CN107118261B - A kind of embryo's secreting type endogenous polypeptide PDBCM and its application - Google Patents
A kind of embryo's secreting type endogenous polypeptide PDBCM and its application Download PDFInfo
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- CN107118261B CN107118261B CN201710333845.6A CN201710333845A CN107118261B CN 107118261 B CN107118261 B CN 107118261B CN 201710333845 A CN201710333845 A CN 201710333845A CN 107118261 B CN107118261 B CN 107118261B
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Abstract
The invention discloses a kind of embryo's secreting type endogenous polypeptide PDBCM and its application.A kind of embryo's secreting type endogenous polypeptide PDBCM, amino acid sequence is as shown in SEQ ID NO.1.Polypeptide PDBCM can increase BIRC6 expression, reduction apoptotic proteins Smac and caspase 9 is expressed, so as to promote Blastocyst formation.Described embryo's secreting type endogenous polypeptide PDBCM can promote Blastocyst formation and/or improve the medicine of quality of blastocysts or prepare to apply in blastocyst culture liquid during IVF preparing.
Description
Technical field
The invention belongs to supplementary reproduction field, is related to a kind of embryo's secreting type endogenous polypeptide PDBCM and its application.
Background technology
Routine IVF-ET technology passes through the development and improvement of more than 30 years, technically solve it is infertile not
The difficult medical problem puzzled for a long time such as educate, but in vitro fertilization-embryo implanting conventional therapy, in order to ensure certain clinical pregnancy
Rate, it has been convention to carry out 2-3 pieces of cleavage stage embryo transfer.Therefore, crowd's totality gemellary pregnancy rate adds 50%- in the past 30 years
70%, triplet pregnancy rate adds 400%, and multifetation serious threat Material and infant safe, and the maternal gestational phase is simultaneously in recent years
Hair disease rises 3-7 times, and infant mortality rate and the incidence of disease rise 4-10 times, and the economy for greatly increasing patient and society is born
Load.Therefore the problem of and can avoids multifetation while improving IVF Clinical Pregnancy Rate ins is always countries in the world supplementary reproduction work
The challenge of author's facing.
Research shows for many years, evades multifetation risk and can and ensures that the most generally acknowledged effective method of Clinical Pregnancy Rate in is exactly real
The single blastaea transplanting of row.Parent genital tract microenvironment can not be still simulated completely yet with current blastocyst culture system, cause body
Outer culture embryo quality and potentiality of development reduce, and embryonic development relatively lags behind.By development for many years, people's Blastocyst formation rate at present
Only about 50% or so, if the patient that all implementation blastocyst culture has 2%-40% can cancel because embryo is not developed to blastaea
Transplanting.Therefore, single blastaea transplantation strategies can not yet be adopted by most of reproductive centers at present, improve In vitro culture system
Improving Blastocyst formation rate and improving the generally development that quality of blastocysts is transplanted to single blastaea has important impetus.And in embryo
In vitro culture system, embryo culture medium is most important of which part, and the composition of culture medium grows to embryo
There is key.Substantial amounts of zoopery confirms that embryo can secrete some cytokine-like materials to adjust before plantation
Embryo's grows, and most scholars think that be obviously promoted effect of these secretion factors to embryonic development mainly appears on blastaea
Formation stages., it is known that secreting type Acrogranin promotes the formation and expansion of blastocoele；Cell factor TGF- α and EGF can pass through
Autocrine and paracrine action promote the expansion of blastocoele to expand；And exogenous TGF-β 1 is added in mice embryonic culture medium can
Effectively improve Blastocyst formation rate, blastaea hatchability and Embryo Implantation Rate in.Although in recent years, people by have found some secretion
The factor constantly improves embryo culture medium, makes some progress, but improves Blastocyst formation rate and improve the effect of quality of blastocysts
Fruit is not satisfactory.Therefore find and identify that simpler, effective new embryo secretes molecule and improves blastocyst culture base, with
It is the target that supplementary reproduction researcher pursues always to improve Blastocyst formation rate, improve quality of blastocysts.
The content of the invention
The purpose of the present invention is the above-mentioned deficiency for prior art, there is provided a kind of embryo's secreting type endogenous polypeptide
PDBCM。
It is a further object of the present invention to provide the application of the polypeptide.
It is yet another object of the invention to provide a kind of blastocyst culture base containing the polypeptide.
A kind of embryo's secreting type endogenous polypeptide PDBCM, amino acid sequence is as shown in SEQ ID NO.1.
Embryo's secreting type endogenous polypeptide PDBCM of the present invention is preparing promotion Blastocyst formation and/or is improving blastaea
Application in the medicine or reagent of quality.
Embryo's secreting type endogenous polypeptide PDBCM capsules during vitro fertilization-embryo implanting is prepared of the present invention
Application in embryo culture liquid.
A kind of blastocyst culture liquid, it is characterised in that contain described embryo's secreting type endogenous polypeptide PDBCM.
Embryo's secreting type endogenous polypeptide PDBCM capsules during vitro fertilization-embryo implanting is prepared of the present invention
Application in idioplasm amount detection reagent.
A kind of quality of blastocysts detection reagent, it is characterised in that contain described embryo's secreting type endogenous polypeptide PDBCM.
Beneficial effect：
Embryo secreting type endogenous polypeptide PDBCM (LGPSVGFDTLRGILISQ), it is HERC2 sources peptide, by 17 amino
Acid composition, there is no its correlation function to report at present.Bioinformatic analysis and experimental result are shown:1. PDBCM derives from HERC2
The 4378-4394 amino acids at PROTEIN C end, the region are also existed on the HERC2 albumen such as mouse, ox, pig, so PDBCM
Sequence is highly conserved in mammal.We equally also detect that the presence of the peptide fragment in Mouse Blastocysts nutrient solution；②
ProtParam on-line analyses find that PDBCM unstability index (Instability index) is 38.16, belongs to stable type
Polypeptide, it can be stabilized；Fat coefficient (Aliphatic index) is 131.76, average hydrophilic and hydrophobic (Grand average
Of hydropathicity) it is 0.635, hydrophobic lipophilic is shown as, is entered easily by diffusion or encytosis intracellular；
3. the PDBCM of FITC marks is added in zygote and 2 cell stage nutrient solutions, glimmering by laser co-focusing observation discovery after 2 hours
Light distribution shows that PDBCM can pass through zona pellucida to play function in zygote and 2 cell oolemma inner cell film surfaces.
PDBCM is added in mice embryonic nutrient solution, there is the biological function for promoting Blastocyst formation；Pull down experiment cards
Real PDBCM is combined with BIRC6；BIRC6 is a kind of IAP, in ox embryo by adjust apoptotic proteins degraded and
Promote Blastocyst formation；BIRC6 expression increases and apoptotic proteins Smac and caspase-9 table in Mouse Blastocysts after PDBCM processing
Used up to reducing, so as to promote Blastocyst formation.Therefore, PDBCM can prepare promotion Blastocyst formation and/or improve quality of blastocysts
Medicine, prepare and applied in blastocyst culture liquid.
By the more poly saccharide peptide standard products of PDBCM, we are compared in the nutrient solution of high-quality blastaea and the nutrient solution of non-prime blastaea
PDBCM content, as a result find that PDBCM contents in high-quality blastocyst culture liquid are significantly higher than non-prime blastaea, it is seen then that PDBCM
It can be applied in quality of blastocysts detection reagent during preparing vitro fertilization-embryo implanting.
Figure of description
The essential characteristic of Blastocyst formation correlation secreting type endogenous polypeptide in Fig. 1, embryo medium.
A：The size distribution of endogenous polypeptide；B：The isoelectric point distribution of endogenous polypeptide；C：The amino acid of endogenous polypeptide
Number distribution.
Fig. 2, differential peptides precursor protein functional analysis
Influence (the * P that Fig. 3,6 polypeptides are formed to Mouse Blastocysts<0.05；**P<0.01vs Control)
Comparision contents (* P of Fig. 4, PDBCM polypeptide in high-quality blastocyst culture liquid and non-prime blastocyst culture liquid<0.05).
A:The mass spectrogram of PDBCM polypeptides；B:PDBCM polypeptides content in height scores blastaea divides blastaea apparently higher than lower assessment.
The PDBCM of Fig. 5, FITC mark adds the distribution (bar after embryo medium on mouse zygote and 2 cell stages
=20 μm)
The influence that Fig. 6, PDBCM are formed to Mouse Blastocysts.
A:PDBCM processing after promote Mouse Blastocysts formed (2 cell mouse embryos are placed in single drop and cultivated,
A drop is put into when taking pictures to be taken pictures；)B:Compared with control group, BIRC6 expression liter in the blastaea formed after PDBCM processing
Height, and Caspase 9 and Smac expression reduces.
Embodiment
Embodiment 1
The screening of the secreting type endogenous polypeptide related to Blastocyst formation in 1.1 people's blastocyst culture liquid
In order to avoid interfering between embryo, we carry out single Embryo Culture to people's cleavage stage embryo, according to blastaea shape
Into final result collect embryo medium (be divided into form blastaea group and do not formed blastaea group) respectively, the Embryo Culture to forming blastaea
Liquid and the embryo medium progress polypeptide group credit analysis for not forming blastaea.Mass spectrometry results, which amount to, identifies polypeptide 3228, its
Middle significant difference has 201 (T-test, P<0.05).Analysis finds that these polypeptides are mainly made up of 5-21 amino acid, point
For sub- size between 500-2500Da, PI is located at 3-10 (Fig. 1).
The amyloid protein precursor functional analysis of 1.2 differential peptides
In view of endogenous cellular polypeptide is mainly derived from degraded of the intracellular protein enzyme body approach to amyloid protein precursor, therefore use
IPA softwares, functional analysis is carried out to the amyloid protein precursor of differential peptides, as a result finds that the function of these amyloid protein precursors is related to cell and sticked
Attached, cell shaping, lipid-metabolism, glycometabolism, proteolysis etc., with embryonic development and implantation closely related (Fig. 2).
The influence that the polypeptide PDBCM of embodiment 2 develops to Preimplantation Embryos of Mouse
It is PDBCM using chemical synthesis synthesis HERC2 sources peptide, amino acid sequence is as shown in SEQ ID NO.1, to small
Mouse embryonated egg carries out single Embryo Culture, and various concentrations (2 μ g/ml, 20 μ g/ml) are added in embryo medium (KSOM)
PDBCM polypeptides stimulate：Take 6-8 weeks ICR mouse, PMSG Injection, inject hCG after 48h, drawn materials after 16h；Under the microscope in defeated ovum
Pipe ampulla obtains ovum group hyaluronic acid enzymic digestion granular cell, operates ova nuda is cleaned in liquid M2 in vitro, place in advance in 37
Spend, in the KSOM nutrient solutions balanced in 5%CO2, be developed to two cells.In morning next day, two cells are added to added with not
With concentration PDBCM KSOM nutrient solutions, continue culture to blastaea.Its influence to Mouse Blastocysts formation rate is observed, is as a result found
Polypeptide PDBCM is remarkably improved Mouse Blastocysts formation rate, and certain concentration dependent (Fig. 3) is presented.
The polypeptide PDBCM of embodiment 3 in high-quality blastocyst culture liquid with the comparision contents in non-prime blastocyst culture liquid
By the more poly saccharide peptide standard products of PDBCM, we compare the nutrient solution of high-quality blastaea and non-prime blastaea by mass spectrum
PDBCM content in nutrient solution, is as a result shown in Fig. 4, and it is non-optimum as a result to find that PDBCM contents in high-quality blastocyst culture liquid are significantly higher than
(blastaea scoring uses Gamder blastaea stagings to matter blastaea, and the blastaea of the 5th day blastaea 3BB above rank is high-quality blastaea, 3BB
The blastaea of following rank is non-prime blastaea).
The PDBCM of the FITC of embodiment 4 marks adds the distribution after embryo medium on mouse zygote and 2 cell stages
Using chemical synthesis synthesis PDBCM FITC fluorescence labeling peptide fragments, it is added in KSOM nutrient solutions (20 μ g/
Ml), the zygote of normal development, two cells are added in nutrient solution, after cultivating 2h, collects zygote and two cells.2%PFA is fixed
After sample cell 20min, washing buffer cleanings, permeable membrane 15min, hoechst (1:1000) nucleus 1min is contaminated.
DABCO mountings, observed after 2 hours by laser co-focusing and find fluorescence distribution in oolemma inner cell film surface (Fig. 5).
Embodiment 5
It is added to and is developed to the blastaea of D5 days in various concentrations group and is counted, counts Blastocyst formation rate, as a result see figure
6A, it is found that after PDBCM is added in 2 cell stage nutrient solutions carries out single Embryo Culture, the rise of Mouse Blastocysts formation rate；Also, receive
Collect blastaea extraction albumen, it is significantly raised to find that BIRC6 is expressed in PDBCM addition groups by westen blot methods, and apoptosis
PROTEIN C aspas-9 and Smac are substantially reduced (Fig. 6 B).
<110>Nanjing Women and Children Healthcare Hospital
<120>A kind of embryo's secreting type endogenous polypeptide PDBCM and its application
<160> 1
<210> 1
<211> 17
<212> PRT
<213>People
<220>
<223>Embryo's secreting type endogenous polypeptide PDBCM
<400> 1
Leu Gly Pro Ser Val Gly Phe Asp Thr Leu Arg Gly Ile Leu Ile Ser
1 5 10 15
Gln
Claims (5)
1. a kind of embryo's secreting type endogenous polypeptide PDBCM, amino acid sequence is as shown in SEQ ID NO.1.
2. the embryo secreting type endogenous polypeptide PDBCM described in claim 1 is preparing promotion Blastocyst formation and/or is improving blastaea
Application in the medicine or reagent of quality.
3. embryo secreting type endogenous polypeptide PDBCM capsules during vitro fertilization-embryo implanting is prepared described in claim 1
Application in embryo culture liquid.
4. embryo secreting type endogenous polypeptide PDBCM capsules during vitro fertilization-embryo implanting is prepared described in claim 1
Application in idioplasm amount detection reagent.
5. a kind of quality of blastocysts detection reagent, it is characterised in that contain embryo's secreting type endogenous polypeptide described in claim 1
PDBCM。
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| CN109913405B (en) * | 2019-03-27 | 2022-12-02 | 南京医科大学第二附属医院 | Application of adenosine in preparation of medicine or reagent for promoting embryo formation or improving embryo quality |
| CN110669123B (en) * | 2019-09-03 | 2020-10-13 | 南京市妇幼保健院 | Secretory endogenous polypeptide PDFF-CO3 and application thereof |
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| CN104140949B (en) * | 2014-08-08 | 2017-10-03 | 山东威高新生医疗器械有限公司 | Blastocyst culture liquid and preparation method thereof |
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