CN110082327A - A kind of egg mother cell H3K27 acetylation index analysis method - Google Patents
A kind of egg mother cell H3K27 acetylation index analysis method Download PDFInfo
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- CN110082327A CN110082327A CN201910359050.1A CN201910359050A CN110082327A CN 110082327 A CN110082327 A CN 110082327A CN 201910359050 A CN201910359050 A CN 201910359050A CN 110082327 A CN110082327 A CN 110082327A
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Abstract
The invention discloses a kind of egg mother cell H3K27 acetylation index analysis methods comprising the steps of: A, is marked using H3K27 acetylation antibody, obtains fluorescent image；The present invention analyzes result compared to H3K27 acetylation fluorescence intensity in original result of study egg mother cell; after H3K27 degree of acetylation index and H3K27 acetylation spatial dispersion index analysis; it is also discrepant for can analyze out H3K27 degree of acetylation index; this illustrates that the difference of fluorescence intensity is rather than the partial region present in the entire ooecium matter.It can be concluded that our data processing method can have more intuitive embodiment to the variation of H3K27 acetylation in the cells such as egg mother cell, egg cell, internal fertilization embryo, nuclear transfer embryo; the variation of H3K27 acetylation is demonstrated from different levels, provides a more accurate biological method for later research.
Description
Technical field
The present invention relates to a kind of index analysis method, specifically a kind of egg mother cell H3K27 acetylation index analysis method.
Background technique
Histone modification refer to histone related enzyme effect issue raw methylation, acetylation, phosphorylation, polyadenylation,
The process of the modifications such as ubiquitination, ADP ribosylation.It is distributed the formational situation that can not only reflect heterochromatin, while
Reflect the change of gene expression pattern and the differentiation situation of subsequent cell.Therefore advantageous to the flow of research of acetylation of histone
In the promotion mankind to the flow of research of gene expression regulation.What is deposited now includes: cell cracking to the research of histone modification,
Histone enrichment, histone purifying, the detection etc. of histone posttranslational modification.The maturation of the process technology of histone modification research
It provides the foundation condition, while is also promoted to amino acid portion specific in acetylation of histone for the research of acetylation of histone
Flow of research of the position to gene expression regulation.
There are many covalent modification modes for histone, and wherein acetylation modification is the important mechanisms of gene transcription regulation.Group egg
White Acetylation status is in diversity, and the acetylation of histone at specific gene position is carried out in a manner of site-specific.Histone acetyl
Change mainly by acetylation of histone enzyme (histone acetylases, HATs) and histon deacetylase (HDAC) (Histone
Deacetylases, HDACs) catalysis completion.HATs makes acetylation of histone, promotes chromatinic opening, make chromatin to
The euchromatin of activation changes；HDACs makes DNA methylase inhibitor, reduces connecing between DNA and transcription factor in nucleosome
Nearly property changes chromatin to the heterochromatin of inhibition.The structure of dynamic equilibrium control tinction matter between HATs and HDACs
With the expression of gene, this balance is strictly controlled, and is a key factor for adjusting gene expression, to participate in determining
Determine the destiny of cell, balance is broken down into the direct inducement for generating certain diseases or even cancer.
On a cellular level, the data of early stage shows that Mechanisms of Histone Acetylation Modification is related to many biological processes of cell
The raising of acetylation of histone level is often along with cell cycle arrest, cell differentiation and apoptosis.The high acetylation of histone can be led
Initial meiosis kataphase is caused to generate more chromosome separation delays and chromosome bridge, this affects Oocyte in Vitro
Maturation, and the maturation in vitro of egg mother cell Oocyte quality and subsequent development in somatic cell clone and Study on Transgenic Animal
Ability is extremely important.At the genetic level, the expression regulation of histone acetylation and deacetylation and gene is closely related, HATs
Between HDACs dynamic equilibrium control tinction matter structure and gene expression, acetylation of histone state it is unbalance with
Tumour occurs closely related.Related data shows to all refer to histone deacetylase activity exception, histone in kinds of tumors
Excessive deacetylation causes expression of tumor suppressor gene inhibition or oncogene activation and overexpression, leads to tumour.Current grinds
Study carefully confirmation, in tumour cell most of histone be in low Acetylation status, and the Acetylation status of histone it is unbalance with it is swollen
The generation of tumor is closely related.Therefore, based on intracellular acetylation of histone regulatory mechanism design and develop anti-tumor drug become grind
Study carefully hot spot.In terms of embryo, acetylation of histone/deacetylation modification is further studied in mammalian gamete formation and embryo
Fetal hair educate during regulating and controlling effect, will have important meaning to the molecular basis for illustrating the infertile cause of disease, embryonic development exception
Justice, also the Mechanism Study for genetic phenotype exception after birth lays the foundation, while to raising supplementary reproduction embryo in vitro culture
Embryo's survival rate and reduction Fetal malformation are all of great significance.
The acetylation of histone all plays an important role in the level such as gene, cell, individual, therefore to histone
The field of the research of the degree of acetylation in all respects is essential.And the presently found level to acetylation of histone is judged
Standard and few and have the shortcomings that more.
The most commonly used is " enzyme-linked immunosorbent assay (ELISA) " for the research of acetylation of histone level, but this method needs
Histone is extracted, then carries out acetylation detection, it is cumbersome, influence factor is more, susceptibility is insufficient.Another detection method
It is that " it is horizontal that Liu Shi method detects acetylation of histone " process is using whole blood and peripheral blood mononuclear cells, by a series of
Comparative experiments compares the experiment conditions convection current such as different sample treatment temperature, rupture of membranes method, antibody dosage and antibody incubation time
The influence of formula cell art detection, finally establishes a set of horizontal detection means of the acetylation of histone based on flow cytometry.This
Kind detection technique needs to carry out the processing of core rupture of membranes and the processing of extracellular rupture of membranes, while should routinely carry out antibody when FCM analysis
Dose titration, antibody dosage is very few cannot sufficiently to combine antigen, but dosage may also will increase greatly very much non-specific binding, simultaneously
Increase experimental cost.
In conclusion problem of the existing technology is: the expression method IOD value of existing histone modification level can only be from
The variation of histone is reacted in single level, this research is by different level come to egg mother cell, egg cell, internal fertilization embryo
And the feature of the histone modification of clone embryos is analyzed.The technology is copolymerized the methods of microscopy using immunofluorescence, laser,
Histone H 3 K37 acetylation index analysis system is created, and utilizes the more different egg mother cell histones of the index analysis system
The difference of H3K37 acetylation.The index number system for further verifying our foundation can be used as the important of evaluation Oocyte quality
Index.
Summary of the invention
The purpose of the present invention is to provide a kind of egg mother cell H3K27 acetylation index analysis methods, to solve the back
The problem of being proposed in scape technology.
In order to achieve the object, the invention provides the following technical scheme:
A kind of egg mother cell H3K27 acetylation index analysis method comprising the steps of:
A, it is marked using H3K27 acetylation antibody, obtains fluorescent image；
B, it is analyzed with image processing techniques, obtains H3K27 acetylation total fluorescence intensity in egg mother cell, in egg mother cell
The H3K27 acetylation gross area, oocyte nuclei equatorial plane area；
C, H3K27 degree of acetylation index and H3K27 acetylation spatial dispersion index are calculated, analysis egg mother cell is used for
The feature of H3K27 acetylation.
As further technical solution of the present invention: described image processing technique passes through Image-Pro Plus6.0 image
Processing software is realized.
As further technical solution of the present invention: the H3K27 degree of acetylation index is equal to H3K27 in egg mother cell
The total luminous intensity of acetylation is divided by H3K27 acetylation total fluorescence intensity in egg mother cell.
As further technical solution of the present invention: the H3K27 acetylation such as described H3K27 acetylation spatial dispersion index is total
Fluorescence intensity is divided by oocyte nuclei equatorial plane area.
As further technical solution of the present invention: the fluorescent image is obtained by laser confocal microscope.
Compared with prior art, the beneficial effects of the present invention are: the present invention is compared to original result of study egg mother cell
Interior H3K27 acetylation fluorescence intensity analyzes result, with H3K27 degree of acetylation index and H3K27 acetylation space
After disperse index analysis, can analyze out H3K27 degree of acetylation index be also it is discrepant, this illustrates the difference of fluorescence intensity
It is rather than the partial region present in the entire ooecium matter.It can be concluded that our data processing method can be female to ovum thin
The variation of H3K27 acetylation has more intuitive embodiment in the cells such as born of the same parents, egg cell, internal fertilization embryo, nuclear transfer embryo, never
Same level demonstrates the variation of H3K27 acetylation, provides a more accurate biological method for later research.
Detailed description of the invention
Fig. 1 is the egg mother cell H3K27 acetylation Raw fluorescence photo that present example provides.
Fig. 2 is Fig. 1 to be converted to the schematic diagram after gray level image.
Fig. 3 is oocyte nuclei equatorial plane area schematic diagram.
Fig. 4 is immature oocyte various configuration H3K27 acetylation influence diagram provided in an embodiment of the present invention.
Fig. 5 is the total fluorescence intensity of immature oocyte various configuration H3K27 acetylation provided in an embodiment of the present invention
It is worth histogram.
Fig. 6 is immature oocyte various configuration H3K27 degree of acetylation index value column provided in an embodiment of the present invention
Shape figure.
Fig. 7 is different chromatin configuration egg mother cell H3K27 acetylation spatial dispersion indexes provided in an embodiment of the present invention
It is worth histogram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1: Fig. 1-7, a kind of egg mother cell H3K27 acetylation index analysis method are please referred to comprising the steps of:
A, it is marked using H3K27 acetylation antibody, fluorescent image is obtained under laser confocal microscope, B, uses Image-Pro
Plus6.0 image processing software is analyzed, and H3K27 acetylation total fluorescence intensity in egg mother cell, H3K27 in egg mother cell are obtained
C, the acetylation gross area, oocyte nuclei equatorial plane area calculate H3K27 degree of acetylation index and H3K27 acetylation sky
Between disperse index, be used for analysis egg mother cell H3K27 acetylation feature.Wherein, H3K27 degree of acetylation index is equal to
The total luminous intensity of H3K27 acetylation is divided by H3K27 acetylation total fluorescence intensity in egg mother cell in egg mother cell.H3K27 acetyl
Change the H3K27 acetylation total fluorescence intensities such as spatial dispersion index divided by oocyte nuclei equatorial plane area.
With the application of the invention, as shown in Figure 1-3, experimental result is as follows in oocyte of mouse: Fig. 1 is that Raw fluorescence shines
Piece can measure H3K27 acetylation total fluorescence intensity in egg mother cell；Fig. 2 is after Fig. 1 is converted to gray level image, according to gray scale
Value puts on black circle, the area in black circle is counted, as the H3K27 acetylation gross area in egg mother cell；Fig. 3: female for ovum in black circle
Nucleus equatorial plane area.
A and B in Fig. 4 respectively refer to the SN configuration and NSN configuration of immature oocyte.
Green represents H3K27 acetylation position, and blue represents the core of egg mother cell, and BF is that corresponding egg mother cell bright field is shone
Piece
Fig. 5 is H3K27 in the egg mother cell of different chromatin configuration egg mother cell H3K27 acetylations provided in an embodiment of the present invention
Degree of acetylation index histogram.
Fig. 6 is the H3K27 acetylation of different chromatin configuration egg mother cell H3K27 acetylations provided in an embodiment of the present invention
Extent index and H3K27 acetylation spatial dispersion index histogram.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (5)
1. a kind of egg mother cell H3K27 acetylation index analysis method, which is characterized in that comprise the steps of:
A, it is marked using H3K27 acetylation antibody, obtains fluorescent image；
B, it is analyzed with image processing techniques, obtains H3K27 acetylation total fluorescence intensity in egg mother cell, in egg mother cell
The H3K27 acetylation gross area, oocyte nuclei equatorial plane area；
C, H3K27 degree of acetylation index and H3K27 acetylation spatial dispersion index are calculated, analysis egg mother cell is used for
The feature of H3K27 acetylation.
2. a kind of egg mother cell H3K27 acetylation index analysis method according to claim 1, which is characterized in that described
Image processing techniques is realized by Image-Pro Plus6.0 image processing software.
3. a kind of egg mother cell H3K27 acetylation index analysis method according to claim 1, which is characterized in that described
H3K27 degree of acetylation index is equal to the total luminous intensity of H3K27 acetylation in egg mother cell divided by H3K27 acetyl in egg mother cell
Change total fluorescence intensity.
4. a kind of egg mother cell H3K27 acetylation index analysis method according to claim 1, which is characterized in that described
The H3K27 acetylation total fluorescence intensities such as H3K27 acetylation spatial dispersion index are divided by oocyte nuclei equatorial plane area.
5. a kind of egg mother cell H3K27 acetylation index analysis method according to claim 1 to 4, feature exist
In the fluorescent image is obtained by laser confocal microscope.
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| CN107664698A (en) * | 2017-12-25 | 2018-02-06 | 阜阳师范学院 | A kind of tri-methylated analysis indexes systems of egg mother cell H3K27 |
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| US20060040336A1 (en) * | 1999-11-29 | 2006-02-23 | Katsuyuki Tamai | Kit for determining the acetylation level of a peptide based on sensitivity of the peptide to peptidase |
| US20020193284A1 (en) * | 2001-06-18 | 2002-12-19 | Chen Lin-Feng | Methods for identifying modulators of NF-KB activity |
| CN103048471A (en) * | 2013-01-09 | 2013-04-17 | 复旦大学 | Method for quantitatively detecting protein acetylation level |
| CN107024588A (en) * | 2016-02-01 | 2017-08-08 | 上海生物芯片有限公司 | Detect the protein chip and kit of protein Acetylation Level |
| CN107664698A (en) * | 2017-12-25 | 2018-02-06 | 阜阳师范学院 | A kind of tri-methylated analysis indexes systems of egg mother cell H3K27 |
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Application publication date: 20190802 |