CN104473664A - Two-material hollow casing pipe for separating primary respiratory tract epithelial cells - Google Patents
Two-material hollow casing pipe for separating primary respiratory tract epithelial cells Download PDFInfo
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- CN104473664A CN104473664A CN201410823161.0A CN201410823161A CN104473664A CN 104473664 A CN104473664 A CN 104473664A CN 201410823161 A CN201410823161 A CN 201410823161A CN 104473664 A CN104473664 A CN 104473664A
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Abstract
The invention relates to a tool for in-vivo separating primary respiratory tract epithelial cells, and particularly relates to a two-material hollow casing pipe for separating primary respiratory tract epithelial cells. The two-material hollow casing pipe is characterized by being formed by bonding a silicone tube (small pipe diameter and high strength) and a Teflon hollow pipe (large pipe diameter, small strength and large flexibility) by virtue of silicone glue.
Description
Technical field
The present invention relates to medical instrument field, in particular to a kind of two material hollow sleeves for separating of primary airway epithelial cell.
Background technology
Airway epithelial cell is the first natural cover for defense of respiratory system and extraneous contact, and its structure and fuction stable state is the cell base that respiratory system maintains normal ventilation function and defense function.Airway epithelial cell is attached to basement membrane growth, the common pseudostratified columnar ciliated epithelium forming respiratory tract, and the cilium on its surface, with having the rhythm and pace of moving things to pharyngeal swing, is got rid of at body in the process of respiratory tract irritation foreign body and played important function.
The primary airway epithelial cell of laboratory animal, as the epithelial experimental model of human respiratory, has for clinical and laboratory work the reference function do not replaced.Constantly improving of airway epithelial cell separation and Culture technology will to virusology, pathology and the toxicology based on respiratory system research, biochemistry, pharmacokinetics, developmental biology, and even clinical treatment produces impetus with application.
At present, domestic and international separating experiment mostly adopts with the primary airway epithelial cell of animal big airways and longitudinally cuts off trachea, direct immersion is containing in the test tube of pronase, 4 DEG C of enzymolysis 18-24 hour, centrifugal enzymolysis solution (centrifugal force 123g) again, the method for last collecting cell group obtains primary airway epithelial cell.But the cell number that this method obtains is less, active poor, and most importantly heteroproteose cell is too much, produces restriction to the cytology research of respiratory tract disease.Therefore, we sum up the experience in experiment, develop the epithelial method of separation big airways based on tracheal intubation.But, in the operating process of the method, there is following problem: if the Material Strength selected is too large and elasticity is little, cause syringe needle to insert to owe tight, and turn back, bulldog clamp clamping difficulty, easily cause pronase to leak outside; If the Material Strength chosen is little and elasticity large, when will cause again surgical thread ligation, should not tightens, pronase also can be caused to leak outside.Though at present existing multiple tracheal intubation method is applied in animal experiment in vitro, these methods not only technical operation 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.So we develop two material hollow sleeve for the problems referred to above.The hollow pipe composition sleeve pipe of two kinds of different qualities, material chosen by this sleeve pipe, meet the different needs in two ends respectively, prevent pronase from leaking outside and the effect effectively reducing pronase enzyme dosage to reach, thus reduce enzymolysis solution to the damaging action of free airway epithelial cell, on the basis ensureing the airway epithelial cell activity be separated, significantly increase cell separation quantity.The present invention realizes this conception by using super 380 silica gel glue of Teflon hollow pipe, GA-1073 silica gel tube and literary composition brocade.
GA-1073 silica gel tube intensity large (hardness 70 ± 5D), and biocompatibility is better.Teflon pipe, makes again politef (Polytetrafluoroethene, PTFE) manage, and is to adopt PTFE material to make, and have intensity little (hardness 50-55D), feature that toughness is large, plasticity is strong.The super 380 silica gel glue chemical analysis of literary composition brocade are cyanoacrylate, the extensive use in orthopaedic trauma of current medical cyanoacrylate adhesive, inanimate object toxicity.Namely this sleeve pipe utilizes GA-1073 silica gel tube intensity very much not yielding, namely the feature not easily collapsed when surgical thread ligation, by choosing the GA-1073 silica gel tube of suitable caliber, while minimizing is to tracheal injury, be more convenient for inserting animal trachea, surgical thread ligation is tight; Utilize again the feature that Teflon hollow pipe intensity is little and toughness is large, be both convenient to connect syringe, and be convenient to turn back when late arterial clamp stopped pipe chamber again.Super 380 silica gel glue of literary composition brocade are the solvent based product based on high molecular polymer, and adhesive strength is high, anti-shearing.This pair of material hollow sleeve not only can be reused, and on the basis ensureing the airway epithelial cell activity be separated, significantly increases cell separation quantity.
At present, commercially available primary airway epithelial cell is expensive, and majority all lost cilium directional swing in body physiological function, active poor.Therefore, more convenient and airway epithelial cell separation equipment is efficiently needed.
Summary of the invention
The present invention relates to a kind of two material hollow sleeves of being convenient to carry out primary airway epithelial cell separation.Described pair of material sleeve pipe can ensure the success rate of laboratory animal tracheal intubation, and guarantee patency and the seal of the tubing be made up of syringe, two material hollow sleeve and trachea, anti-tubing is here soaked in the medium, occur enzymolysis solution seepage in the process of enzymolysis, while the contact ensureing pronase and airway epithelial cell and effect, the consumption of effective minimizing pronase, thus reduce enzymolysis solution to the damage of free airway epithelial cell.To obtain more more number and active better primary airway epithelial cell.
Described two material hollow sleeves by,
(1) inner layer sleeve of the insertion trachea that the silica gel tube that caliber is thin, intensity is high is formed, and
(2) caliber is thick, toughness is large, can doubling the outer layer sleeve that the Teflon hollow pipe closed by mosquito forceps is formed be socketed to form;
And the external diameter of silica gel tube equals the internal diameter of Teflon hollow pipe.
Described socket is for bonding by can be used for operating adhesive of medical, and described adhesive of medical is preferably super 380 silica gel of literary composition brocade and glues silica gel glue.
Described silica gel tube is preferably GA-1073 silica gel tube, intensity is very much not yielding, not easily collapse when surgical thread ligation, by choosing the GA-1073 silica gel tube of suitable caliber, while minimizing is to tracheal injury, can be more convenient for inserting animal trachea, and do not subside during surgical thread ligation thus ensure that the smooth inflow trachea chamber of pronase digestion liquid;
The described little toughness of Teflon hollow pipe intensity is large, has both been convenient to connect syringe, is convenient to again folding and by mosquito forceps pinch seal when the later stage closes tube chamber.
This sleeve pipe not only can be reused, and the success rate of laboratory animal tracheal intubation can be ensured, and guarantee patency and the seal of the pipeline be made up of syringe, two material hollow sleeve and laboratory animal trachea, reduce the consumption of pronase, effective activity promoting the airway epithelial cell that separation obtains, increases the separation quantity of airway epithelial cell.
The caliber size of described silica gel tube and Teflon hollow pipe and length experimentally can carry out choose reasonable by animal model, so that intubation.
Described silica gel tube socket is depended on the length in Teflon hollow pipe and is separated the animal pattern kind of primary airway epithelial cell and the length of two kinds of material pipes, is preferably 1/4th of silica gel tube length.
The present invention is about the research of airway epithelial cell, teaching, medical application provide favourable material conditions, our culture medium described in patent (ZL 201110440894.2) of having authorized of use in conjunction, effectively can increase the quantity of airway epithelial cell and vigor that are separated and obtain, and retain the cilium directional swing of airway epithelial cell in body physiological function.
The invention enables primary airway epithelial cell lock out operation simple, repeatable strong, easy to utilize, there is promotional value widely.
Accompanying drawing explanation
Fig. 1. the axonometric chart (for mice special pair of material hollow sleeve) of two material hollow sleeve.(scale 5:1)
Fig. 2. the sectional view of two material hollow sleeve.(scale 5:1)
Fig. 3. the sectional view of two material hollow sleeves A-A ' along the line.(scale 50:1)
Fig. 4. two material hollow sleeve connects the free retrotracheal pictorial diagram of BALB/c mouse, 1. a surgical thread; 2. mice trachea; 3.GA-1073 silica gel tube; 4. Teflon hollow pipe.
Fig. 5. before injecting chain pheron, two material hollow sleeve connects 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. Teflon hollow pipe.
Fig. 7. the mouse primary airway epithelial cell under inverted microscope
Detailed description of the invention
For separating mouse airway epithelial cell special pair of material hollow sleeve
The making of embodiment 1. pairs of material sleeve pipes
(1) get 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) 20mm is got and two ends are 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 being coated super 380 silica gel glue of 10 microlitre literary composition brocade inserts in Teflon hollow pipe, fixes 15s and namely obtain described pair of material sleeve pipe after inserting 5mm.
Figure 1 shows that the axonometric chart (for mice special pair of material hollow sleeve) 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 GA-1073 silica gel tube insertion 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) get BALB/c mouse to put to death through disconnected neck, tilt head, by alcohol-pickled for its thorax abdomen sterilization 30s, note protection oral cavity and nasal meatus, stimulate from ethanol.Be fixed on operating-table, the downward hunter's line of aseptic condition cuts off skin, exposes ventral aorta, cuts blood-letting to flowing to end, and its object is to avoid being mixed into too much erythrocyte when being separated airway epithelial cell;
(2) blunt separation trachea, after exposing trachea, and it is fully free;
(3) bifilar No. 1 surgical thread 100mm is worn, below trachea cricoid cartilage, a small gap is cut at 5mm place, inserts two material sleeve pipe GA-1073 silica gel tube 45 degree of oblique ends, and it is dark that insertion trachea half is about 5mm, outside trachea, make a call to a tight knot with No. 1 surgical thread, silica gel sleeve pipe is fixed on tracheal strips;
(4) connect 1ml gauge hypodermic device by Teflon hollow pipe, injection PBS buffer washing trachea in conductive pipe, and observe lung and have N/D;
(5) make a call to a tight knot again at the crotch surgical thread that trachea downstream silica gel tube is not deep and close trachea end, remove excess tissue and surgical thread around trachea, 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 to the tracheal strips that cannula tip is fixed by two material sleeve pipe, the PBS of washing removes by lavation inner surface of trachea;
(2) 0.05% pronase is filled whole section of trachea, trachea is slightly expanded in balloon sample, clamps with 30mm bulldog clamp after the Teflon hollow tube portion of doubling, close the combination of whole trachea-sleeve pipe;
(3) whole section of trachea is fixed infiltration in the MEM being added with penicillin, digestion 8h;
(4) cut off lower end trachea, collect Digestive system, and draw PBS flushing trachea with 1ml syringe;
(5) collect flushing liquor and be about 1.5ml (collect liquid+PBS and rinse trachea liquid), be transferred in EP pipe after merging, the centrifugal 4min of 500X g removes supernatant;
(6) add erythrocyte cracked liquid (Sigma R7757-100ML) 150 μ l, after the mixing of 1min gentleness, PBS is diluted to 1.5ml, the centrifugal 6min of 500g; Remove supernatant, then use the abundant re-suspended cell of appropriate complete medium, get 10 μ l for cell counting, through counting, every mice can be extracted cell number and is about 5*10
5individual cell.
The original cuiture of embodiment 4. airway epithelial cell and qualification
Airway epithelial cell is cultivated
(1) for 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 according to actual experiment require determine), note mixing, avoid that cell is in heaps cannot launch;
(2) after cross shakes up, after laboratory table places 30min, be transferred in incubator, carry out every day changing liquid process (identical system), can subsequent experimental 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 flakes, and generally adherent and launch completely, Microscopic observation at about 24h visible cell, purity is about 95%, only has a small amount of fibroblast to mix wherein;
(2) cultivate cell fusion degree after 5d to reach higher level and namely form typical cell island (Fig. 7), and can see fibre swing fast, this is the exclusive biological behaviour of airway epithelial cell.
Because the cell area be separated derives from inner surface of trachea, this position uniquely has the cell of rhythmic exercise to only have airway epithelial cell, simultaneously, the swing of this rule is again the typical characteristic of airway epithelial cell, therefore using this feature as the mark of this cell, and illustrating that Growth of Cells is good, its physiological function is close in body level.
Claims (5)
1. carry out two material hollow sleeves for primary airway epithelial cell separation, it is characterized in that, described two material hollow sleeves by,
(1) inner layer sleeve of the insertion trachea that the silica gel tube that caliber is thin, intensity is high is formed, and
(2) caliber is thick, toughness is large, can doubling the outer layer sleeve that the Teflon hollow pipe closed by mosquito forceps is formed be socketed to form;
And the external diameter of silica gel tube equals the internal diameter of Teflon hollow pipe.
2. according to claim 1 pair of material hollow sleeve, is characterized in that, described socket is for bonding by can be used for operating adhesive of medical, and described adhesive of medical is preferably super 380 silica gel of literary composition brocade and glues silica gel glue.
3. according to claim 1 and 2 pair of material hollow sleeve, is characterized in that, described silica gel tube is preferably GA-1073 silica gel tube.
4. according to claim 3 pair of material hollow sleeve, it is characterized in that, described silica gel tube socket is depended on the length in Teflon hollow pipe and is separated the animal pattern kind of primary airway epithelial cell and the length of two kinds of material pipes, is preferably 1/4th of silica gel tube length.
5. according to claim 4 pair of material hollow sleeve, it is characterized in that: described hollow sleeve is used for mouse primary airway epithelial cell and is separated, the external diameter of GA-1073 silica gel tube equals the internal diameter of Teflon pipe, for mice trachea mean inside diameter 0.8mm, GA-1073 silica gel tube length is 20mm, and the length of Teflon pipe is 25mm.
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| 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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| 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974429A (en) * | 2017-11-29 | 2018-05-01 | 宁夏医科大学总医院 | A kind of method and Optimal Medium of quick separating culture human airway epithelial cells |
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|---|---|---|---|---|
| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974429A (en) * | 2017-11-29 | 2018-05-01 | 宁夏医科大学总医院 | A kind of method and Optimal Medium of quick separating culture human airway epithelial cells |
| 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 |
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