CN104198720A - Kit used for apoptosis detection of mammal blastocyst cell - Google Patents
Kit used for apoptosis detection of mammal blastocyst cell Download PDFInfo
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- CN104198720A CN104198720A CN201410355901.2A CN201410355901A CN104198720A CN 104198720 A CN104198720 A CN 104198720A CN 201410355901 A CN201410355901 A CN 201410355901A CN 104198720 A CN104198720 A CN 104198720A
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Abstract
The invention provides a kit used for apoptosis detection of mammal blastocyst cells. The kit includes: a PBS containing 0.5% of BSA, a PBS containing 2% of paraformaldehyde, a PBS containing 0.5% of Triton X-100 and 0.05% of Tween20, a PBS solution containing 10% of goat serum and 0.05% of Tween20, a rabbit anti-caspase-3 primary antibody, a goat anti-rabbit-IgG secondary antibody having an Alexa Fluor 594 red fluorescein marker, and a PBS containing 20 [mu]M H33342. The kit is mainly based on immunofluorescence technology. A cysteine protease 3 (the caspase-3), which is important in the apoptosis process, is employed as a target point and an antigen-antibody reaction is carried out with the primary antibody aiming to the caspase-3 and the secondary antibody having the fluorescein for carrying out fluorescence labeling and counting to an apoptotic cell in an embryo, and then cell nucleus in all of the cells are stained with a DNA dye so that an apoptosis rate of the blastocyst cells is calculated. With combination of a fluorescence microscope, accurate qualitative and quantitative detection of the apoptotic cell can be carried out. The kit is high in sensitivity, is strong in specificity, is easy to operate, is good in repeatability and is high in efficiency.
Description
Technical field
The present invention relates to cell biology field and immunofluorescence technique, specifically, relate to a kind of kit detecting for mammal blastomere apoptosis.
Background technology
Apoptosis (apoptosis) be cell for maintaining homeostasis, adapt to better living environment and a kind of self-death process that initiatively forms, it relates to the effects such as activation, expression and regulation and control of series of genes.A large amount of light microscopics and the Ultrastructural embryo who researchs and analyses before attached the planting of proof mammal just can observe apoptotic generation, the apoptosis of embryonic cell is conducive to embryo and removes dysplastic cell, but result of study finds that Apoptosis is relevant to the stagnation of embryonic development, and the apoptosis that exceeds a certain degree is unfavorable for embryo's growth.Therefore the apoptosis of studying preimplantation embryos cell will contribute to correct understanding embryo's growth course, improves culture system in vitro, improves embryo quality, solves the high problem of mammal embryo abortion ratio.
The apoptosis degree of blastomere is the important indicator of passing judgment on embryo quality, and usually dUTP fracture mark (TUNEL) method of conventional terminal deoxynucleotidyl transferase mediation detects the apoptosis of embryonic cell.TUNEL method does not need specific installation, is current widely used method.But because non-viable non-apoptotic cell also has DNA break point, form, be also TUNEL reacting positive; DNA restructuring, reparation, mitosis in cellular metabolism, the human factor in experimentation all can cause false positive as sample is fixed, proteinase pre-service etc., and therefore experiment must be established positive and negative contrast.In addition, in operating process, need to grope the fixing and digestion time of cell, to improve the susceptibility of this technology and to obtain lower background value.
Programmed cellization is dead removes some special cells in predetermined time and site exactly, and the form that these removed cells show and the variation of biochemical aspect are just defined as " apoptosis ".The apoptosis of mammal embryo cell has important effect at mammalian reproduction with in growing.
In growth course before embryo nidation, a vital role of apoptosis is to remove abnormal, the harmful or superfluous cell of minority, with this, controls the quality and quantity of embryonic cell.In vitro fertilization and blastocyst rate body-cell neucleus transplanting embryo are low, can not implantation after many embryo transfers, and embryonic cell apoptosis may be one of its reason.Pomar etc. study discovery, and the embryo of produced in vitro compares and has higher apoptosis rate with the embryo who obtains in body, and clone's embryo has higher apoptosis ratio than embryo in vitro fertilization.The proofs such as Betts, whether the apoptosis of embryonic cell can indicate embryo to cultivate under appropriate environment and pressure, and the increase of apoptosis indication Embryo Culture is under unconformable environment, as unaccommodated nutrient solution composition or unaccommodated cultivation density.
The blastomere of apoptosis is discharged in all gaps of ovum or blastocoele, apoptotic cell generation shrinkage, chromatic agglutination, DNA break.Terminal deoxynucleotidyl transferase mediation the fracture of marker DNA effectively of dUTP fracture mark (TUNEL) method, demonstrate the cell of apoptosis, so it is the common method that detects at present embryonic cell apoptosis.To in ox body and the blastaea of in vitro culture, the embryo that TUNEL method detects 91-100% is contained apoptotic cell; In the blastaea of growing in pig body, have the embryo of 56-7l% to contain apoptotic cell, and in the blastaea of in vitro culture, 90% embryo is contained apoptotic cell.About the type of apoptotic cell, for the blastaea of ox, Mouse and rat, the apoptosis rate of inner cell mass is higher; For the blastaea of people and pig, apoptotic cell is distributed in inner cell mass and trophoderm equably.The employing TUNEL method such as Xu Wei proof heat stress can increase the lonely female activation embryo's of ox apoptosis number, and the researchs such as Sun Guojie show, the apoptosis rate of transgenic embryo is also significantly higher than non-transgenic embryo.In a word, compare with the blastaea of growing in body, it is high that the apoptosis rate of the blastaea of in vitro culture is wanted.
Along with deepening continuously to Apoptosis Study on Molecular Mechanism, find that there is the apoptosis of the Gene regulation mammalian cell of Liang Ge family, it is cysteine proteinase enzyme (cysteinyl aspartate specific proteinase, the caspases) family of Bcl-2 family and aspartic acid specific.The former comprises short apoptosis member (Bax, Bcl-Xs, Bak, Bad etc.) and the large class of anti-apoptosis member (Bcl-2, Bcl-XL, Bcl-W) two.Bcl-2 family member is mainly combined with nuclear membrane, mitochondrial membrane and endoplasmic reticulum, and the gene of this family starts by dead enzyme-specific and regulates Apoptosis.The member of the dead enzyme-specific Dou Shi caspase of majority family, it is present in the halfcystine/aspartate specific protease in endochylema or core, wherein caspase-3 is the key element in apoptosis process, it activates with extraordinary expression and all causes Apoptosis, can be by the interaction regulating cell apoptosis with numerous protein factors.In addition, the downstream of Caspase-3 in the orderly cascade reaction of apoptosis, is most important effect type Caspase, is one of most important apoptosis executor in Caspase family, so Caspase-3 is often used as the mark that Apoptosis detects.
Summary of the invention
The object of this invention is to provide a kind of kit detecting for mammal blastomere apoptosis.
In order to realize the object of the invention, the kit detecting for mammal blastomere apoptosis of the present invention, comprising containing the PBS liquid of 0.5%BSA, containing the PBS liquid of 2% paraformaldehyde, containing the PBS liquid of 0.5%Triton X-100 and 0.05%Tween20, containing the anti-caspase-3 primary antibodie of PBS liquid, rabbit of 10% lowlenthal serum and 0.05%Tween20, anti-with the goat anti-rabbit igg two of Alexa Fluor594 red fluorescence element mark, and containing the PBS liquid of 20 μ M H33342.
Wherein, the anti-caspase-3 primary antibodie of rabbit is the polyclonal antibody with the polypeptide antigen immune rabbit acquisition of the n terminal residue of close Asp175 in synthetic people caspase-3 albumen.The anti-caspase-3 primary antibodie of rabbit is diluted in confining liquid by the volume ratio of 1:500.Described confining liquid is the PBS liquid containing 10% lowlenthal serum and 0.05%Tween20.
Goat anti-rabbit igg two is anti-to be diluted in above-mentioned confining liquid by the volume ratio of 1:500.
The mammal relating in the present invention includes but not limited to ox, pig, mouse etc.Be preferably ox.
The present invention is mainly based on immunofluorescence technique, the important caspase-3 (caspase-3) of take in apoptosis process is target spot, employing is for caspase-3 primary antibodie and carry two of fluorescein and anti-carry out antigen-antibody reaction, and the apoptotic cell in embryo is carried out to fluorescence labeling and counting; Use again the nucleus of DNA dye coloring all cells, thereby calculate the apoptosis rate of blastomere.Combined with fluorescent microscope of the present invention, can carry out accurate quantitative and qualitative analysis detection to apoptotic cell, highly sensitive, high specificity, and simple to operate, reproducible, efficiency is high.
Accompanying drawing explanation
Fig. 1 is the apoptotic cell coloration result in ox blastaea in the embodiment of the present invention 1.
Fig. 2 is the total cell dyeing result of ox blastaea in the embodiment of the present invention 1.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The anti-caspase-3 primary antibodie of rabbit of using in following examples is the polyclonal antibody with the polypeptide antigen immune rabbit acquisition of the n terminal residue of close Asp175 in synthetic people caspase-3 albumen, purchased from Cell Signal Technology company limited, article No. 9661.
Kit and application that embodiment 1 detects for ox blastomere apoptosis
Based on immunofluorescence technique, develop the kit detecting for ox blastomere apoptosis, described kit comprises following reagent:
1, washing lotion: containing the PBS liquid of 0.5%BSA;
2, immobile liquid: containing the PBS liquid of 2% paraformaldehyde (PFA);
3, penetrating liquid: containing the PBS liquid of 0.5%Triton X-100,0.05%Tween20;
4, confining liquid: containing the PBS liquid of 10% lowlenthal serum, 0.05%Tween20;
5, primary antibodie: the anti-caspase-3 antibody of rabbit (volume ratio by 1:500 is diluted in confining liquid);
6, two is anti-: the goat anti-rabbit igg (volume ratio by 1:500 is diluted in confining liquid) that is marked with Alexa Fluor594 red fluorescence element;
7, H33342 dyeing liquor: containing the PBS liquid of 20 μ M H33342.
Differential dyeing program following (following all dying operations all complete in four orifice plates):
A. ox blastaea is washed 3 times in 37 ℃ of washing lotions;
B. use fixedly 20min of immobile liquid room temperature, the many 25 pieces of blastaeas of the every Kongzui of four orifice plates;
C. room temperature treatment 30min in penetrating liquid, the penetrating liquid of every hole 500 μ L, the many 25 pieces of blastaeas of every Kongzui;
D. take out blastaea, in washing lotion, clean 3 times;
E. room temperature treatment 20min in 2M HCl;
F. room temperature treatment 20min in 100mM Tris-HCl;
G. take out blastaea, in washing lotion, clean 3 times;
H. put into confining liquid, 4 ℃ are spent the night;
I. take out blastaea and move in the anti-caspase-3 primary antibodie of rabbit, incubated at room 2h;
J. take out blastaea, in washing lotion, wash 3 times, be more than each 5min;
K. blastaea is put into the goat anti-rabbit igg of mark Alexa Fluor594, incubated at room 2h, lucifuge;
L. take out blastaea, in washing lotion, wash 3 times, be more than each 5min;
M. blastaea is put into H33342 dyeing liquor, room temperature dyeing 10min;
N. take out blastaea, washing lotion is washed 3 times;
O. use DABCO mounting, compressing tablet, takes pictures under fluorescent microscope.
Ox blastomere counting: add up respectively apoptosis cell and the total cell number of blastaea under fluorescent microscope, calculate apoptosis rate (apoptosis cell/total cell number) and assess quality of blastocysts.
Ox blastomere apoptosis rate statistics: experiment repeats 3 times, chooses at random 10 ox blastaeas at every turn, under green, burst of ultraviolel irradiation, counts apoptotic cell (Fig. 1), the total cell of blastaea (Fig. 2) respectively, and both compare and draw apoptosis rate.The mean value of the total cell number of ox blastaea is 64.50, and apoptosis rate is 5.67%, and visible embryo's apoptosis ratio is lower, and embryo quality is outstanding, and later stage developmental potency is better.(table 1)
Table 1 ox blastomere apoptosis rate statistics
Note: the total cell number in apoptosis rate=apoptosis cell * 100/
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
List of references
Pomar?FJ,Teerds?KJ,Kidson?A,Colenbrander?B,Tharasanit?T,Aguilar?B,Roelen?BA.Differences?in?the?incidence?of?apoptosis?between?in?vivo?and?in?vitro?produced?blastocysts?of?farm?animal?species:a?comparative?study.Theriogenology.2005,63:2254-2268.
Betts?DH,King?WA.Genetic?regulation?of?embryo?death?and?senescence.Theriogenology.
2001,55:171-91.
Xu Wei, Tang Shuan, Lv Zhi is first-class. the impact of heat shock damage on the follow-up growth of the lonely female activation embryo of ox. journal of animal science and veterinary medicine.
2010,41:1253-1259.
Sun Guojie, Li Rong, wears and accumulates equality. transgenosis and cloning again the apoptotic impact of clone embryos. and natural science progress .2009,19:266-274.
Luo Ting etc. the attached progress of planting front embryonic cell apoptosis of mammal. Chinese animal and veterinary .2012,39:125-130.
Claims (6)
1. the kit detecting for mammal blastomere apoptosis, it is characterized in that, described kit comprises containing the PBS liquid of 0.5%BSA, containing the PBS liquid of 2% paraformaldehyde, containing the PBS liquid of 0.5%Triton X-100 and 0.05%Tween20, containing the anti-caspase-3 primary antibodie of PBS liquid, rabbit of 10% lowlenthal serum and 0.05%Tween20, anti-with the goat anti-rabbit igg two of Alexa Fluor594 red fluorescence element mark, and containing the PBS liquid of 20 μ M H33342.
2. kit according to claim 1, is characterized in that, the anti-caspase-3 primary antibodie of described rabbit is the polyclonal antibody with the polypeptide antigen immune rabbit acquisition of the n terminal residue of close Asp175 in synthetic people caspase-3 albumen.
3. kit according to claim 2, is characterized in that, the anti-caspase-3 primary antibodie of described rabbit is diluted in confining liquid by the volume ratio of 1:500; Wherein, described confining liquid is the PBS liquid containing 10% lowlenthal serum and 0.05%Tween20.
4. kit according to claim 2, is characterized in that, described goat anti-rabbit igg two is anti-to be diluted in confining liquid by the volume ratio of 1:500; Wherein, described confining liquid is the PBS liquid containing 10% lowlenthal serum and 0.05%Tween20.
5. according to the kit described in claim 1-4 any one, it is characterized in that, described mammal includes but not limited to ox, pig, mouse.
6. kit according to claim 5, is characterized in that, described mammal is ox.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104807788A (en) * | 2014-12-18 | 2015-07-29 | 中山大学 | Method for monitoring vascular remodeling phenomenon and kit |
| CN106124755A (en) * | 2016-06-17 | 2016-11-16 | 广州杰特伟生物科技有限公司 | AnnexinV FITC apoptosis kit |
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| CN101897699A (en) * | 2010-06-13 | 2010-12-01 | 中国人民解放军第三军医大学第一附属医院 | Application in preparation of medicament for preventing and controlling lipopolysaccharide-induced acute liver damage |
| CN102183651A (en) * | 2011-02-22 | 2011-09-14 | 沈阳药科大学 | Caspase-3 proenzyme protein immunohistochemical diagnosis kit and application |
| WO2016079146A1 (en) * | 2014-11-17 | 2016-05-26 | Genome Research Limited | In vitro production of expanded potential stem cells |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101302256A (en) * | 2007-05-11 | 2008-11-12 | 中国人民解放军第二军医大学 | Novel recombinant fusion molecule and antineoplastic treatment function thereof |
| CN101897699A (en) * | 2010-06-13 | 2010-12-01 | 中国人民解放军第三军医大学第一附属医院 | Application in preparation of medicament for preventing and controlling lipopolysaccharide-induced acute liver damage |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807788A (en) * | 2014-12-18 | 2015-07-29 | 中山大学 | Method for monitoring vascular remodeling phenomenon and kit |
| CN106124755A (en) * | 2016-06-17 | 2016-11-16 | 广州杰特伟生物科技有限公司 | AnnexinV FITC apoptosis kit |
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Application publication date: 20141210 |