CN109055298A - A kind of isolated culture method of primary dog vascular endothelial cell - Google Patents
A kind of isolated culture method of primary dog vascular endothelial cell Download PDFInfo
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- CN109055298A CN109055298A CN201810908934.3A CN201810908934A CN109055298A CN 109055298 A CN109055298 A CN 109055298A CN 201810908934 A CN201810908934 A CN 201810908934A CN 109055298 A CN109055298 A CN 109055298A
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Abstract
The present invention relates to biological field more particularly to a kind of isolated culture methods of primary dog vascular endothelial cell.The isolated culture method is the following steps are included: S1: taking the aorta of dog；S2: overturning aorta is simultaneously cleaned；S3: blood vessel monolayer surface cell is obtained；S4: it obtains primary dog vascular endothelial cell and carries out in vitro culture.Isolated culture method of the invention has the advantage that easy to operate, favorable repeatability, and experimental period is short；Vascular endothelial cell derives from the dog aorta of traffic death, avoids dispute of ethic；It is combined using Mechanical Method scraping cells, then with collagenase digestion, a large amount of single vascular endothelial cells with strong proliferative capacity can be obtained；The vascular endothelial cell purity is high of acquisition；The probability of cell contamination is very low, and the success rate of the method turning operation is close to 100%；Removing is attached to the knot hoof tissue of blood vessel surface, greatly reduces the opportunities for contamination of other cells such as fibroblast.
Description
Technical field
The present invention relates to biological field more particularly to a kind of isolated culture methods of primary dog vascular endothelial cell.
Background technique
In cardiovascular and cerebrovascular disease high-incidence today, the foundation of disease model and medicament research and development are particularly important.And blood vessel
Endothelial cell has separating difficulty big as the most suitable model cell of cardiovascular and cerebrovascular disease, the low feature of purity, to a certain degree
On constrain cardiovascular and cerebrovascular diseases medicament research and development development.Although having numerous Blood vessel endothelial cell lines in the market, itself and original
For cell compared to certain variation has occurred, there is larger difference with the vascular endothelial cell under body physiological environment.Dog is as most
Welcome companion pets, blood vessel kind disease supreme people's court equally annoying pet doctor and owner, and in the market temporarily without dog source
Blood vessel endothelial cell line, therefore, therefore primary separation dog vascular endothelial cell technology is particularly important.
Current separating blood vessel endothelium cell mainly applies tissue block method and enzyme digestion, and is carried out with immunomagnetic beads method pure
Change, this method complex steps and to obtain cell quantity few constrain vascular endothelial cell and isolate and purify and its answer in disease model
Development.
Summary of the invention
In order to solve above-mentioned existing technical problem, the present invention provides a kind of being separately cultured for primary dog vascular endothelial cell
Method, this method is simple, and obtained primary cell purity is high and activity is good.
Technical problem solved by the invention is realized using following technical scheme:
One aspect of the present invention discloses a kind of isolated culture method of primary dog vascular endothelial cell, comprising the following steps:
S1: the aorta of dog is taken；
S2: overturning aorta is simultaneously cleaned；
S3: blood vessel monolayer surface cell is obtained；
S4: it obtains primary dog vascular endothelial cell and carries out in vitro culture.
Preferably, in S1 the following steps are included:
S11: taking the doggie of rigid traffic death, carries out thorax abdomen shaving after clearing up corpse；
S12: thoracic cavity, coring aorta are opened；
S13: extrusion remains in endovascular extravasated blood, takes out and is placed in superclean bench after being impregnated with 75% alcohol.
Preferably, in S2 the following steps are included:
S21: removing is attached to the knot hoof tissue of blood vessel surface, then using sterile tip tweezers by blood vessel one end to internal plug
Enter lumen, it is slowly that intraluminal vascular is mobile until whole extra vascular is overturn to the other end；
S22: blood vessel is cleaned.
Preferably, in S3 specifically includes the following steps:
Blood vessel is transferred in the culture dish equipped with PBS, blood vessel monolayer surface cell is gently scraped, is collected intravascular
Wall surface cell monolayer is into PBS solution.
It is furthermore preferred that gently scraping blood vessel monolayer surface cell with aseptic operation blade in S3.
Preferably, in S4 the following steps are included:
S41: it will collect after the PBS solution for having blood vessel monolayer surface cell is centrifuged and discard supernatant, centrifugation is obtained
Precipitating carries out digestion reaction after being resuspended with clostridiopetidase A；
S42: with being centrifuged after the screen to filtrate after the completion of digestion reaction, the cell precipitation that centrifugation obtains is cultivated completely with ECM
It is seeded in Tissue Culture Flask and is cultivated after base weight is outstanding；
S43: after growing cell clone in culture bottle, careful picking individual cells clone be transferred in new culture bottle into
Row expands culture and obtains primary dog vascular endothelial cell, and primary dog vascular endothelial cell can be passed on after covering with.
It is furthermore preferred that in S41, with collagenase type I under conditions of 37 DEG C digestion reaction 30-50min.
It is furthermore preferred that containing 5% serum, 1% dual anti-and 1% endothelial cell growth in ECM complete medium in S42
The factor.
The second aspect of the present invention discloses the dog vascular endothelial cell that above-mentioned isolated culture method obtains.
Third aspect of the present invention discloses application of the above-mentioned dog vascular endothelial cell in the research of dog blood vessel kind disease.
Compared with prior art, the invention has the following advantages:
(1) operation of the present invention is simple, favorable repeatability, and experimental period is short；
(2) Endothelial Cell of the present invention derives from the dog aorta of traffic death, avoids dispute of ethic, and artery
Vascular tissue is larger, can disposable isolated a large amount of vascular endothelial cell；
(3) present invention uses Mechanical Method scraping cells, convenient and efficient, then combines with collagenase digestion, can obtain big
The single vascular endothelial cell with strong proliferative capacity of amount, the vascular endothelial cell can be used for the biological characteristics of the subsequent cell
Property, build and be and the research such as disease model；
(4) its Endothelial Cell amplification is cloned from primary individual cells, and dedicated by using endothelial cell
Culture medium culture, the vascular endothelial cell purity is high of acquisition；
(5) endangium is flipped out using sterile tip tweezers, intercellular friction can be avoided to the greatest extent
And damage, the probability of cell contamination is very low, and the success rate of the method turning operation is close to 100%；
(6) before turning operation, removing is attached to the knot hoof tissue of blood vessel surface, greatly reduce fibroblast etc. its
The opportunities for contamination of his cell.
Detailed description of the invention
The present invention will be further described with reference to the accompanying drawings, but the embodiment in attached drawing is not constituted to any limit of the invention
System.
Fig. 1 is photo of the dog vascular endothelial cell under * 40 microscopes in the embodiment of the present invention；
Fig. 2 is the growth curve chart of the 6th generation dog vascular endothelial cell in the embodiment of the present invention；
Fig. 3 is dog vascular endothelial cell in the embodiment of the present invention respectively to skeletonization and the result figure broken up at rouge；
Fig. 4 is photo of the identified by immunofluorescence of dog vascular endothelial cell in the embodiment of the present invention under * 100 microscopes.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments be only used for the present invention without
For limiting the scope of the invention.
Embodiment 1
Present embodiment discloses a kind of isolated culture methods of primary dog vascular endothelial cell, comprising the following steps:
(1) doggie for taking rigid traffic death, cleans out bloodstain and dirt on corpse, in sterile hand after thorax abdomen shaving
The indoor tincture of iodine of art carries out after the cleaning disinfection of thorax abdomen again with 75% alcohol by tincture of iodine wiped clean.Thoracic cavity is opened, it is sterile to cut
Knife clip cardiac aorta about 4cm remains in endovascular extravasated blood with tweezers extrusion, is placed in taking out after 75% alcohol immersion 10s
In superclean bench；
(2) blood vessel is impregnated with the PBS solution containing 5% dual anti-solution (Pen .- Strep mixed solution), it is careful to remove
It is attached to the knot hoof tissue of blood vessel surface, lumen is inwardly then filled in into blood vessel one end using sterile tip tweezers, it slowly will pipe
Endoluminal vascular is mobile to the other end until the overturning of whole extra vascular, cleans blood vessel；
(3) blood vessel is transferred in the culture dish equipped with 5ml PBS solution, gently scrapes blood vessel using aseptic operation blade
Inner wall surface cell monolayer collects PBS containing cell, and (purchase is in Sigma public affairs with 1mg/ml Type I collagen enzyme for centrifuged deposit
Department, article No. C0130) in 37 DEG C of digestion 40min, with 200 mesh net filtrations.It collects filtrate 1200rpm to be centrifuged 5 minutes, abandons supernatant,
6ml is added, cell is made containing 5% serum, 1% dual anti-, 1%ECGS (endothelial growth factor) ECM complete medium mixing
Suspension is seeded in two T25 Tissue Culture Flasks, with 5%CO2Concentration, 37 DEG C of environment are cultivated in incubator；
(4) cell growth condition is observed daily, until cell grows clone, careful picking individual cells clone is transferred to
It is expanded culture in new culture bottle and obtains primary dog vascular endothelial cell, primary dog vascular endothelial cell can be into after covering with
Row passage.
From microscopically observation as can be seen that the cellular morphology of isolated primary dog vascular endothelial cell is in short shuttle shape
Or paving stone shape, as shown in Figure 1.
Reagent used in the present embodiment includes:
The preparation formula of PBS solution are as follows:
Endothelial cell special culture media (ECM) be purchased from SclenCell company (article No.: 1001), according to operation instruction by its
It is made into complete culture solution, is formulated are as follows:
Wherein, fetal calf serum and ECGS are that above-mentioned culture medium (is purchased from SclenCell company, article No.: 1001) mating in
Including reagent.
Isolated culture method disclosed in the present embodiment has the advantage that easy to operate, favorable repeatability, and when experiment
Between it is short；Vascular endothelial cell derives from the dog aorta of traffic death, avoids dispute of ethic, and arterial vascular tissue is larger,
It can disposable isolated a large amount of vascular endothelial cell；It is convenient and efficient using Mechanical Method scraping cells, then disappear with clostridiopetidase A
Change method combines, and can obtain a large amount of single vascular endothelial cells with strong proliferative capacity；The amplification of its Endothelial Cell
It is cloned from primary individual cells, and by using endothelial cell special culture media culture, the vascular endothelial cell of acquisition is pure
Degree is high；Endangium is flipped out using sterile tip tweezers, intercellular friction and damage can be avoided to the greatest extent,
The probability of cell contamination is very low, and the success rate of the method turning operation is close to 100%；Before turning operation, remove attached
Blood vessel surface knot hoof tissue, greatly reduce the opportunities for contamination of other cells such as fibroblast.
Embodiment 2
The present embodiment depicts the growth curve of dog vascular endothelial cell (VEC), and concrete operations are as follows:
(1) the primary dog vascular endothelial cell in embodiment 1 is subjected to cell and was passaged to for the 6th generation, take well-grown blood
Endothelial cell, with cell suspension is made after 0.25% trypsin digestion；
(2) cell is diluted to 2 × 104/ mL is seeded in 24 orifice plates, and every hole 0.5mL is placed in containing 5%CO2, 37 DEG C of cultures
It is cultivated in case；
(3) the same time randomly selects 3 hole cells daily, counts under the microscope after suspension is made with trypsin digestion
Number, every hole meter are averaged three times；
(4) continuous counter eight days, according to count results, using cell density as ordinate (a/mL), the time is abscissa,
Drafting obtains growth curve chart as shown in Figure 2.Figure it is seen that primary dog vascular endothelial cell reaches the 6th generation cell
Still there is activity well, grow into top in the 7th day cell.
Embodiment 3
The present embodiment carries out identified by immunofluorescence experiment to the 6th generation dog vascular endothelial cell, the specific steps are as follows:
(1) the primary dog vascular endothelial cell in embodiment 1 is subjected to cell and was passaged to for the 6th generation, take well-grown blood
Endothelial cell is seeded on the culture plate that gelatin was coated with, and is made the adherent 12h of cell, is discarded culture medium, with (the tire containing 5%FBS
Cow's serum) PBS solution wash 2 times；
(2) it is added and fixes 5 minutes containing 4% paraformaldehyde room temperature of 0.1%Triton × 100, remove fixer, with containing
The PBS solution of 5%FBS is washed 3 times；
(4) confining liquid (PBS containing 10%FBS) is added, 37 DEG C of closing 1h remove confining liquid, molten with the PBS containing 5%FBS
Liquid washs 3 times；
(5) it is separately added into CD31 (being purchased from Bioss company, article No. bs-0915R), CD34 (is purchased from abcam company, article No.
Ab81289) the primary antibody of two kinds of surface markers albumen, 4 DEG C are protected from light overnight incubation, discard after liquid with the PBS solution containing 5%FBS
Washing 3 times；
(6) it is separately added into the corresponding secondary antibody (being purchased from beyotime company, article No. A0568) of FITC label, 4 DEG C are protected from light
It is incubated for 30 minutes, discards liquid, washed 3 times with the PBS solution containing 5%FBS；
(7) 1 μ g/mL DAPI (being purchased from Solarbio company, article No. C0065) of addition, which is protected from light, redyes 5 minutes, discards liquid,
It is washed 3 times with the PBS containing 5%FBS；
(8) anti-fluorescence quenching (being purchased from Solarbio company, article No. S2100) is added, in inverted fluorescence microscope (100
×) under observe and coloration result and take pictures to obtain qualification result figure as shown in Figure 3.
As can be seen from Figure 3 the equal positive expression of CD31 and CD34 in dog vascular endothelial cell identified by immunofluorescence.
Embodiment 4
Chosen in the present embodiment the good 6th generation dog vascular endothelial cell of growth conditions in embodiment 2 carry out at rouge and at
Self-bone grafting differentiation.Wherein, the 6th generation dog vascular endothelial cell of any induction processing is not carried out as a control group.
Reagent needed for the present embodiment includes:
Dog vascular endothelial cell Osteoinductive differentiation culture medium is purchased from Gibco company, article No.: A1007201 dog blood vessel endothelium
Cell adipogenic induction differential medium is purchased from Cyagen company, article No. 90031, formula specifically:
(1) induction is at rouge differentiation step:
(1) when dog vascular endothelial cell culture to 80% convergence degree, with cell suspension is made after trypsin digestion；
(2) cell density is adjusted to 2 × 104/cm2, it is added in six orifice plates, every hole adds 2ml cell suspension；
(3) 37 DEG C, 5%CO are placed in2Under the conditions of cultivate cell, change the liquid once within every 3 days；
(4) when cell reaches 100% convergence degree, the culture medium in hole is discarded, is rinsed 2 times with PBS solution, every hole is added
2ml A liquid；
(5) after cultivating three days, original fluid is discarded, is rinsed 2 times with PBS solution, 2ml B liquid is added in every hole
(6) after cultivating 24 hours, original fluid is discarded, is rinsed 2 times with PBS solution, 2ml A liquid is added in every hole
(7) step (5) and (6) are repeated 3-5 times, finally cell is supported in B liquid 7 days, is changed the liquid once within every 3 days；
(8) oil red O (being purchased from Solarbio company, article No. G1260) staining analysis: culture solution in hole is discarded, PBS solution is used
4% formalin of 2ml is added fixed cell 30 minutes in cleaning 2 times；Then liquid is discarded, PBS is cleaned 2 times, with 1ml oil red O
Working solution is cleaned 2 times after dyeing 30 minutes with PBS solution, and microscopically observation is simultaneously taken pictures, and microscope magnification is 400 herein
Times.
(2) induced osteogenesis differentiation step:
(1) when dog vascular endothelial cell culture to 80% convergence degree, with cell suspension is made after trypsin digestion；
(2) cell density is adjusted to 3 × 104/cm2, it is added in six orifice plates, every hole adds 2ml cell suspension；
(3) 37 DEG C, 5%CO are placed in2Under the conditions of cultivate and discard original fluid afterwards for 24 hours, be added after cleaning 2 times with PBS solution
2ml Osteoblast Differentiation culture solution；
(4) not good liquor is changed within every 3 days after, until carrying out alizarin red after 2-3 weeks (is purchased from Solarbio company, article No.
G1450) staining analysis；
(5) Alizarin red staining is analyzed: discarding original culture solution, it is molten that 4% formaldehyde of 2ml is added in every hole after cleaning 2 times with PBS
Liquid fixes 30 minutes；Then it discards liquid to be washed 2 times with PBS, is added after 1ml alizarin red working solution reacts five minutes and cleans 2 with PBS
Secondary, under an optical microscope, microscope multiple is 200 times herein, observes and takes pictures, obtains differentiated result figure as shown in Figure 4.
Figure 4, it is seen that the dog vascular endothelial cell in the 6th generation still has stronger differentiation performance, it can be to skeletonization
Break up at rouge direction, there is stronger application value, can be used for clinical research.
Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and
Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and
Modification, all should be contained within the scope of the invention.
Claims (10)
1. a kind of isolated culture method of primary dog vascular endothelial cell, which comprises the following steps:
S1: the aorta of dog is taken；
S2: overturning aorta is simultaneously cleaned；
S3: blood vessel monolayer surface cell is obtained；
S4: it obtains primary dog vascular endothelial cell and carries out in vitro culture.
2. the isolated culture method of primary dog vascular endothelial cell according to claim 1, which is characterized in that wrapped in S1
Include following steps:
S11: taking the doggie of rigid traffic death, carries out thorax abdomen shaving after clearing up corpse；
S12: thoracic cavity, coring aorta are opened；
S13: extrusion remains in endovascular extravasated blood, takes out and is placed in superclean bench after being impregnated with 75% alcohol.
3. the isolated culture method of primary dog vascular endothelial cell according to claim 1, which is characterized in that wrapped in S2
Include following steps:
S21: removing is attached to the knot hoof tissue of blood vessel surface, and pipe is inwardly then filled in blood vessel one end using sterile tip tweezers
Chamber, it is slowly that intraluminal vascular is mobile until whole extra vascular is overturn to the other end；
S22: blood vessel is cleaned.
4. the isolated culture method of primary dog vascular endothelial cell according to claim 1, which is characterized in that specific in S3
The following steps are included:
Blood vessel is transferred in the culture dish equipped with PBS, blood vessel monolayer surface cell is gently scraped, collects blood vessel table
Face cell monolayer is into PBS solution.
5. the isolated culture method of primary dog vascular endothelial cell according to claim 4, which is characterized in that in S3,
Blood vessel monolayer surface cell is gently scraped with aseptic operation blade.
6. the isolated culture method of primary dog vascular endothelial cell according to claim 1, which is characterized in that wrapped in S4
Include following steps:
S41: will collect after the PBS solution for having blood vessel monolayer surface cell is centrifuged and discard supernatant, the precipitating that centrifugation is obtained
Digestion reaction is carried out after being resuspended with clostridiopetidase A；
S42: with being centrifuged after the screen to filtrate after the completion of digestion reaction, obtained cell precipitation ECM complete medium weight will be centrifuged
It is seeded in Tissue Culture Flask and is cultivated after outstanding；
S43: after growing cell clone in culture bottle, careful picking individual cells clone, which is transferred in new culture bottle, to be expanded
Big culture obtains primary dog vascular endothelial cell, and primary dog vascular endothelial cell can be passed on after covering with.
7. the isolated culture method of the primary dog vascular endothelial cell according to claim 6, which is characterized in that
In S41, with collagenase type I under conditions of 37 DEG C digestion reaction 30-50min.
8. the isolated culture method of the primary dog vascular endothelial cell according to claim 6, which is characterized in that
In S42,5% serum, 1% dual anti-and 1% endothelial growth factor are contained in ECM complete medium.
9. the dog vascular endothelial cell that any one of -8 isolated culture methods obtain according to claim 1.
10. application of the dog vascular endothelial cell according to claim 9 in the research of dog blood vessel kind disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110484491A (en) * | 2019-08-20 | 2019-11-22 | 北京京蒙高科干细胞技术有限公司 | The endothelial progenitor cells acquisition methods and its purification cultivation method of amnion and amniotic fluid-derived |
| CN113025566A (en) * | 2020-12-30 | 2021-06-25 | 无锡市第九人民医院 | Endothelial cell osteogenesis induced differentiation culture medium and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484491A (en) * | 2019-08-20 | 2019-11-22 | 北京京蒙高科干细胞技术有限公司 | The endothelial progenitor cells acquisition methods and its purification cultivation method of amnion and amniotic fluid-derived |
| CN110484491B (en) * | 2019-08-20 | 2022-03-18 | 北京京蒙高科干细胞技术有限公司 | Method for obtaining amniotic membrane and amniotic fluid derived endothelial progenitor cells and purification culture method thereof |
| CN113025566A (en) * | 2020-12-30 | 2021-06-25 | 无锡市第九人民医院 | Endothelial cell osteogenesis induced differentiation culture medium and preparation method thereof |
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Application publication date: 20181221 |