CN104473664B - A kind of double material hollow sleeves for separating primary airway epithelial cell - Google Patents
A kind of double material hollow sleeves for separating primary airway epithelial cell Download PDFInfo
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- CN104473664B CN104473664B CN201410823161.0A CN201410823161A CN104473664B CN 104473664 B CN104473664 B CN 104473664B CN 201410823161 A CN201410823161 A CN 201410823161A CN 104473664 B CN104473664 B CN 104473664B
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- silica gel
- epithelial cell
- gel tube
- airway epithelial
- teflon
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Abstract
The present invention relates to separate at body the instrument of primary airway epithelial cell, be specifically related to a kind of double material hollow sleeves for separating primary airway epithelial cell.It is characterized in that the silica gel tube (caliber is thin, intensity is high) by different tube diameters and Teflon hollow pipe (caliber is thick, intensity is little, toughness big) are bonded by silica gel glue.
Description
Technical field
The present invention relates to medical instrument field, in particular to a kind of double materials for separating primary airway epithelial cell
Hollow sleeve.
Background technology
Airway epithelial cell is the first natural cover for defense of respiratory system and extraneous contact, and its structure and function stable state are to breathe system
System maintains normal ventilation function and the cell base of defense function.Airway epithelial cell is attached to basement membrane growth, collectively forms
The pseudostratified columnar ciliated epithelium of respiratory tract, the cilium on its surface to pharyngeal swing, gets rid of respiratory tract at body with having the rhythm and pace of moving things
Important function has been played during property foreign body.
The primary airway epithelial cell of laboratory animal is as the experimental model of human respiratory epithelial cell, for clinical and laboratory
Work has the reference function do not replaced.Constantly improving of airway epithelial cell separation and Culture technology will be to respiratory system
Virusology, pathology and toxicology based on research, biochemistry, pharmacokinetics, developmental biology, even clinic are controlled
Treat and produce impetus with application.
At present, domestic and international separating experiment mostly uses with the primary airway epithelial cell of animal big airways and longitudinally cuts off trachea, directly
Immerse in the test tube containing pronase, 4 DEG C of enzymolysis 18-24 hour, then it is centrifuged enzymolysis solution (centrifugal force 123g), finally collect thin
The method of born of the same parents group obtains primary airway epithelial cell.But, the cell number that this method obtains is less, active poor,
For it is important that heteroproteose cell is too much, the cytology research of respiratory tract disease is produced restriction.Therefore, the warp during we sum up experiment
Test, the method being developed based on the separation big airways epithelial cell of tracheal intubation.But, exist in the operating process of the method
Problems with: if the Material Strength selected is too big and elastic little, causes syringe needle to insert and owe tight, and turn back, bulldog clamp clamping difficulty,
Easily cause pronase to leak outside；If the Material Strength chosen is little and elastic greatly, when surgical thread will be caused again to ligature, should not prick
Tightly, will also result in pronase to leak outside.Though the most existing multiple tracheal intubation method is applied in animal experiment in vitro, but this
A little method not only technical operations are complicated and need special installation, and often cause cell dissociation insufficient because pronase leaks outside,
Thus affect the success or failure of whole experiment.Then we develop double material hollow sleeve for the problems referred to above.This sleeve pipe chooses two kinds
Different qualities, the hollow pipe composition sleeve pipe of material, meet two ends difference needs, respectively to reach to prevent pronase from leaking outside also
Effectively reduce the effect of pronase enzyme dosage, thus reduce the enzymolysis solution damaging action to free airway epithelial cell, protecting
On the basis of the airway epithelial cell activity that card separates, cell separation quantity is significantly increased.The present invention is by using in Teflon
Blank pipe, GA-1073 silica gel tube and literary composition super 380 silica gel glue of brocade realize this conception.
GA-1073 silica gel tube intensity is big (hardness 70 ± 5D), and biocompatibility is preferable.Teflon pipe, is again politef
(Polytetrafluoroethene, PTFE) manages, and is to use PTFE material to make, has intensity little (hardness 50-55D), toughness
Big feature, plasticity is strong.The literary composition super 380 silica gel glue chemical analysis of brocade are cyanoacrylate, current medical cyanoacrylate
Acid esters binding agent is extensively applied in orthopaedic trauma, inanimate object toxicity.This sleeve pipe i.e. utilizes GA-1073 silica gel tube intensity very much not
Yielding, i.e. it is difficult to the feature collapsed when surgical thread ligatures, by choosing the GA-1073 silica gel tube of suitable caliber, is reducing
While tracheal injury, being more convenient for inserting animal trachea, surgical thread ligation is closely；Utilize again Teflon hollow pipe intensity little and
The feature that toughness is big, had both been easy to connect syringe, and had been easy to turn back when late arterial clamp stopped pipe chamber again.Literary composition super 380 silicon of brocade
Glue glue is the solvent based product based on high molecular polymer, and adhesive strength is high, anti-shearing.This pair of material hollow sleeve is not
Only can reuse, and on the basis of ensureing the airway epithelial cell activity separated, cell separation quantity is significantly increased.
At present, commercially available primary airway epithelial cell is expensive, and majority the most lost cilium directional swing at body physiology
Function, activity is poor.Accordingly, it would be desirable to more convenient and airway epithelial cell separation equipment efficiently.
Summary of the invention
The present invention relates to a kind of double material hollow sleeves being convenient for the separation of primary airway epithelial cell.Described pair of material sleeve pipe
Ensure that the success rate of laboratory animal tracheal intubation, and guarantee to be made up of syringe, double material hollow sleeve and trachea
The patency of tubing and seal, anti-tubing here is soaked in the medium, occur that enzymolysis solution oozes during enzymolysis
Leakage, while ensureing the contacting and act on of pronase and airway epithelial cell, the consumption of effective minimizing pronase,
Thus reduce the enzymolysis solution damage to free airway epithelial cell.To obtain on greater number and the more preferable primary respiratory tract of activity
Chrotoplast.
Described double material hollow sleeves by,
(1) inner layer sleeve inserting trachea that the silica gel tube that caliber is thin, intensity is high is constituted, and
(2) caliber is thick, toughness is big, can be socketed to form by the outer layer sleeve that constitutes of doubling the Teflon hollow pipe closed by mosquito forceps；
And the external diameter of silica gel tube is equal to the internal diameter of Teflon hollow pipe.
Described socket is for by can be used for the bonding of operating adhesive of medical, and described adhesive of medical is preferably Wen Jinchao
Can 380 silica gel glue.
Described silica gel tube is preferably GA-1073 silica gel tube, and intensity is unlikely to deform greatly, is difficult to collapse when surgical thread ligatures, passes through
Choose the GA-1073 silica gel tube of suitable caliber, while reducing tracheal injury, can be more convenient for inserting animal trachea, and hands
Do not subside during the ligation of art line thus ensure that the inflow trachea chamber smoothly of pronase digestion liquid；
The described Teflon little toughness of hollow pipe intensity is big, has both been easy to connect syringe, has been easy to again folding when the later stage closes tube chamber
Fold and pass through mosquito forceps pinch seal.
This sleeve pipe is possible not only to reuse, and can guarantee that the success rate of laboratory animal tracheal intubation, and guarantee by syringe,
The patency of the pipeline that double material hollow sleeves and laboratory animal trachea are formed and seal, reduce the consumption of pronase,
Effectively promote the activity of the airway epithelial cell of isolated, increase the separation quantity of airway epithelial cell.
Described silica gel tube and the caliber size of Teflon hollow pipe and length rationally can select according to experimental animal model, with
It is easy to intubation.
The animal pattern separating primary airway epithelial cell is depended in described silica gel tube socket with the length in Teflon hollow pipe
Kind and the length of two kinds of material pipes, preferably 1/4th of silica gel tube length.
The present invention is that the research about airway epithelial cell, teaching, medical application provide favourable material conditions, and combining should
The culture medium described in patent (ZL 201110440894.2) authorized with us, can be effectively increased on the respiratory tract of isolated
The quantity of chrotoplast and vigor, and retain airway epithelial cell cilium directional swing in body physiological function.
The invention enables primary airway epithelial cell lock out operation simple, repeatable strong, it is simple to popularization and application, have
Promotional value widely.
Accompanying drawing explanation
Fig. 1. the axonometric chart (as a example by special pair of material hollow sleeve of mice) of double material hollow sleeves.(scale 5:1)
Fig. 2. the sectional view of double material hollow sleeves.(scale 5:1)
Fig. 3. the sectional view of double material hollow sleeve A-A ' along the line.(scale 50:1)
Fig. 4. double material hollow sleeves connect the free retrotracheal pictorial diagram of BALB/c mouse, a 1. surgical thread；2. mice trachea；
3.GA-1073 silica gel tube；4. Teflon hollow pipe.
Fig. 5. before injecting chain pheron, double material hollow sleeves connect trachea pictorial diagram, 1. mice trachea；2.GA-1073 silica gel tube；
3. Teflon hollow pipe.
Fig. 6. the pictorial diagram that after injecting chain pheron, trachea balloon sample changes, 1. mice trachea；2.GA-1073 silica gel tube；3. ferrum fluorine
Dragon hollow pipe.
Fig. 7. the mouse primary airway epithelial cell under inverted microscope
Detailed description of the invention
As a example by special pair of material hollow sleeve of separating mouse airway epithelial cell
The making of 1. pairs of material sleeve pipes of embodiment
(1) take two ends and be respectively 45 degree of oblique end peace ends, the GA-1073 silica gel tube of internal diameter 0.6mm, external diameter 0.8mm；
(2) take 20mm and two ends be flat end, the Teflon hollow pipe 25mm of internal diameter 0.8mm, external diameter 1.0mm,
(3) the flat end of GA-1073 silica gel tube is coated 10 super 380 silica gel glue of microlitre literary composition brocade and inserts in Teflon hollow pipe, insert
Fix 15s after entering 5mm and i.e. obtain described pair of material sleeve pipe.
Fig. 1 show the axonometric chart (as a example by special pair of material hollow sleeve of mice) of of the present invention pair of material hollow sleeve, wherein
1 is Teflon hollow pipe, length: 25mm
2 is GA-1073 silica gel tube, length: 20mm
BB ' is Teflon hollow pipe external diameter: 1.0mm
CC ' is that GA-1073 silica gel tube inserts Teflon hollow pipe length 5mm
DD ' is GA-1073 silica gel tube internal diameter: 0.6mm
EE ' is Teflon hollow pipe internal diameter, GA-1073 silica gel tube external diameter: 0.8mm
The separation of embodiment 2. mice trachea
(1) take BALB/c mouse to put to death through disconnected neck, tilt head, by alcohol-pickled for its thorax abdomen sterilization 30s, note protecting oral cavity
And nasal meatus, stimulate from ethanol.Being fixed on operating-table, the downward hunter's line of aseptic condition cuts off skin, exposes abdomen actively
Arteries and veins, incision blood-letting, to flowing to end, its object is to avoid being mixed into too much erythrocyte when separating airway epithelial cell；
(2) blunt separation trachea is after exposing trachea and it is the most free；
(3) wear bifilar No. 1 surgical thread 100mm, below trachea cricoid cartilage, at 5mm, cut a small gap, insert double material sleeve pipe
45 degree of oblique ends of GA-1073 silica gel tube, insert trachea half about 5mm deep, make a call to a tight knot with No. 1 surgical thread outside trachea, will
Silica gel sleeve pipe is fixed on tracheal strips；
(4) in connecting 1ml gauge hypodermic device, conductive pipe by Teflon hollow pipe, injection PBS washes one's hands and face trachea, and observes lung
There is N/D；
(5) make a call to a tight knot again at the crotch surgical thread that trachea downstream silica gel tube is the most deep and close trachea end, go around gas removing pipe
Excess tissue and surgical thread, free trachea.
The digestion of embodiment 3. tracheal tissue and erythrocytic removal
(1) injected 0.05% pronase of a little 4 DEG C of pre-coolings by double material sleeve pipes to the tracheal strips that cannula tip is fixing, fill
The PBS of washing is removed by scrubber tube inwall；
(2) 0.05% pronase is filled whole section of trachea, make trachea slightly expand in balloon sample, the Teflon hollow pipe portion of doubling
Clamp with 30mm bulldog clamp after Fen, close the combination of whole trachea-sleeve pipe；
(3) whole section of trachea is fixed infiltration in the MEM added with penicillin, digest 8h；
(4) cut off lower end trachea, collect Digestive system, and draw PBS flushing trachea with 1ml syringe；
(5) collecting flushing liquor about 1.5ml (collect liquid+PBS and rinse trachea liquid), be transferred in EP pipe after merging, 500X g is centrifuged
4min removes supernatant；
(6) adding erythrocyte cracked liquid (Sigma R7757-100ML) 150 μ l, after the mixing of 1min gentleness, PBS is diluted to 1.5ml,
500g is centrifuged 6min；Remove supernatant, then with the abundant re-suspended cell of appropriate complete medium, take 10 μ l for cell counting,
Through counting, every mice can extract about 5*105 cell of cell number.
The original cuiture of embodiment 4. airway epithelial cell and qualification
Airway epithelial cell is cultivated
(1) as a example by 24 orifice plates, every hole 500 μ l complete medium cultivating system (culture medium is shown in patent CN102505005A), add
Appropriate cell (concrete cell quantity requires to determine according to actual experiment), notes mixing, it is to avoid cell is in heaps cannot launch；
(2) after cross shakes up, after laboratory table places 30min, it is transferred in incubator, carries out changing liquid every day and process (identical
System), subsequent experimental can be carried out after cell attachment.
Airway epithelial cell identification of morphology
(1) human airway epithelial cells just separated is rounded, some Cheng little Tuan or in blocks, typically pastes at about 24h visible cell
Wall is the most fully deployed, Microscopic observation, and purity is about 95%, and the most a small amount of fibroblast mixes wherein；
(2) cultivate cell degrees of fusion after 5d to reach higher level and i.e. form typical cell island (Fig. 7), it is possible to see quick fibre
Hair swings, and this is the biological behaviour that airway epithelial cell is exclusive.
Cell area owing to separating derives from inner surface of trachea, and this position uniquely has the cell of rhythmic exercise to only have airway epithelial
Cell, meanwhile, the swing of this rule is again the typical characteristic of airway epithelial cell, therefore using this feature as the mark of this cell
Will, and explanation cell well-grown, its physiological function is close in body level.
Claims (6)
1. the double material hollow sleeves carrying out primary airway epithelial cell separation, it is characterised in that described double material hollow sleeves
By,
(1) inner layer sleeve inserting trachea that the silica gel tube that caliber is thin, intensity is high is constituted, and
(2) caliber is thick, toughness is big, can be socketed to form by the outer layer sleeve that constitutes of doubling the Teflon hollow pipe closed by mosquito forceps；
And the external diameter of silica gel tube is equal to the internal diameter of Teflon hollow pipe；
Described socket is for by can be used for the bonding of operating adhesive of medical.
The most according to claim 1 pair of material hollow sleeve, it is characterised in that described adhesive of medical is literary composition brocade super 380
Silica gel glue.
The most according to claim 1 and 2 pair of material hollow sleeve, it is characterised in that described silica gel tube is GA-1073 silica gel
Pipe.
The most according to claim 3 pair of material hollow sleeve, it is characterised in that described silica gel tube socket and Teflon hollow pipe
Interior length depends on animal pattern kind and the length of two kinds of material pipes separating primary airway epithelial cell.
The most according to claim 4 pair of material hollow sleeve, it is characterised in that in described silica gel tube socket and Teflon hollow pipe
A length of silica gel tube length 1/4th.
The most according to claim 4 pair of material hollow sleeve, it is characterised in that: described hollow sleeve is breathed for mouse primary
Tract epithelial cell separates, and the external diameter of GA-1073 silica gel tube is equal to the internal diameter of Teflon hollow pipe, for mice trachea mean inside diameter 0.8
The a length of 20mm of mm, GA-1073 silica gel tube, a length of 25mm of Teflon hollow pipe.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410823161.0A CN104473664B (en) | 2014-12-24 | 2014-12-24 | A kind of double material hollow sleeves for separating primary airway epithelial cell |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410823161.0A CN104473664B (en) | 2014-12-24 | 2014-12-24 | A kind of double material hollow sleeves for separating primary airway epithelial cell |
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| Publication Number | Publication Date |
|---|---|
| CN104473664A CN104473664A (en) | 2015-04-01 |
| CN104473664B true CN104473664B (en) | 2016-08-17 |
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| CN201410823161.0A Expired - Fee Related CN104473664B (en) | 2014-12-24 | 2014-12-24 | A kind of double material hollow sleeves for separating primary airway epithelial cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107974429B (en) * | 2017-11-29 | 2021-06-29 | 宁夏医科大学总医院 | Method for rapidly separating and culturing human airway epithelial cells and optimized culture medium |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0425361A1 (en) * | 1989-10-23 | 1991-05-02 | Ovi | Catheter for artificial pulmonary ventilation of a patient |
| CN201783048U (en) * | 2010-07-30 | 2011-04-06 | 田亮 | Novel trachea cannula |
| CN103690306A (en) * | 2014-01-07 | 2014-04-02 | 魏永祥 | Specimen sampling swab |
| CN203693550U (en) * | 2014-01-17 | 2014-07-09 | 苏州朗开医疗技术有限公司 | Turning extending catheter for bronchoscope |
-
2014
- 2014-12-24 CN CN201410823161.0A patent/CN104473664B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0425361A1 (en) * | 1989-10-23 | 1991-05-02 | Ovi | Catheter for artificial pulmonary ventilation of a patient |
| CN201783048U (en) * | 2010-07-30 | 2011-04-06 | 田亮 | Novel trachea cannula |
| CN103690306A (en) * | 2014-01-07 | 2014-04-02 | 魏永祥 | Specimen sampling swab |
| CN203693550U (en) * | 2014-01-17 | 2014-07-09 | 苏州朗开医疗技术有限公司 | Turning extending catheter for bronchoscope |
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| CN104473664A (en) | 2015-04-01 |
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Granted publication date: 20160817 Termination date: 20211224 |