CN109402220A - A method of collecting blastocoele liquid - Google Patents
A method of collecting blastocoele liquid Download PDFInfo
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- CN109402220A CN109402220A CN201811470251.0A CN201811470251A CN109402220A CN 109402220 A CN109402220 A CN 109402220A CN 201811470251 A CN201811470251 A CN 201811470251A CN 109402220 A CN109402220 A CN 109402220A
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Abstract
The invention discloses a kind of methods for collecting blastocoele liquid, include the following steps: the preparation of microneedle, and with needle instrument is drawn, broken needle instrument and card grinding instrument make the injection needle that internal diameter is 10 μm and the fixed pin that needle mouth is 40 μm, injection needle are top-notch by needle point；The collection of blastocoele liquid, under micromanipulation instrument, inner cell mass side is fixed with fixed pin, the liquid in blastocoele in absorption blastocoele is needled into as in 1 μ LDPBS drop with injection, and the liquid in blastocoele is transferred in the centrifuge tube of 200 μ L of import with thinner mouth suction spindle after the completion of collection；The detection of DNA content in blastocoele liquid.This collection method easily punctures blastocoele using homemade thinner injection needle, smaller on influencing caused by blastaea subsequent development, and more precisely, can collect the liquid in single blastocoele, provide new method for science of heredity screening before embryo implantation.
Description
Technical field
The invention belongs to cell biology, specifically a kind of method for collecting blastocoele liquid.
Background technique
Before embryo nidation in growth course, need by the spilting of an egg several times, blastomere densification and Blastocyst formation, expansion and
Stages, the height of quality of blastocysts such as hatching determine the potentiality of embryo's subsequent development ability.Blastaea is mainly by two kinds of
Cell composition, volume is larger and the cell of negligible amounts, referred to as inner cell mass, and this cell develops into various groups of fetus in the future
It knits and organ；And prolong the extension of oolemma inner wall and arrangement, small volume and a fairly large number of cell, referred to as trophocyte,
These cells develop into embryophoric membrane and placenta in the future.Quality of blastocysts is to influence the key factor of embryo transfer success or failure.Therefore, pass through shape
State observation, cell or molecular marker detection technology come evaluate quality of blastocysts help to improve the pregnancy rate after animal embryo transplanting and
Birth rate.On morphology, the tightness degree of blastaea inner cell mass cells number and the combination of these inner cell mass cells is nourished
Whether whether confluent monolayer cells combine close by more cell composition, between cell, these morphological observations are all to determine blastaea matter
Measure the standard of superiority and inferiority.In addition, also can be determined that blastaea by analysis embryonic cell number, embryo metabolism activity, chromosome abnormality etc.
Whether there are the potentiality for continuing development.However, these detection methods be all it is amount of activated for cost to damage blastaea, it is such
Its developmental potency will significantly reduce after blastaea transplanting.
In human reproduction's medicine, the sample that science of heredity screening mainly uses before traditional embryo implantation is that cleavage stage is single
Blastomere 1-2 or blastula stage trophocyte 5-10.It is this by taking cleavage stage or blastula stage unicellular progress embryo's plant
Entering prochromosome screening may have a certain impact to the development of late embryogenesis.Therefore, a kind of pair of embryonic development is invented relatively to pacify
Full quality of blastocysts detection method is particularly important.Blastocoele liquid contains some DNA moleculars of blastomere secretion, certain
DNA molecular is the important molecular markers for evaluating quality of blastocysts height, therefore passes through DNA molecular content in evaluation blastocoele liquid
Whether there is or not or height can reflect quality of blastocysts indirectly, this be it is a kind of not will cause the reduction of blastomere quantity without infringement blastaea matter
Measure assessment technology.Amount of liquid that is very small in view of blastaea volume, containing is also very low, and urgent need, which develops one kind, can accurately collect capsule
The method of embryo chamber liquid.The liquid in blastocoele is sucked out by micromanipulative technique by the present invention, then detects blastocoele liquid
In the feasibility of such method whether is concluded containing DNA, and then open up a kind of new method for detection quality of blastocysts.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for collecting blastocoele liquid, to solve to propose in above-mentioned background technique
The problem of.
To achieve the above object, the invention provides the following technical scheme:
A method of blastocoele liquid is collected, is included the following steps:
(1) prepared by micromanipulation injection needle: thin glass tube one for being passivated both ends is taken, is installed on and needle instrument is drawn to carry out drawing needle,
After pulling needle, broken needle instrument is opened, internal diameter is made and is 10 μm of flat mouth needle, then carries out card grinding, so that needle mouth wears into angle, by milled
Needle tubing carry out broken needle, looper, make needle point be bent 20 ° it is spare；
(2) prepared by micromanipulation fixed pin: the good needle tubing of above-mentioned drawing being mounted on broken needle on disconnected instrument, disconnected good rear needle carries out again
Looper makes needle tubing be bent 20 °, needle is unloaded, is placed in spare in box for needle or pin；
(3) collection of blastocoele liquid
Blastaea is sucked with micromanipulation fixed pin first, stirring blastaea with micromanipulation injection needle makes inner cell mass and show
The tight-lipped patch of microoperation fixed pin needle sucks blastaea at this time and pricks after passing through oolemma with injection needle into blastocoele, blastaea is sucked out
Liquid in chamber is simultaneously transferred in the DPBS of 1 μ L on right side, tries not to be drawn to any cellular material.Collect 10 pieces of capsules
The blastaea liquid being mixed in 1 μ L DPBS is sucked out with thinner mouth suction spindle, is put into 200 μ L imports of import by the liquid in embryo chamber
In centrifuge tube；
(4) liquid for the blastocoele being collected into is subjected to DNA content detection.
Preferably, in step (1), needle specific steps are drawn are as follows:
It opens and draws needle instrument, adjust and draw needle instrument parameter: P=500, PULL=200, VEL=80, TIME=100.It takes and is passivated
The thin glass tube at both ends one is installed on and draws needle instrument.PULL key is pressed, the needle tubing pulled down is carefully unloaded after system end of run, is put
Enter box for needle or pin and carries out broken needle in case concentrating.
Preferably, in step (1), broken needle specific steps are as follows:
Open broken needle instrument switch.10 × object lens adjust glass pin position, will be at 10 μm of glass tube tip internal diameter and bead
Just contact.Heating knob is adjusted to 35 DEG C or so, steps on floor push, when seeing that glass needle and bead stick together,
And glass tube tip unclamps floor push when having the tendency that bent, and since cooling is shunk, bead can breaking glass pipe
Tip, to obtain the flat mouth needle that satisfactory internal diameter is 10 μm.
Preferably, in step (1), card grinding specific steps are as follows:
Card grinding instrument switch is opened, and distilled water will be piled in water injection pipe above grinding stone, with water-soaked grinding stone, adjusts grinding stone
Revolving speed is 30rpm.Card grinding angle is located in 45 °, and the glass needle tubing to have broken is installed on fixed handle.Turn rough quasi-coil,
Make needle point rapid decrease, when close to grinding stone, uses thin quasi- burnt spiral instead, observed by eyepiece, careful needle point is fallen on grinding stone.Contact
After grinding stone, liquid level rises in needle tubing, and thin quasi- burnt spiral is turned 90 ° again.After continuing 1-2min, needle mouth can be worn into tiltedly
Mouthful.If not wearing into angle, can continue to grind.The needle tubing of milled is removed.
Preferably, in step (1), top-notch and looper specific steps are as follows:
The needle tubing of milled is installed on broken needle instrument, glass tube and bead relative position are adjusted, it is mutual when avoiding operating
Contact.Needle is holded up under 10 × object lens, is located at needle point above bead, heating knob is adjusted to 40 DEG C or so, steps on and steps on
Plate falls needle point with thin spiral, after needle point contacts softening with bead, rapidly lifts needle point, unclamps after forming needle point
Pedal.Length of needlepoint is 11-13um, it is preferable that length of needlepoint 12um.After the completion of top-notch, the needle of point will be pulled out under 5 × object lens
It is placed on the left of bead in same plane at 100 μm of calibers, first makes bead far from needle tubing, heating knob is adjusted to 60 DEG C,
Pedal is slammed simultaneously by glass marble close to needle tubing, when needle tubing is bent to 20 °, unclamps floor push, remove glass needle.
Preferably, in step (2), micromanipulation fixed pin prepares specific steps are as follows:
The good needle tubing of above-mentioned drawing is mounted on broken needle instrument, heating knob is transferred to 40 DEG C, breaks at 100 μm under 10 × object lens
Needle holds up needle after breaking well, and needle mouth is placed in the top of glass marble, first makes glass marble far from needle mouth, heating knob is adjusted to 60
DEG C, step on pedal, while by needle point close to glass marble, when needle necking to unclamped at 40 μm and when needle mouth smooths out pedal stop plus
The needle for pulling out point is placed on the left of bead 100 μm or so calibers in same plane under 5 × object lens by fixed pin from holding up by heat
Place.Needle tip is placed in glass marble left downward, and heating knob is adjusted to 60 DEG C and steps on switch, so that needle tubing is bent 20 °, needle is unloaded
Under, it is placed in spare in box for needle or pin.
Preferably, in step (3) before the collection of blastocoele liquid, first by above-mentioned ready-made micromanipulation injection needle and
Fixed pin is mounted on micromanipulation instrument and adjusts angle, be placed in 0.25% trypsase drop check fixed pin and
Whether injection needle use is normal.The 35mm culture dish ware lid for taking a clean import, with preheated DPBS among ware lid
By left side, vertical setting of types does three 10 μ L DPBS drops, and the drop of a 1 μ L is done on the right side of each drop, is covered with paraffin oil
DPBS drop.The 7th day Blastocysts are moved into mouth suction spindle in the DPBS drop of an intermediate 10 μ L, micromanipulation is injected
Needle lifts and culture dish is placed on objective table, puts down micromanipulation injection needle, 10 μ that micromanipulation injection needle is placed over
In LDPBS drop, micromanipulation injection needle is cleaned, the trypsase in micromanipulation injection needle is avoided to have an impact blastaea.
Compared with prior art, the beneficial effects of the present invention are:
1. collect blastocoele liquid, smaller to the injury of blastaea, blastaea can carry out subsequent development.
2. the DNA damage in pair blastocoele being sucked out is smaller, DNA long chain break not will cause.
3. carry out science of heredity screening before traditional embryo implantation, only need a small amount of blastocoele liquid that can go to detect institute
The information needed.
4. more economical convenience is received by vast scientific research institution.
Detailed description of the invention
Fig. 1 is the collection schematic diagram of blastocoele liquid of the present invention.
Fig. 2 is the DNA content testing result figure of DPBS.
Fig. 3 is the DNA content testing result figure of the DPBS containing blastocoele liquid.
Specific embodiment
1. material
Microscope (OLYMPUS, IX51)；Micromanipulation handle (eppendorf, CellTram vario)；Microelectrode control
Instrument (Sutter, P-97) processed；Card grinding instrument (NARISHIGE, EG-400)；It forges needle instrument (NARISHIGE, MF-900)；Spectrophotometric
It counts (Thermo, NanoDrop 2000)；BJ-40 thin glass tube (1.0 × 0.8 × 100,2.5 × 2.0 × 100)；Centrifuge tube (200 μ
L, 1.5mL, Axygen)；10 μ L (Dragon Lab, 0.5-10 μ L)；200 μ L (Dragon Lab, 10-100 μ L)；Import culture
Ware: diameter 35mm (Corning, USA)；200 μ L centrifuge tubes (BBI life sciences)
2. reagent
DPBS (gibco, 14190-144)；RNAse-Water；Embryo medium is PZM-3；Trypsase (HyClone,
SH30042.01)；Mineral oil (SIGMA, M8410-1L)
Embodiment 1: the liquid in the 7th day blastocoele is collected
The preparation of 1.1 micromanipulation injection needles
Micromanipulation injection needle
Draw needle
It opens and draws needle instrument, adjust and draw needle instrument parameter: P=500, PULL=200, VEL=80, TIME=100.It takes and is passivated
The thin glass tube at both ends one is installed on and draws needle instrument.PULL key is pressed, the needle tubing pulled down is carefully unloaded after system end of run, is put
Enter box for needle or pin and carries out broken needle in case concentrating.
Broken needle
Open broken needle instrument switch.10 × object lens adjust glass pin position, will be at 10 μm of glass tube tip internal diameter and bead
Just contact.Heating knob is adjusted to 35 DEG C or so, steps on floor push, when seeing that glass needle and bead stick together,
And glass tube tip unclamps floor push when having the tendency that bent, and since cooling is shunk, bead can breaking glass pipe
Tip, to obtain the flat mouth needle that satisfactory internal diameter is 10 μm.
Card grinding
Card grinding instrument switch is opened, and distilled water will be piled in water injection pipe above grinding stone, with water-soaked grinding stone, adjusts grinding stone
Revolving speed is 30rpm.Card grinding angle is located in 45 °, and the glass needle tubing to have broken is installed on fixed handle.Turn rough quasi-coil,
Make needle point rapid decrease, when close to grinding stone, uses thin quasi- burnt spiral instead, observed by eyepiece, careful needle point is fallen on grinding stone.Contact
After grinding stone, liquid level rises in needle tubing, and thin quasi- burnt spiral is turned 90 ° again.After continuing 1-2min, needle mouth can be worn into tiltedly
Mouthful.If not wearing into angle, can continue to grind.The needle tubing of milled is removed.
Top-notch and looper
The needle tubing of milled is installed on broken needle instrument, glass tube and bead relative position are adjusted, it is mutual when avoiding operating
Contact.Needle is holded up under 10 × object lens, is located at needle point above bead, heating knob is adjusted to 40 DEG C or so, steps on and steps on
Plate falls needle point with thin spiral, after needle point contacts softening with bead, rapidly lifts needle point, unclamps after forming needle point
Pedal.Length of needlepoint is 11-13 μm, it is preferable that length of needlepoint is 12 μm.After the completion of top-notch, the needle of point will be pulled out under 5 × object lens
It is placed on the left of bead in same plane at 100 μm of calibers, first makes bead far from needle tubing, heating knob is adjusted to 60 DEG C,
Pedal is slammed simultaneously by glass marble close to needle tubing, when needle tubing is bent to 20 °, unclamps floor push, remove glass needle.
Micromanipulation fixed pin
The good needle tubing of above-mentioned drawing is mounted on forging needle instrument, heating knob is transferred to 40 DEG C, breaks at 100 μm under 10 × object lens
Needle holds up needle after breaking well, and needle mouth is placed in the top of glass marble, first makes glass marble far from needle mouth, heating knob is adjusted to 60
DEG C, step on pedal, while by needle point close to glass marble, when needle necking to unclamped at 40 μm and when needle mouth smooths out pedal stop plus
The needle for pulling out point is placed on the left of bead 100 μm or so calibers in same plane under 5 × object lens by fixed pin from holding up by heat
Place.Needle tip is placed in glass marble left downward, and heating knob is adjusted to 60 DEG C and steps on switch, so that needle tubing is bent 20 °, needle is unloaded
Under, it is placed in spare in box for needle or pin.
The collection (Fig. 1) of 1.2 blastocoele liquid
Above-mentioned ready-made micromanipulation injection needle and fixed pin are mounted on micromanipulation instrument and are adjusted angle, is put
Check whether fixed pin and injection needle use are normal in 0.25% trypsase drop.Take a clean import
35mm culture dish ware lid does three 10 μ L DPBS drops by left side vertical setting of types among ware lid with preheated DPBS, in each liquid
The drop that a 1 μ L is on the right side of drop covers DPBS drop with paraffin oil.It is with mouth suction spindle that Blastocysts immigration in the 7th day is intermediate
In the DPBS drop of one 10 μ L, micromanipulation injection needle is lifted and culture dish is placed on objective table, puts down micromanipulation
Injection needle in the 10 μ LDPBS drops for being placed over micromanipulation injection needle, cleans micromanipulation injection needle, avoids micro- behaviour
The trypsase made in injection needle has an impact blastaea.Micromanipulation injection needle is moved into intermediate DPBS by moving stage
Start to collect the liquid in blastocoele in drop.Blastaea is sucked with fixed pin first, stirring blastaea with injection needle makes inner cell
Group and the tight-lipped patch of fixed pin needle, such as Fig. 1, shown in A；Then it sucks blastaea and pricks after passing through oolemma with injection needle into blastocoele
In, such as Fig. 1, shown in B, the liquid that is sucked out in blastocoele is simultaneously transferred in the DPBS of 1 μ L on right side, tries not to be drawn to
Any cellular material.The liquid in 10 pieces of blastocoeles is collected, is inhaled the blastaea liquid being mixed in 1 μ L DPBS with thinner mouth suction spindle
Out, it is put into 200 μ L import centrifuge tubes of import.
The liquid for the blastocoele being collected into is carried out DNA content detection by 1.3
By the DPBS wink containing blastocoele liquid in 200 μ L import centrifuge tubes from rear progress DNA concentration detection, by Fig. 3
, it is apparent that the DPBS containing blastocoele liquid plays peak at 260nm, illustrate this collection side for collecting blastocoele liquid
Method be it is feasible, Fig. 2 be DPBS blank test.
It can be obtained through experimental result, it is feasible, and this method that the liquid in blastocoele is collected by such method
More accurate, the liquid that can be collected into single blastocoele measures the quality of single blastaea, and after measuring quality of blastocysts,
Blastaea can also continue to be developed.Be compared to it is traditional puncture blastocoele by means of the method for laser boring and Piezo,
Piezo applies the vibration of ultra-high frequency to injection needle in operation, may generate physical damnification to the DNA of injection needle, make
The fracture of growth chain DNA.In addition, the special installation of Piezo is more expensive, installation instruction is inconvenient, frequent operation and is not easy to slap
It holds.In the method for carrying out laser boring to oolemma, oolemma is also provided other than supporting to protect blastaea for blastaea necessary
Substance channel, in many albumen of oolemma surface inserting, the missing of any albumen can all influence oolemma function and
The development of blastaea, therefore, laser boring is deeper on oolemma, bigger to the development impact of blastaea.If laser boring is deeper, and
Injection is needled into shallower, easily leads to the liquid outflow of blastocoele, and also have certain damage to trophocyte.
In summary, blastocoele is easily punctured using homemade thinner injection needle, to produced by blastaea subsequent development
Influence it is smaller, and be compared to the more cheap economy of Piezo and laser boring both methods, can be vast scientific research machine
Structure is received.
Claims (7)
1. a kind of method for collecting blastocoele liquid, which comprises the steps of:
(1) prepared by micromanipulation injection needle: taking thin glass tube one for being passivated both ends, is installed on and needle instrument is drawn to carry out drawing needle, pull
After needle, broken needle instrument is opened, internal diameter is made and is 10 μm of flat mouth needle, then carries out card grinding, so that needle mouth wears into angle, by the needle of milled
Pipe carries out broken needle, top-notch, looper, keeps 20 ° of needle tubing bending spare；
(2) prepared by micromanipulation fixed pin: the good needle tubing of above-mentioned drawing being mounted on broken needle on broken needle instrument, disconnected good rear needle carries out curved again
Needle makes needle tubing be bent 20 °, needle is unloaded, is placed in spare in box for needle or pin；
(3) collection of blastocoele liquid: blastaea being sucked with micromanipulation fixed pin first, stirs capsule with micromanipulation injection needle
Embryo makes inner cell mass and the tight-lipped patch of micromanipulation fixed pin needle, sucks blastaea at this time and pricks after passing through oolemma with injection needle into capsule
In embryo chamber, be sucked out blastocoele in liquid and be transferred to right side 1 μ L DPBS in, try not to be drawn to any cell
Substance collects the liquid in 10 pieces of blastocoeles, and the blastaea liquid being mixed in 1 μ L DPBS is sucked out with thinner mouth suction spindle, is put into
In 200 μ L import centrifuge tubes of import；
(4) liquid for the blastocoele being collected into is subjected to DNA content detection.
2. the method according to claim 1 for collecting blastocoele liquid, which is characterized in that in step (1), draw needle set body step
Suddenly are as follows: open and draw needle instrument, adjust and draw needle instrument parameter: P=500, PULL=200, VEL=80, TIME=100, take passivation two
The thin glass tube at end one is installed on and draws needle instrument, presses PULL key, the needle tubing pulled down is carefully unloaded after system end of run, is put into
Box for needle or pin carries out broken needle in case concentrating.
3. the method according to claim 1 for collecting blastocoele liquid, which is characterized in that in step (1), broken needle is specifically walked
Suddenly are as follows: open broken needle instrument switch, 10 × object lens adjust glass pin position, will be rigid with bead at 10 μm of glass tube tip internal diameter
Heating knob is adjusted to 35 DEG C or so, steps on floor push by good contact, when seeing that glass needle and bead stick together, and
And glass tube tip unclamps floor push when having the tendency that bent, since cooling is shunk, bead can breaking glass pipe point
End, to obtain the flat mouth needle that satisfactory internal diameter is 10 μm.
4. the method according to claim 1 for collecting blastocoele liquid, which is characterized in that in step (1), card grinding is specifically walked
Suddenly are as follows: open card grinding instrument switch, and distilled water will be piled in water injection pipe above grinding stone, with water-soaked grinding stone, adjust turning for grinding stone
Speed is 30rpm, and card grinding angle is located in 45 °, the glass needle tubing to have broken is installed on fixed handle, and turn rough quasi-coil makes
Needle point rapid decrease when close to grinding stone, is used thin quasi- burnt spiral instead, is observed by eyepiece, careful needle point is fallen on grinding stone, contact mill
Shi Hou, liquid level rises in needle tubing, and thin quasi- burnt spiral is turned 90 ° again, after continuing 1-2min, needle mouth can be worn into angle,
The needle tubing of milled is removed.
5. the method according to claim 1 for collecting blastocoele liquid, which is characterized in that in step (1), top-notch and looper
Specific steps are as follows: the needle tubing of milled is installed on broken needle instrument, adjusts glass tube and bead relative position, avoids operation phase
It mutually contacts, holds up needle under 10 × object lens, be located at needle point above bead, heating knob is adjusted to 40 DEG C or so, is stepped on
Pedal falls needle point with thin spiral, after needle point contacts softening with bead, rapidly lifts needle point, forms pine after needle point
Pedal is opened, length of needlepoint is 11-13 μm, it is preferable that length of needlepoint is 12 μm, after the completion of top-notch, and point will be pulled out under 5 × object lens
Needle is placed on the left of bead in same plane at 100 μm of calibers, first makes bead far from needle tubing, and heating knob is adjusted to 60
DEG C, it slams pedal simultaneously by glass marble close to needle tubing, when needle tubing is bent to 20 °, unclamps floor push, remove glass needle.
6. the method according to claim 1 for collecting blastocoele liquid, which is characterized in that in step (2), micromanipulation is solid
Determine needle preparation specific steps are as follows: the good needle tubing of above-mentioned drawing is mounted on broken needle instrument, heating knob is transferred to 40 DEG C, under 10 × object lens
The broken needle at 100 μm is holded up needle, needle mouth is placed in the top of glass marble, heats glass marble will far from needle mouth after breaking well
Knob is adjusted to 60 DEG C, steps on pedal, while by needle point close to glass marble, when needle necking to pine at 40 μm and when needle mouth smooths out
It opens pedal and stops heating, by fixed pin from holding up, the needle for pulling out point is placed on the left of bead in same plane under 5 × object lens
At 100 μm or so calibers, needle tip is placed in glass marble left downward, and heating knob is adjusted to 60 DEG C and steps on switch, makes needle tubing
20 ° of bending, needle is unloaded, is placed in spare in box for needle or pin.
7. the method according to claim 1 for collecting blastocoele liquid, which is characterized in that blastocoele liquid in step (3)
Collection before, first above-mentioned ready-made micromanipulation injection needle and fixed pin are mounted on micromanipulation instrument and adjust angle
Degree is placed in 0.25% trypsase drop and checks whether fixed pin and injection needle use normal, take one it is clean into
The 35mm culture dish ware lid of mouth does three 10 μ L DPBS drops by left side vertical setting of types among ware lid with preheated DPBS, every
The drop that a 1 μ L is on the right side of a drop covers DPBS drop with paraffin oil, is moved into the 7th day Blastocysts with mouth suction spindle
In the DPBS drop of the one 10 μ L in centre, micromanipulation injection needle is lifted and culture dish is placed on objective table, is put down micro-
Injection needle is operated, in the 10 μ LDPBS drops that micromanipulation injection needle is placed over, micromanipulation injection needle is cleaned, avoids showing
Trypsase in microoperation injection needle has an impact blastaea.
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