CN107805644A - A kind of cell based on minimum strain is pierced into method - Google Patents
A kind of cell based on minimum strain is pierced into method Download PDFInfo
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- CN107805644A CN107805644A CN201711104600.2A CN201711104600A CN107805644A CN 107805644 A CN107805644 A CN 107805644A CN 201711104600 A CN201711104600 A CN 201711104600A CN 107805644 A CN107805644 A CN 107805644A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
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Abstract
The invention provides a kind of cell based on minimum strain to be pierced into method.The characteristics of this method is during micropin is pierced into cell, is pierced into using different speed, analyzes the cell strain under friction speed by optical flow method, obtains the piercing speed under minimum strain, realizes that the cell based on minimum strain is pierced into method.This method includes：Cell position and micropin tip position are obtained using image processing method；Piercing operation is carried out to cell using different speed；Using the different cell strains being pierced under speed of optical flow method analysis, piercing speed during minimum strain is obtained, the piercing speed is selected and is pierced into service speed as conventional, realize the cell piercing method based on minimum strain.
Description
Technical field
The invention belongs to the other micromanipulative technique field of cell grade.
Background technology
The piercing process of cell is the committed step of the biological operations such as nuclear transfer, clone.By study, it is understood that working as
Preceding cell is pierced into method and has mainly been primarily focused in the success rate being pierced into and the accuracy of piercing.And for cell
Stress Study on Problems during piercing is less, but the growth that the stress problem during cell piercing again can be follow-up to cell
Development produces considerable influence, such as Patricia R.Chess have found that mechanical strain can cause the propagation of pulmonary epithelial cells；
It is related to cell interior deformation that S.J.Gladman also demonstrates spinal nerve cell development.It thus can further have influence on cell
The success rate and survival rate of the operations such as nuclear transfer, clone, therefore design a cell based on minimum strain and be pierced into method ten
Divide necessity.
The content of the invention
The present invention seeks to design a kind of cell based on minimum strain to be pierced into method, to reduce cell during piercing
Internal strain, improve the cell development potentiality after cell is pierced into and the clone in later stage, the success rate of nuclear transfer operation.
The invention provides a kind of cell based on minimum strain to be pierced into method, and methods described is different using optical flow method detection
The cell interior strain being pierced under speed, the cell interior strain for having obtained minimum are pierced into speed, finally realize minimum strain
Cell be pierced into.
Methods described specifically includes following steps：
1st, cell position and micropin tip position are obtained using the method for image procossing.
Concrete operations：After being pierced into cell and fixing the centre coordinate of cell will be being obtained using the method for image procossing
Position, the radius of cell and the needle point coordinate position for being pierced into micropin；
2nd, piercing operation is carried out to cell using different speed.
Concrete operations：After the location parameter of cell and micropin needle point is obtained, control micropin is at different rates to thin
Born of the same parents are pierced into, and are pierced into cell centre position in the horizontal direction.
3rd, the different cell strains being pierced under speed of optical flow method analysis are utilized.
Concrete operations：The cell interior under different piercing speed is obtained using optical flow method to strain, and is tried to achieve under friction speed
Cell be pierced into during maximum strain.
4th, the cell obtained under minimum strain is pierced into speed, selectes the speed and is pierced into service speed as conventional, realizes
Cell piercing method based on minimum strain.
Concrete operations：Maximum strain under the friction speed tried to achieve, minimum strain is selected, then under the strain
Speed is the piercing speed under minimum strain, selectes the speed and carries out the cell piercing then achievable cell based on minimum strain
It is pierced into.
The advantages and positive effects of the present invention：
The present invention realizes a kind of cell based on minimum strain and is pierced into method.The more conventional cell piercing side of this method
Method, the success rate and accuracy of piercing are basically unchanged, and methods described is detected intracellular under different piercing speed using optical flow method
Portion strains, and the cell interior strain for having obtained minimum is pierced into speed, and the inside for reducing cell during actual cell is pierced into should
Become, improve the cell development potentiality after cell is pierced into and the clone in later stage, the success rate of nuclear transfer operation.
Brief description of the drawings
Fig. 1 is the FB(flow block) of the inventive method.
Fig. 2 is that (Matching Template are to obtain for acquisition cell centre position, cell radius and micropin tip position
Take the matching template of tip position).
Fig. 3 is that friction speed is pierced into cell schematic diagram, and figure is that frame (a that micropin has just punctured cell:10μm/s；b:20μ
m/s；c:30μm/s；d:40μm/s；e:50μm/s).
Fig. 4 is that the cell interior that optical flow method is tried to achieve under friction speed strains (a:10μm/s；b:20μm/s；c:30μm/s；d:
40μm/s；e:50μm/s).
Fig. 5 is the cell interior maximum strain contrast under different piercing speed.
Embodiment
Embodiment 1
Cell used in the present embodiment is the family's porcine oocytes taken from local butchery site.The acquisition side of egg mother cell
Method is as follows：
After ovary takes out from slaughterhouse, the vacuum flask equipped with 35 ° to 37 ° of physiological saline is used to transport within two hours
To laboratory.Then the sterile saline at once with 37 ° of the penicillin containing 100IU/L and 50mg/L streptomysin is clear
Wash twice.Egg mother cell extracts from the folliculus of 2 to 6 mm dias on ovary, by the cell TL-Hepes-PVA of extraction
After flushing three times in 39 °, the incubator of gas concentration lwevel 5% In-vitro maturation (IVM) 42 hours., will after IVM
Cell carries out de- ovum with 0.1% hyaluronidase.Finally cell is cleaned three times with M199, these obtained cells are this example
In egg mother cell used.
The experiment of the present invention is carried out using NK-MR601 micromanipulation systems, and the apparatus system framework includes will be micro-
The moving stage of cell mass is placed within sweep of the eye, includes the motion arm of two Three Degree Of Freedoms, including it is female thin for fixing ovum
The sticking micropin of born of the same parents, including for being pierced into the piercing micropin of cell.Wherein, the pin port radius scope of sticking micropin is 50 to 80 micro-
Rice, be pierced into micropin pin port radius scope be 9 to 12 microns, be pierced into micropin hysterisis error be 1 μm, moving range be 50mm ×
50mm×50mm。
Accompanying drawing 1 gives the FB(flow block) that cell of the realization based on minimum strain provided by the invention is pierced into method, specifically
Performance is as follows：
1st, Fig. 2 show the first step of specific steps, the centre coordinate position of cell is obtained by image processing method, carefully
The radius of born of the same parents and the needle point coordinate position for being pierced into micropin：
The method of acquisition cell centre position used and cell radius is Hough detection methods in the present embodiment, can be same
When obtain cell centre coordinate position and cell radius；Acquisition tip position method used is template matches in the present embodiment
Method, the position coordinates of needle point can be obtained；
2nd, Fig. 3 show the second step of specific steps, and control micropin is pierced into cell at different rates：
We are respectively with 10 μm/s in the present embodiment, and 20 μm/s, 30 μm/s, 40 μm/s and 50 μm/s speed is entered to cell
Assassinate into because obtained cell radius and center and be pierced into micropin tip position, we can realize with
Fixed speed is pierced into cell in the horizontal direction, and micropin is pierced into the centre bit of cell by we with friction speed in the present embodiment
Put；
3rd, Fig. 4 show the 3rd step of specific steps, utilizes the different cell strains being pierced under speed of optical flow method analysis：
Strain analysis is carried out to the cell being pierced into friction speed respectively using optical flow method, each group cell is obtained and was puncturing
Maximum strain in journey；
4th, Fig. 5 show the result of the 4th step in specific steps, and the cell that com-parison and analysis is obtained under minimum strain is pierced into
Speed, select the speed and be pierced into service speed as conventional, realize the cell piercing method based on minimum strain：
After obtaining the cell interior maximum strain during friction speed is pierced into, each group result is counted, compared
Cell under to minimum strain is pierced into speed, selectes the speed and is pierced into service speed as conventional, realizes based on minimum strain
Cell piercing method.
In the present embodiment, we respectively to a batch of porcine oocytes with 10 μm/s, 20 μm/s, 30 μm/s,
40 μm/s and 50 μm/s speed has carried out being pierced into operation.10 cells, the maximum of every group of cell are operated under every group speed
The average value of strain is respectively 0.6950 ± 0.0508,0.6400 ± 0.0970,0.6130 ± 0.0536,0.5980 ± 0.0834
With 0.5410 ± 0.1006.It can be seen that when speed is 50 μm/s, the maximum strain of cell interior is minimum, selectes 50 μ
M/s is follow-up actual clone, the piercing speed of nuclear transfer operation.
Embodiment of above is merely to illustrate the present invention, and not limitation of the present invention, about the common of technical field
Technical staff, the situation of the spirit and scope of the present invention is not being departed from, can also made a variety of changes, thus it is all equivalent
Technical scheme falls within scope of the invention, and scope of patent protection of the invention should be defined by the claims.
Claims (8)
1. a kind of cell based on minimum strain is pierced into method, it is characterised in that this method includes：
1st, cell position and micropin tip position are obtained using the method for image procossing；
2nd, piercing operation is carried out to cell using different speed；
3rd, the different cell strains being pierced under speed of optical flow method analysis are utilized；
4th, the cell obtained under minimum strain is pierced into speed, selectes the speed and is pierced into service speed as conventional, realization is based on
The cell piercing method of minimum strain.
2. according to the method for claim 1, it is characterised in that cell is handled by the following method first：Ovum is female thin
Born of the same parents flushed three times with TL-Hepes-PVA after in 39 °, the incubator of gas concentration lwevel 5% In-vitro maturation (IVM) 42
Hour；After IVM, cell is subjected to de- ovum with 0.1% hyaluronidase；Finally cell is cleaned three times with M199.
3. according to the method for claim 1, it is characterised in that in the 1st step, need to obtain cell centre coordinate position, half
The coordinate position of footpath size and micropin needle point.
4. according to the method for claim 1, it is characterised in that in the 2nd step concrete operations be：Obtaining cell and micropin
After the location parameter of needle point, control micropin is pierced into cell at different rates, is pierced into cell centre in the horizontal direction
Position.
5. according to the method for claim 1, it is characterised in that in the 3rd step concrete operations be：Difference is obtained using optical flow method
The cell interior strain being pierced under speed, and try to achieve the maximum strain during the cell under friction speed is pierced into.
6. according to the method for claim 1, it is characterised in that in the 4th step concrete operations be：That has tried to achieve is not synchronized
Maximum strain under degree, minimum strain is selected, then the speed under the strain is the piercing speed under minimum strain, and selecting should
Speed carries out cell and is pierced into the cell piercing that then can be achieved based on minimum strain.
7. according to the method for claim 3, it is characterised in that be both needed to micropin needle point during friction speed piercing and be pierced into
To cell centre position, to ensure the uniformity tested every time.
8. according to the method for claim 7, it is characterised in that the Strain Distribution frame number tried to achieve is previous to be punctured in cell
The distribution of frame.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711104600.2A CN107805644A (en) | 2017-11-10 | 2017-11-10 | A kind of cell based on minimum strain is pierced into method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711104600.2A CN107805644A (en) | 2017-11-10 | 2017-11-10 | A kind of cell based on minimum strain is pierced into method |
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| CN107805644A true CN107805644A (en) | 2018-03-16 |
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| CN201711104600.2A Pending CN107805644A (en) | 2017-11-10 | 2017-11-10 | A kind of cell based on minimum strain is pierced into method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517871A (en) * | 2018-10-31 | 2019-03-26 | 南开大学 | A kind of determination method of best stoning pressure |
| CN111595508A (en) * | 2020-05-07 | 2020-08-28 | 南开大学 | Method for measuring intracellular pressure |
-
2017
- 2017-11-10 CN CN201711104600.2A patent/CN107805644A/en active Pending
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517871A (en) * | 2018-10-31 | 2019-03-26 | 南开大学 | A kind of determination method of best stoning pressure |
| CN111595508A (en) * | 2020-05-07 | 2020-08-28 | 南开大学 | Method for measuring intracellular pressure |
| CN111595508B (en) * | 2020-05-07 | 2021-06-25 | 南开大学 | Method for measuring intracellular pressure |
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Application publication date: 20180316 |
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