR signal path activator I is 50-200 μ M phosphatidic acid (phosphatidic acid, PA), mTOR signal path activator II is 20-50 μ M Proprasylyte (Propranolol, PRO).
Described phosphatidic acid is 200 μ M, and Proprasylyte is 50 μ M.
Described mTOR signal path activator activates primordial follicle and promotes the application in follicular development medicine in preparation.
PI3K signal path activator, comprise PI3K signal path activator III and IV, PI3K signal path activator III is 30-100 μ M bpV (bpV (pic)-Dipotassium Bisperoxo (picolinato) oxovanadate, or bpV (HOpic)-Dipotassium Bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate), PI3K signal path activator IV is that (740Y-P is polypeptide for the 740Y-P of 250-500 μ g/mL, its aminoacid sequence is: RQIKIWFQNRRMKWKKSDGGYMDMS (Modifications: Tyr-25=pTyr)).
Described bpV is 30 μ M, and 740Y-P is 250 μ g/mL.
Described PI3K signal path activator and mTOR signal path activator combined utilization activate primordial follicle and promote the application in follicular development medicine in preparation.
Kit for in-vitro primordial follicle activation comprises following component:
1) mTOR signal path activator I and II;
2) PI3K signal path activator III and IV;
3) basic activating cells nutrient solution;
4) L-15 nutrient solution (GIBCO, invitrogen).
Described test kit, a kind of composition proposal is, and mTOR signal path activator I is phosphatidic acid, and its concentration is: 50-200 μ M; Wherein, mTOR signal path activator II is Proprasylyte, and its concentration is: 20-50 μ M; A kind of composition proposal is, PI3K signal path activator III is bpV, and its concentration is: 30-100 μ M, and wherein, PI3K signal path activator IV is the 740Y-P of 250-500 μ g/mL; Basic activating cells nutrient solution formula: MEMa (GIBCO
tMminimum Essential Medium (MEM) Alpha Medium (1X) liquid, Invitrogen) be mother liquor, also contain Sodium.alpha.-ketopropionate 0.23mM, human serum albumin 10wt.%, L-AA (vitamins C) 50 μ g/mL, Streptomycin sulphate 50mg/L and penicillin 75mg/L.
Described test kit, wherein another kind of reagent composition proposal is:
Solution I: basic activating cells nutrient solution;
Reagent I:mTOR signal path activator I and II, the Proprasylyte of the phosphatidic acid that contains 50-200 μ M and 20-50 μ M, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL;
Reagent II:PI3K signal path activator III and IV, the 740Y-P of the bpV that contains 30-100 μ M and 250-500 μ g/mL, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL;
Solution II: L-15 nutrient solution is mother liquor, also contains human serum albumin 10wt.%.
Described test kit, the reagent set of the third composition proposal becomes:
Solution I: basic activating cells nutrient solution;
Reagent I: the mTOR signal path activator I and the II that mix in advance, the Proprasylyte of the phosphatidic acid that contains 50-200 μ M and 20-50 μ M, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL.
Reagent II, PI3K signal path activator III and IV, the 740Y-P of the bpV that contains 30-100 μ M and 250-500 μ g/mL, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL;
Solution II: L-15 nutrient solution is mother liquor, also contains human serum albumin 10wt.%.
Described test kit, packs solution I, reagent I, reagent II, solution II respectively, is more together packaged in same packing box.
The preparation method of described test kit, wherein basic activating cells nutrient solution compound method is as follows:
MEMa is mother liquor, also contains Sodium.alpha.-ketopropionate 0.23mM, human serum albumin 10wt.%, L-AA 50 μ g/mL, Streptomycin sulphate 50mg/L and penicillin 75mg/L;
Described reagent I contains mTOR signal path activator and comprises:
(1) mTOR signal path activator I, the phosphatidic acid of 50-200 μ M,
(2) mTOR signal path activator II, the Proprasylyte of 20-50 μ M;
Described reagent II contains PI3K signal path activator and comprises:
(1) PI3K signal path activator III, the bpV of 30-100 μ M,
(2) PI3K signal path activator IV, the 740Y-P of 250-500 μ g/mL;
Wherein, the compound method of solution II is as follows:
L-15 nutrient solution is mother liquor, also contains human serum albumin 10wt.%.
The application method of test kit, step is as follows:
1) get ovary cortex in 5mL solution II, ovary cortex is cut into 1mm
3fritter, by solution II, repeatedly clean;
2) get 1mL solution I and mix with reagent I, after candidate agent I dissolves completely, by solution I, be mixed with the activating cells nutrient solution of 10mL;
3) ovary cortex is incubated at contain the little net that suspends (Millicell cell culture inserts,, 24-orifice plate Millipore), little side off the net, add the basic activating cells nutrient solution of 400 μ L, cultivate 24 hours, culture condition is 37 ℃, 5%CO
2;
4), after 24h, get 1mL solution I and mix with reagent II, after candidate agent II dissolves completely,, by solution I, be mixed with the activating cells nutrient solution of 10mL;
5) nutrient solution in culture plate is blotted, add the activation nutrient solution of new configuration, continue to cultivate 24 hours, culture condition is 37 ℃, 5%CO
2;
6) after 24 hours, activation process completes, and tissue can be used for subsequent operations.
Wherein, mTOR signal path activator PA, Chinese name phosphatidic acid, for a kind of micromolecular compound, be selected from: 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt), belong to prior art, can buy and obtain from the market, its consumption be: 50-200 μ M; Wherein the structural formula of PA is as follows:
Wherein, mTOR signal path activator PRO, the general naphthalene Nore of Chinese name, is a kind of micromolecular compound, is selected from: Propranolol hydrochloride, belong to prior art, can buy and obtain from the market, its consumption is: 20-50 μ M;
Wherein, PI3K activator bpV, comprise: bpV (pic) (Dipotassium Bisperoxo (picolinato) oxovanadate, or bpV (HOpic) (Dipotassium Bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate, or other PTEN molecule inhibitor, these PTEN inhibitor, belong to prior art, can buy and obtain from the market, its consumption be: 30-100 μ M; Wherein the structural formula of bpV (pic) and bpV (HOpic) is as follows::
Wherein, PI3K activator 740Y-P, the peptide molecule for a kind of synthetic, belongs to prior art, can buy and obtain from the market, and its consumption is: 50-500 μ g/mL; Wherein its aminoacid sequence of 740Y-P polypeptide is: RQIKIWFQNRRMKWKKSDGGYMDMS ((Modifications:Tyr-25=pTyr)) Tocris Bioscience CAS No:1236188-16-1;
Above test kit and method are through evidence successful, and relevant effect test is as follows::
1) mTOR signal path activator individual curing can promote the growth of primordial follicle in newborn mice ovary
Newborn the 3rd day mouse ovarian activated and cultivates with kit for in-vitro primordial follicle activation after 24 hours, extracted the variation of ovarian protein check mTOR signal path marker molecule phosphorylation level.As Figure 1A,, through mTOR signal path activator PA and propranolol (PRO), process after 24h, the downstream molecules that mTOR signal path activates, the phosphorylation level of S6K1 and rps6 significantly raises.After mTOR signal path activator is processed, the expression of phosphorylation rps6 is mainly positioned at the ovocyte (Figure 1B, arrow) of primordial follicle.In order further to evaluate the developmental state that primordial follicle activates rear ovarian follicle, newborn the 3rd day mouse ovarian, after Activation In Vitro test kit processes 24, enters ovarian transplantation under the kidney tunicle of acceptor mouse, simultaneously by the Bilateral oophorectomy of acceptor mouse.Second day after operation, every day is to injected in mice FSH to the 14 days,, then collect ovary.As shown in Figure 1 C, at every pictures, be control group ovary at the middle and upper levels, lower floor is the ovary after activating.No matter compare with control group is the growth that PA or PRO can promote ovary, and has synergy.The ovary of collection is carried out after serial section ovarian follicle counting, in the ovary that PA/PRO processes, its ovarian follicle ratio showed increased in growth and development stage, and the shared ratio of primordial follicle obviously reduces (Fig. 1 D), show that mTOR signal path activator can activate the primordial follicle in mouse ovarian and promote the growth of ovarian follicle.2) after mTOR activator individual curing activation primordial follicle, do not affect the quality of ovum
By the newborn mice ovary of crossing by mTOR signal path activator individual curing, be implanted under the kidney tunicle of acceptor mouse, simultaneously by the Bilateral oophorectomy of acceptor mouse.Second day after operation, to injected in mice FSH to the 18 days, award disposable celiac injection hCG (10IU/ mouse) every day, after 12 hours, collects ovary, and acupuncture ovulation ovarian follicle is also collected ripe MII ovum, carries out in vitro fertilization.Wherein a part of ovum and zygote are by the structure of spindle body and the variation of epigenetics in the method evaluation ovum of immunofluorescence, and the method that remaining zygote is transplanted by vitro culture and 2-cell stage is further evaluated the quality of ovum.As shown in a-c in Fig. 2, immunofluorescence result shows, the mature egg producing after primordial follicle Activation In Vitro (in Fig. 2 a) have normal spindle body structure (b in Fig. 2) and methylation level (c in Fig. 2)), and at after fertilization, female-male pronucleus has the variation of methylating normally, be that female pronucleus presents hyper-methylation state,, and male pronucleus presents demethylation state (d in Fig. 2).Vitro culture is tested and is shown, zygote is cultivated in vitro and can be grown blastaea (e, f in Fig. 2), and wherein a part of 2 cell stages carry out after embryo transfer, has obtained the healthy offspring with reproductive performance (g in Fig. 2).The above results shows, primordial follicle Activation In Vitro step can't affect the quality of ovum except accelerating the growth of ovarian follicle.
3) PI3K signal path activator individual curing can activate the primordial follicle in newborn mice ovary
Mouse ovarian is processed and is cultivated after 48 hours with PI3K signal path activator, and fixedly the go forward side by side immunohistochemical methods of line flag molecule of ovary is detected.As A in Fig. 3, left side (a, c, e) is contrast, and right side (b, d, f) is the ovary after activating.A, b, the ImmunohistochemistryResults Results of FOXO3, arrow indication is the primordial follicle after activating, the dyeing of FOXO3 is transferred to cytoplasm by nucleus (seeing contrast).C, d, the coloration result of BrdU shows, cells a large amount of in the ovary tissue after activation are in vegetative state.E, f, the ImmunohistochemistryResults Results of AMH shows, the ovary after activation, after cultivating 48 hours, compared to control group ovary, has more ovarian follicle to enter the secondary follicle stage.By ovary independent with two kinds of PI3K activator or that merging was processed, be implanted under the kidney tunicle of acceptor mouse, simultaneously by the Bilateral oophorectomy of acceptor mouse.Second day after operation, to injected in mice FSH to the 14 days, then put to death acceptor mouse every day, gets ovary, as shown in B in Fig. 3, at every pictures, is control group ovary at the middle and upper levels, and lower floor is the ovary after activating.The ovary of comparing each treatment group with control group is all obviously bigger than normal, and two kinds of PI3K activator combined utilization can obviously promote the growth of mouse ovarian.
4) mTOR and PI3K signal path activator can be worked in coordination with activation and the growth that promotes mouse and people's primordial follicle
In order to verify whether mTOR and PI3K signal path activator have synergy, newborn mice ovary is processed through the activator of various combination respectively: PI3K activator bpV directly mixes application with mTOR activator PA/PRO; First apply PI3K activator bpV and 740Y-P and process 48 hours, then apply mTOR activator PA/PRO and process 6 hours; First apply mTOR activator PA/PRO and process 24 hours, then apply PI3K activator bpV and 740Y-P and process 24 hours; The ovary of control group is applied respectively mTOR or PI3K activator individual curing.By the ovarian transplantation after processing under the kidney tunicle of acceptor mouse, second day after operation, to injected in mice FSH to the 14 days, then put to death acceptor mouse the developmental state of getting ovary and observing ovary every day.As shown in Figure 4, only have and add in order in ovary combination nutrient solution when mTOR activator and PI3K activator, just can observe the synergy (Fig. 4, lower group) for ovarian growth.
Further, in research, we select people's ovary cortex, are cut into 1-2mm
3after the fritter of left and right, apply the activator of various combinations and process, the ovary tissue after processing continues to cultivate to collecting after the 6th day and carrying out subsequent analysis again.As Fig. 5 A a and b, before cultivation, in ovary cortex, be mainly primordial follicle.Ovary tissue is cultivated after 6 days in vitro, and each treatment group comprises that the ovarian follicle in control group has growth, but in each treatment group, the developmental state of ovarian follicle is obviously better than control group.As c and d in Fig. 5 A, in the stage of the ovarian follicle great majority in control group in transforming to primary follicle, even if grow to the secondary follicle stage, also only has layer 2-3 granulosa cell.And no matter be the secondary follicle that can find that in mTOR activator (e and f in Fig. 5 A) or the ovary tissue of PI3K activator (g in Fig. 5 A) individual curing cluster exists, and there is multilayer particle cell.This situation has obtained better embodiment in mTOR and PI3K activator combined utilization, finds the appearance (h in Fig. 5 A) of ovarian follicle before large chamber in the ovary tissue of its cultivation.Application BrdU mark proliferative cell, has also found that in the ovary tissue of mTOR and the processing of PI3K activator combined utilization, around ovarian follicle, the propagation (c and d in Fig. 5 B) of granulosa cell is apparently higher than control group (a and b in Fig. 5 B).Ovarian follicle count results has further been verified the result of morphological analysis, as Fig. 5 C, compared to control group, no matter is mTOR activator, PI3K activator individual curing or combined utilization, can promote the activation of primordial follicle, and promote the growth of secondary follicle.Wherein mTOR activator and PI3K activator combined utilization have synergy, can further promote the growth of secondary follicle, and are reduced in the degeneration of ovarian follicle in culturing process.Show to apply separately compared to two kinds of activator in mTOR and the PI3K activator combined utilization pattern of mouse discovery, can better promote the growth of primordial follicle in people's ovary cortex.
Sequence table
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<213> artificial sequence
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Claims (10)
1. primordial follicle activator, it is characterized in that being formed by mTOR signal path activator and PI3K signal path activator, described mTOR signal path activator comprises mTOR signal path activator I and II, mTOR signal path activator I is 50-200 μ M phosphatidic acid, and mTOR signal path activator II is 20-50 μ M Proprasylyte; Described PI3K signal path activator comprises PI3K signal path activator III and IV, and PI3K signal path activator III is the bpV of 30-100 μ M, and PI3K signal path activator IV is the 740Y-P of 250-500 μ g/mL.
2. primordial follicle activator according to claim 1, is characterized in that described phosphatidic acid is 200 μ M, and Proprasylyte is 50 μ M, and described bpV is 30 μ M, and 740Y-P is 250 μ g/mL.
3. described in claim 1 or 2, primordial follicle activator activates primordial follicle and promotes the application in follicular development medicine in preparation.
4. kit for in-vitro primordial follicle activation, is characterized in that, comprises following component:
1) mTOR signal path activator I and II;
2) PI3K signal path activator III and IV;
3) basic activating cells nutrient solution;
4) L-15 nutrient solution.
5. kit for in-vitro primordial follicle activation according to claim 4, is characterized in that mTOR signal path activator I is phosphatidic acid, and its concentration is: 50-200 μ M; MTOR signal path activator II is Proprasylyte, and its concentration is: 20-50 μ M; PI3K signal path activator III is bpV, and its concentration is 30-100 μ M; PI3K signal path activator IV is 740Y-P, and its concentration is 250-500 μ g/mL; Basic activating cells nutrient solution formula: MEMa is mother liquor, includes Sodium.alpha.-ketopropionate 0.23mM, human serum albumin 10 wt. %, L-AA 50 μ g/mL, Streptomycin sulphate 50mg/L and penicillin 75mg/L.
6. kit for in-vitro primordial follicle activation according to claim 5, is characterized in that, reagent set becomes:
solution I:basic activating cells nutrient solution;
solution II: L-15 nutrient solution is mother liquor, includes human serum albumin 10 wt.%;
reagent I: mTOR signal path activator I and II, the Proprasylyte of the phosphatidic acid that contains 50-200 μ M and 20-50 μ M, according to the packing in advance respectively of the dosage of the basic activating cells nutrient solution of 10mL;
reagent II: PI3K signal path activator III and IV, contain 30-100 μ M bpV and 250-500 μ g/mL 740Y-P, according to the packing in advance respectively of the dosage of the basic activating cells nutrient solution of 10mL.
7. kit for in-vitro primordial follicle activation according to claim 5, is characterized in that, reagent set becomes:
solution I:basic activating cells nutrient solution;
solution II:l-15 nutrient solution is mother liquor, includes human serum albumin 10 wt.%;
reagent I:the mTOR signal path activator I and the II that mix in advance, the Proprasylyte of the phosphatidic acid that contains 50-200 μ M and 20-50 μ M, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL;
reagent II:the PI3K signal path activator III and the IV that mix in advance, the 740Y-P of the PA that contains 30-100 μ M and 250-500 μ g/mL, according to the dosage packing in advance of the basic activating cells nutrient solution of 10mL.
8. according to the kit for in-vitro primordial follicle activation described in claim 6 or 7, it is characterized in that solution I, reagent I, reagent II, solution II to pack respectively, be more together packaged in same packing box.
9. the preparation method of kit for in-vitro primordial follicle activation described in claim 6 or 7, is characterized in that
solution I:basic activating cells nutrient solution compound method is as follows: MEMa is mother liquor, contains Sodium.alpha.-ketopropionate 0.23mM, human serum albumin 10wt.%, L-AA 50 μ g/mL, Streptomycin sulphate 50mg/L and penicillin 75mg/L; Described reagent I contains mTOR signal path activator and comprises: (1) mTOR signal path activator I, the phosphatidic acid of 50-200 μ M, (2) mTOR signal path activator II, the Proprasylyte of 20-50 μ M; Described reagent II contains PI3K signal path activator and comprises: (1) PI3K signal path activator I, the PA of 30-100 μ M, (2) PI3K signal path activator II, the 740Y-P of 250-500 μ g/mL; The compound method of solution II is as follows: L-15 nutrient solution is mother liquor, also contains human serum albumin 10wt.%.
10. the application method of non-diagnoses and treatment object kit for in-vitro primordial follicle activation described in claim 6 or 7, is characterized in that step is as follows:
1) get ovary cortex in 5mL solution II, ovary cortex is cut into 1mm
3fritter, by solution II, repeatedly clean;
2) get 1mL solution I and mix with reagent I, after candidate agent I dissolves completely, by solution I, be mixed with the activating cells nutrient solution of 10mL;
3) ovary cortex is incubated to the 24-orifice plate that contains the little net that suspends, little side off the net, adds 400 μ L activating cells nutrient solutions, cultivate 24 hours, culture condition is 37 ℃, 5%CO
2;
4) after 24h, get 1mL solution I and mix with reagent II, after candidate agent II dissolves completely, by solution I, be mixed with the activating cells nutrient solution of 10mL;
5) nutrient solution in culture plate is blotted, add the activation nutrient solution of new configuration, continue to cultivate 24 hours, culture condition is 37 ℃, 5%CO
2;
6) after 24 hours, activation process completes, and tissue can be used for subsequent operations.
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Effective date of registration: 20171227 Address after: New Kumho 210000 Nanjing Road, Jiangsu province high tech development 3-1, Danish Ecological Life Science Industrial Park building A 2005-2007 Patentee after: Nanjing Ivy biotech Co. Ltd. Address before: 211166 Tianyuan East Road, Jiangning District, Jiangsu, China, No. 818, No. Patentee before: Nanjing Medical University |