CN107236698A - The method of separating mouse inner ear hair cells - Google Patents
The method of separating mouse inner ear hair cells Download PDFInfo
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- CN107236698A CN107236698A CN201610190542.9A CN201610190542A CN107236698A CN 107236698 A CN107236698 A CN 107236698A CN 201610190542 A CN201610190542 A CN 201610190542A CN 107236698 A CN107236698 A CN 107236698A
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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Abstract
The present invention relates to the method for separating mouse inner ear hair cells.The present invention takes out Cochlea of Mouse and removes surface sclerotin, separation obtains cochlea labyrinthus membranaceus, remove perienchyma, isolate the basilar memebrane with organ of Corti, it is micro- after collagenase digesting effect to be partitioned into inner hair cell area and external hair cell area, segment cell cluster is obtained, the interior external hair cell cluster after segmentation is drawn respectively with the glass pipette with buffer solution, blow out again on slide, obtaining from single interior external hair cell.Instant invention overcomes blow and beat the defect that basement membrane tissue separation screening hair cell is brought with machinery after Trypsin Induced.Accurate positioning of the present invention, tissue damage is small, time-consuming short, and clostridiopetidase A is more suitable for the fibrous connective tissue around digestion separation hair cell, damages small in itself to hair cell, single cell active quality is high, screens unicellular workload and greatly reduces, operating efficiency raising.
Description
Technical field
The present invention relates to a kind of audiology Basic Experiment Study alternative, more particularly to separating mouse inner ear capillary
The method of born of the same parents.
Background technology
In view of the special anatomical position and number of human inner ear's hair cell are very few, application model animal inner ear hair cells
Replacement is the basis of audiology research.Because ripe hair cell is unable to division growth, sets up and improve in single
The separation method of tragus cell be beneficial to it is more deeper into scientific experiment study.
Before the present invention makes, studied primarily directed to the separation method of cavy, gerbil jird inner ear hair cells, and
The genotype research of mouse is most thorough, it has also become most widely used model animal.At present for cochlea volume compared with
The high murine inner ear hair cell separation method of small technical operation difficulty there is no System describe.Cavy, gerbil jird isotype
Animal volume is larger, and operation difficulty is relatively small, and previous research method is mainly with after Trypsin Induced
Machinery piping and druming basement membrane tissue separation screening hair cell.Though trypsase is widest cell dissociation agent, its
Tissue damage is big, and machine to sensory epithelial cell (especially sensitive fragile inner ear hair cells) for chemical enzyme effect
Tool blows and beats method poor location, must be screened in volume cell and search the extremely low hair cell of number ratio, time-consuming, work
Work amount is big, while big to hair cell mechanical injuries, the cell quantity of acquisition is few, and quality is low, so as to influence to grind
The reliability and authenticity studied carefully.
The content of the invention
It is an object of the invention to overcome the above-mentioned clear defect of the fact, separating mouse inner ear capillary is described in detail
The method of born of the same parents.
The technical scheme is that：
The method of separating mouse inner ear hair cells, it is as follows that it is mainly characterized by step：
(1) take out Cochlea of Mouse and remove surface sclerotin, separation obtains cochlea labyrinthus membranaceus；
(2) perienchyma is removed, the basilar memebrane with organ of Corti is isolated；
(3) it is micro- after collagenase digesting effect to be partitioned into inner hair cell area and external hair cell area, obtain segment cell cluster；
(4) the interior external hair cell cluster after segmentation is drawn respectively with the glass pipette with buffer solution, then blow out in slide
On, you can obtain from single interior external hair cell.
Advantages of the present invention and effect are accurate positioning, and tissue damage is small, take short, single cell active quality
Height, screens unicellular workload and greatly reduces, operating efficiency is improved.
Brief description of the drawings
Fig. 1 --- Cochlea of Mouse exemplary plot in the present invention.
Fig. 2 --- divest top in the present invention and turn cochlea exemplary plot after bone wall.
Fig. 3 --- cochlea labyrinthus membranaceus exemplary plot in the present invention.
Fig. 4 --- basilar memebrane exemplary plot in the present invention.
Fig. 5 --- row's inner hair cell schematic diagram is obtained in the present invention after micro- segmentation.
Fig. 6 --- from single interior external hair cell (IHC in the present invention：Inner hair cell, OHC：External hair cell).
Embodiment
The present invention technical thought be：
The present invention combines micro-dissections and collagenase digesting isolates the more higher-quality murine inner ear hairs of acquisition
Cell.Compare previous separation method overcome it is above-mentioned time-consuming, the low many defects of quantity-quality.The present invention
Advantage and effect be：Clear and definite using the micro- segmentation positioning of micro- filament row, separation cell individual is accurate, no
Lookup need to be screened in a large amount of cells, mechanical damage is small；Clostridiopetidase A mainly acts on the knot such as iuntercellular collagen simultaneously
Tissue is formed, digestion chorista damage is small, to acting on the single inner ear hair cells of basilar membrane digestion separation more
Targetedly, preferably maintain the activity of individual cells, it is ensured that the purity of sample, more conducively carry out reliable
Experimental study.
The present invention is specifically described below.
The technical step of the present invention is as follows：
First, by taking C57BL/6 mouse as an example, break neck after deep anaesthesia, peels off external auditory meatus and otic capsule, takes out band vestibular
The cochlea of base；
2nd, remove after the posterior bony wall of cochlea top, completely divest cochlea outer layer bone wall, separation obtains cochlea labyrinthus membranaceus；
3rd, the perienchymas such as spiral ligament, stria vascularis, epiphragma are removed, the basilar memebrane with organ of Corti is isolated；
4th, micro- filament is partitioned into row's inner hair cell and three exclusive hairs after 37 DEG C of preheating IV Collagenase Type digestions
Cell, makes into segment cell cluster；
5th, with tip diameter about 50um glass pipette, first suck after one section of buffer solution, then draw respectively in segment
External hair cell cluster, then blow out on slide, you can obtain from single interior external hair cell.
Inner ear hair cells are one of main objects of audiology experimental study, and in vitro hair cell can only be cultivated several days
It can not pass on.So it is the base for carrying out audiology related mechanism experimental study to efficiently separate inner ear hair cells technology
Plinth.The cochlea volume such as cavy, gerbil jird is relatively large, and hair cell separation is easy and convenient, but gene-correlation molecule machine
System research is not thorough.Mouse is current most widely used most common model animal, many human diseases animal moulds
Type is all that the sound gene order of dependent Mice is set up, and same increasing audiology research also depends on small
Mouse is used as animal model.Efficiently separate certain amount and the good inner ear hair cells of activity are effective progress morphology
The basis of observational study and molecular biology research.
It is contemplated that introducing, one kind combines micro-dissections and collagenase digesting is isolated, and faster obtains more more excellent
The method of the murine inner ear hair cell of matter.
By taking 2 monthly age C57BL/6 mouse as an example (regardless of sex), skin of pinna is cut off after disconnected neck, along external auditory meatus
Choose except otic capsule, take out cochlea, such as Fig. 1 is the right mouse cochlea with vestibular base.
On rear side of cochlea top and top is removed in low temperature (on ice) isotonic buffer solution under body formula disecting microscope and turns thin cut bone
Wall, is obtained as removed the cochlea after local bone wall in accompanying drawing 2, it is high-visible that its top turns the exposure of stria vascularis spiral ligament.
Method, is turned to divest cochlea surface bone wall, you can obtain the removal periphery sclerotin ear such as Fig. 3 by the apical turn bottom of to according to this
Snail labyrinthus membranaceus, removes the perienchymas such as spiral ligament, stria vascularis, epiphragma, along cupula cochleae downward spiral, segmentation point
Separate out as to push up in basilar memebrane, such as accompanying drawing 4 and turn (mainly exclusive including row's inner hair cell and three with an organ of Corti
Hair cell) basilar memebrane.Basilar memebrane is laid on small plate (a diameter of 3.5cm), a small amount of preheating is added dropwise
In after 37 DEG C of IV Collagenase Types (1mg/ml), 37 DEG C be incubated 2-4 minutes after under inverted phase contrast microscope
External hair cell region in being split respectively with micro- filament (a diameter of 30 μm of stainless steel metal wires).Accompanying drawing 5
For row's inner hair cell after micro- segmentation.This micro- segmentation step is the key of the technology, is also this technology phase
Part is greatly improved compared with prior method, the method that basilar memebrane is integrally blown and beaten after previous enzymic digestion, operation is blindly fixed
Potential difference, tissue damage is big, and the cell cluster for needing screening all to break up afterwards, and screening efficiency is substantially reduced, expended
Time is long, while being unfavorable for preserving cytoactive, the unicellular quality filtered out is low.In inversion phase in the present invention
The poor descending micro- cutting operation of surgical microscope is trickle, and precisely microscopic metal filament diameter is small, damages small, it is only necessary to
Screened in cutting zone cell, it is time-consuming short, raising separative efficiency can be reached, separation damage is reduced,
Cytoactive is kept, and obtains the purpose of the higher-quality single hair cell of more.
After the segmentation of micro- filament, then with the tip diameter of connection miniature displacer (filter diameter is 0.22 μm) about
50 μm be about 5cm glass pipette, first suck one section of buffer solution after, respectively draw segmentation after inside and outside capillary
Born of the same parents' cluster, then blow out on slide, you can obtain such as the interior external hair cell in Fig. 6 from list, interior external hair cell top
Hold epidermis plate flat and thick, there is short cilium to adhere to, inner hair cell likeness in form flask shape, core is centrally located；Outer capillary
Born of the same parents are cylindrical, and core is located at cell space bottom.The connection of miniature displacer can increase resistance when drawing cell, be beneficial to
Transfer individual cells are drawn in observation.
Micro- filament segmentation is combined after the partition method i.e. application collagenase digesting and is significantly better than Trypsin Induced
Fine needle blows and beats separation method afterwards.Trypsase is that current most widely used vitellophag interstitial is used for passage
The digestive pharmaceutical of culture, but it is applied to the less soft tissue of vitellophag interstitial.And clostridiopetidase A has very to collagen
Strong digestion, only has digestion on cytoplasm and influences little to epithelial cell, it is adaptable to digestion point
From epithelial cell, primary cell is damaged in itself small.Inner ear hair cells belong to sensory epithelial cell, in contrast,
Clostridiopetidase A is more suitable for the fibrous connective tissue around digestion separation hair cell, hair cell is damaged in itself small.Together
Fine needle blindly blows and beats monoblock tissue easy damaged hair cell in Shi Xianqian separation method and loss cell is more, screening rate
Low, workload is big.
The present invention combines micro- filament and splits the basement membrane tissue after collagenase digesting, and inverted phase contrast microscope is descending
External hair cell in the subregion segmentation in situ of micro- filament, positioning is clear and definite, and cellular damage is small, then to connect miniature displacer
Glass pipette pressure-vaccum is obtained from single hair cell, and this method cell loss is few, and screening acquisition amount is more, substantially increases
Operating efficiency.The method can efficiently separate murine inner ear hair cell into interior external hair cell that is single, being separated
Available for the specific molecular Mechanism Studies such as single or single cytogene or protein level and basic morphology
Observation.In addition, because the inventive method takes short, individual cells vital preservation is good.Due to hair cell
Specific properties, the single hair cell isolated can be used to observe shadow of the different environmental stimuluses to hair cell form
Ring.As different osmotic solution stimulates the change of fore-and-aft observing cellular morphology to detect the work(of cytoskeleton related protein
(it can be confirmed using a cytoskeleton related protein hair cell specific knockdown mouse).This separation method
Applications well the unicellular correlative study method of more inner ear hair cells is established, promote hearing
Learn the developing of the deep and research field of research.
New model and regulation that the method that the present invention is innovated is formed in a research field, that is, establish one kind
The model of separating mouse inner ear hair cells, is conducive to the progress of research activities according to this model regulation.
Claims (1)
1. the method for separating mouse inner ear hair cells, it is characterised in that step is as follows：
(1) take out Cochlea of Mouse and remove surface sclerotin, separation obtains cochlea labyrinthus membranaceus；
(2) perienchyma is removed, the basilar memebrane with organ of Corti is isolated；
(3) it is micro- after collagenase digesting effect to be partitioned into inner hair cell area and external hair cell area, obtain segment cell cluster；
(4) the interior external hair cell cluster after segmentation is drawn respectively with the glass pipette with buffer solution, then blow out in slide
On, you can obtain from single interior external hair cell.
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| CN201610190542.9A CN107236698A (en) | 2016-03-28 | 2016-03-28 | The method of separating mouse inner ear hair cells |
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| CN201610190542.9A CN107236698A (en) | 2016-03-28 | 2016-03-28 | The method of separating mouse inner ear hair cells |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182250A (en) * | 2018-09-30 | 2019-01-11 | 山东省立医院 | A kind of Cochlea of Mouse hair cell cultural method |
| CN113005087A (en) * | 2021-03-22 | 2021-06-22 | 澎立生物医药技术（上海）有限公司 | Adherent culture method of cochlear organ of mouse |
| CN114788508A (en) * | 2021-08-13 | 2022-07-26 | 南京鼓楼医院 | Method for constructing sudden deafness mouse model by using inflammation as characteristic |
Citations (1)
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| CN104313131A (en) * | 2014-09-24 | 2015-01-28 | 浙江大学 | Labelled molecules for detecting mouse inner ear hair cells and application thereof |
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2016
- 2016-03-28 CN CN201610190542.9A patent/CN107236698A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104313131A (en) * | 2014-09-24 | 2015-01-28 | 浙江大学 | Labelled molecules for detecting mouse inner ear hair cells and application thereof |
Non-Patent Citations (3)
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| EMMERT-BUCK MR 等: "Laser Capture Microdissection", 《SCIENCE》 * |
| 杨仕明 等: "内毛细胞的分离和形态学特点", 《中华耳鼻咽喉科杂志》 * |
| 田洁 等: "新生小鼠内耳前庭毛细胞分离方法", 《上海医科大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182250A (en) * | 2018-09-30 | 2019-01-11 | 山东省立医院 | A kind of Cochlea of Mouse hair cell cultural method |
| CN113005087A (en) * | 2021-03-22 | 2021-06-22 | 澎立生物医药技术（上海）有限公司 | Adherent culture method of cochlear organ of mouse |
| CN114788508A (en) * | 2021-08-13 | 2022-07-26 | 南京鼓楼医院 | Method for constructing sudden deafness mouse model by using inflammation as characteristic |
| CN114788508B (en) * | 2021-08-13 | 2024-05-03 | 南京鼓楼医院 | Method for constructing sudden deafness mouse model characterized by inflammation |
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Application publication date: 20171010 |