CN107828721A - The culture of chicken embryo cardiac muscle cell a kind of and immunohistochemistry authentication method - Google Patents
The culture of chicken embryo cardiac muscle cell a kind of and immunohistochemistry authentication method Download PDFInfo
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- CN107828721A CN107828721A CN201711106879.8A CN201711106879A CN107828721A CN 107828721 A CN107828721 A CN 107828721A CN 201711106879 A CN201711106879 A CN 201711106879A CN 107828721 A CN107828721 A CN 107828721A
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- cardiac muscle
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention belongs to biological technical field, a kind of more particularly to culture and the immunohistochemistry authentication method of chicken embryo cardiac muscle cell, using the chicken embryo of 11 ages in days as separation object, the progress simple to operate for more mainly embodying animal welfare, fibroblastic pollution is reduced using differential attachment method, this method is simple and easy, the higher cardiac muscle cell of purity can be obtained, so that cardiac muscle cell is adherent.The present invention is improved with reference to cardiac muscle of mammal cell culture and authentication method, forms more stable reliable chicken embryo myocardial cells culture and identification technology, using the teaching of the invention it is possible to provide enough cardiac muscle cells are used for research work.
Description
Technical field
The invention belongs to biological technical field, more particularly to the culture of chicken embryo cardiac muscle cell a kind of and immunohistochemistry mirror
Determine method.
Background technology
Cardiac muscle cell is the pathology such as research cardiomegaly, heart failure, myocardial ischemia-reperfusion injury and radical damage
The key link of process occurrence and development.The culture of cardiac muscle cell helps to further investigate the pathomechanism of heart disease.In the mankind
Cultured myocardial is studied the Main Means for turning into disease of cardiovascular system research in medical science.
With being deepened continuously to birds disease of cardiovascular system research, studied using the cardiac muscle cell of culture as material
Critical role will be accounted for.Many scholars update to the Cardiac myocytes method of muroid, and its culture report is more, still
Poultry primary cell myocardial cells culture is rarely reported, and the chicken embryo cardiac muscle cell for cultivating purity and high vigor is remained one
Determine difficulty.There is larger difference in poultry cardiac muscle, and 9-11d cardiac muscular tissue is in and do not divided completely with cardiac muscle of mammal tissue
The state of change, the original cuiture of chicken embryo cardiac muscle cell is the related poultry angiocardiopathy myocardial structural of research and changes of function
One ideal model.How to obtain the chicken embryo cardiac muscle cell of higher degree and higher vigor turns into this area urgent problem to be solved
One of.
The content of the invention
The present invention is directed to many weak points existing for prior art, there is provided the culture of chicken embryo cardiac muscle cell a kind of and exempts from
Epidemic disease histochemistry authentication method, it is simple to operate more mainly to embody animal good fortune using the chicken embryo of 11 ages in days as separation object
The progress of profit, fibroblastic pollution is reduced using differential attachment method, this method is simple and easy, and it is higher can to obtain purity
Cardiac muscle cell, so that cardiac muscle cell is adherent.The present invention is improved with reference to cardiac muscle of mammal cell culture and authentication method,
Form more stable reliable chicken embryo myocardial cells culture and identification technology, using the teaching of the invention it is possible to provide enough cardiac muscle cells are used to study work
Make.
The concrete technical scheme of the present invention is as follows：
A kind of cultural method of chicken embryo cardiac muscle cell, specific preparation process are as follows：
(1) the SPF chicken embryos of 11 ages in days are chosen, eggshell is successively wiped with Iodophor and 75% alcohol；Tweezers knock egg gas open
Room end, tears film off, and tweezers pull out chicken embryo；
(2) tweezers clamp carina, and scissors is cut off from carina both sides, exposure heart；The clip apex of the heart, it is in have PBS to be placed in
25mL small beakers in, successively wash five times, untill PBS is substantially without color；
(3) press from both sides out the apex of the heart after above-mentioned processing to be placed in culture dish, scissors is shredded to fine sand shape, every 10 apexes of the heart and added
8mL NTx enzymes, enzyme concentration 1mg/mL, with 0.01M pH 7.2PBS solution allocations；4 DEG C of digestion 14-16h；
(4) culture dish is taken, DMEM in high glucose of the 2mL containing 20vt%FBS is added and terminates digestion；Bus suction pipe dispels mixing heart
Particle, filtering is suctioned out, be placed in centrifuge tube, 800r/min centrifugation 10min, abandon supernatant；
(5) it is resuspended with DMEM in high glucose of the 10mL containing 20vt%FBS, is distributed into culture dish, 37 DEG C of differential velocity adherent 50-60min,
Then caused supernatant after differential velocity adherent is transferred in centrifuge tube；
(6) after the supernatant in above-mentioned centrifuge tube is mixed with the piping and druming of bus suction pipe, according to 2 × 105Individual/ml density is added to
In 6 porocyte plates, per hole 2mL, add BRDU and suppress fibroblast, BRDU final concentration of 0.1Mm/L, 24-48 are small in every hole
When after cell grow up to individual layer, be separately cultured end, observe cell growth condition.
Wherein step (5) mainly utilizes poly-D-lysine, stronger to fibroblast adhesion, and cardiac muscle cell is glued
The attached weaker principle of power, and fibroblast is adhered on slide, the supernatant containing non-adhering cardiac muscle cell is collected, culture.
All reagent articles for use are laboratory conventional chemical reagent material in the above method, can be bought in Reagent Company.One
As for mammal cultured myocardial use newborn young animal, it is more painful during execution.And used in this method
Be 11 ages in days chicken embryo, will with respect to tissue differentiation for chicken main reason is that mid-term stage of the chicken embryo also in development
Rudimentary is more, and the sensory system of chicken embryo is also complete without development, and chicken is also far below to the extent of reaction of environmental stimuli.From this meaning
Said in justice, substituting chicken using chicken embryo more meets the requirement of animal welfare, and with respect to chick or mammal the step of operate
Less, sterile working is more easy, and because chicken embryo heart development degree is relatively low, cell culture fluid influences relatively small on it.
Present invention digestion clostridiopetidase A used is a kind of enzyme extracted from bacterium, has very strong digestion to make to collagen
With suitable for digestion fibroid tissue, epithelial tissue and cancerous tissue, it has preferable digestion to cytoplasm, to cell
Itself influence less, cell can be made with collagen component to depart from and preserve from.The enzyme good separating effect, even if having calcium, magnesium ion
In the presence of still active, therefore PBS and the nutrient solution containing serum can be used to prepare, i.e., easy to operate and cell survival rate can be improved, this enzyme
Digestion relaxes, without mechanical oscillation.In the prior art for the heart culture of mammal, usually using trypsase
37 DEG C are digested, and are digested using constant temperature blender with magnetic force mechanical assistant to ensure digestion effect；It is and used in the present invention
Clostridiopetidase A carries out 4 DEG C and is digested overnight, it is not necessary to by mechanical digestion, in the selection of digestion time, such as time too short tissue digestion
Not thoroughly, long then cell survival rate is relatively low, and digestion 12-14h is optimal.Although digestion time is compared with cardiac muscle of mammal cell culture
Method time length, but the cell damaged is less, time easy control, while the cardiac muscle cell's quality better finally obtained.4℃
When cell is blown and beaten after digestion, bus suction pipe used can reduce the damage to cell, and play raising cellular activities
The effect of rate.For differential velocity adherent time control between 50min -1h, the time is too short, and fibroblast is adherent insecure,
Overlong time, cardiac muscle cell's also adherent growth, for adding BRDU in cells and supernatant, can suppress fibroblastic
Growth.
Indirect immunohistochemistry identification can be carried out after above-mentioned chick-embryo cell is obtained, is comprised the following steps that：Under
It is volume fraction to state percentage；
(1) bed board：By the chicken embryo cardiac muscle cell obtained by the above method with 2 × 105It is thin that individual/mL density is added to 24 holes
On born of the same parents' plate, cell grows up to individual layer after every hole 500 μ L, about 48h；
(2) 0.01M pH 7.2PBS are washed 3 times；Then 4% paraformaldehyde of 200 μ l precoolings is added per hole, room temperature is fixed
15min, finally washed again 3 times using 0.01M pH 7.2PBS；
(3) the μ L/ holes of 2%Triton X-100 (Beijing Suo Laibao Science and Technology Ltd) 200 are used, room temperature 10min, afterwards
PBS is washed 3 times again.
(4) rabbit-anti chicken α-actin polyclonal antibodies (abcam) 200 μ L are added per hole, 1h is acted in 37 DEG C of incubators；
(5) PBS washs 5min × 4 time；Goat-anti rabbit secondary antibody (Beijing Bo Aosen of 100 μ L FITC marks is added per hole afterwards
Bioisystech Co., Ltd), 37 DEG C of incubator lucifuges act on 1h；
(6) PBS is washed, 5min × 4 time；Four steam washing 10min × 1 time, microscopy after water removal lucifuge is dried：
Cardiac muscle cell's cell space of culture is larger, and in variforms such as fusiformis, star, triangles, karyon ellipse occupies center,
Iuntercellular often has aixs cylinder to be connected, core subcircular；Cell is linked to be piece after culture 3-5d, and 95% anti alpha-actin is immunized glimmering indirectly
Cardiac muscle cell's synchronous in blocks of photochemistry stained positive, it was demonstrated that have been set up good being separately cultured chicken embryo cardiac muscle cell
Method.
In summary, the present invention using 11 ages in days chicken embryo as separation object, it is simple to operate more mainly embody it is dynamic
The progress of thing welfare, fibroblastic pollution is reduced using differential attachment method, this method is simple and easy, can obtain purity compared with
High cardiac muscle cell, so that cardiac muscle cell is adherent.The present invention is subject to reference to cardiac muscle of mammal cell culture and authentication method
Improve, form more stable reliable chicken embryo myocardial cells culture and identification technology, using the teaching of the invention it is possible to provide enough cardiac muscle cells are used to grind
Study carefully work.
Brief description of the drawings
Fig. 1 is to be schemed using the cardiac muscle cell after the inventive method culture 48h；
Wherein left side is the form (200 ×) of cardiac muscle cell under white light；
Right side is the result (200 ×) of indirect immunofluorescence identification of cell, and its cardiac myocyte cytoplasm can be marked as
Green (grey parts in figure), are not cardiac muscle cells, cell does not have green fluorescence.
Embodiment
The technical scheme in the embodiment of the present invention is clearly and completely described below, it is clear that described embodiment
Only part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the common skill in this area
The every other embodiment that art personnel are obtained under the premise of creative work is not made, belong to the model that the present invention protects
Enclose.
Embodiment 1
(1) the SPF chicken embryos of 11 ages in days are chosen, eggshell is successively wiped with Iodophor and 75% alcohol；Tweezers knock egg gas open
Room end, tears film off, and tweezers pull out chicken embryo；
(2) tweezers clamp carina, and scissors is cut off from carina both sides, exposure heart；The clip apex of the heart, it is in have PBS to be placed in
25mL small beakers in, successively wash five times, untill PBS is substantially without color；
(3) press from both sides out the apex of the heart after above-mentioned processing to be placed in culture dish, scissors is shredded to fine sand shape, every 10 hearts and added
8mL NTx enzymes, enzyme concentration 1mg/mL, with 0.01M pH 7.2PBS solution allocations；4 DEG C of digestion 14-16h；
(4) culture dish is taken, DMEM in high glucose of the 2mL containing 20vt%FBS is added and terminates digestion；Bus suction pipe dispels mixing heart
Particle, filtering is suctioned out, be placed in centrifuge tube, 800r/min centrifugation 10min, abandon supernatant；
(5) it is resuspended with DMEM in high glucose of the 10mL containing 20vt%FBS, is distributed into culture dish, 37 DEG C of differential velocity adherent 50-60min,
Then caused supernatant after differential velocity adherent is transferred in centrifuge tube；
(6) after the supernatant in above-mentioned centrifuge tube is mixed with the piping and druming of bus suction pipe, according to 2 × 105Individual/ml density is added to
In 6 porocyte plates, per hole 2mL, add BRDU and suppress fibroblast, BRDU final concentration of 0.1Mm/L, 24-48 are small in every hole
When after cell grow up to individual layer, be separately cultured end, observe cell growth condition.
Embodiment 2
(1) bed board：By the chicken embryo cardiac muscle cell obtained by the above method with 2 × 105It is thin that individual/mL density is added to 24 holes
On born of the same parents' plate, cell grows up to individual layer after every hole 500 μ L, about 48h；
(2) 0.01M pH 7.2PBS are washed 3 times；Then the 4vt% paraformaldehydes of 200 μ L precoolings are added per hole, room temperature is solid
Determine 15min, finally washed again 3 times using 0.01M pH 7.2PBS；
(3) the μ l/ holes of 2vt%Triton X-100 (Beijing Suo Laibao Science and Technology Ltd) 200 are used, room temperature 10min, it
PBS is washed 3 times again afterwards.
(4) rabbit-anti chicken α-actin polyclonal antibodies (abcom) 200 μ L are added per hole, 1h is acted in 37 DEG C of incubators；
(5) PBS washs 5min × 4 time；Goat-anti rabbit secondary antibody (Beijing Bo Aosen of 100 μ L FITC marks is added per hole afterwards
Bioisystech Co., Ltd), 37 DEG C of incubator lucifuges act on 1h；
(6) PBS is washed, 5min × 4 time；Four steam washing 10min × 1 time, microscopy after water removal lucifuge is dried：
As a result as shown in figure 1, cardiac muscle cell's cell space of culture is larger, in the variforms such as fusiformis, star, triangle, born of the same parents
Core ellipse occupies center, and iuntercellular often has aixs cylinder to be connected, core subcircular (as shown in the left side of accompanying drawing 1)；Cell after culture 3-5d
It is linked to be piece, cardiac muscle cell's synchronous in blocks (such as accompanying drawing 1 of the 95% anti alpha-actin indirect immunofluorescences chemical staining positive
Shown in right side), it was demonstrated that have been set up the good method for being separately cultured chicken embryo cardiac muscle cell.
Claims (1)
1. a kind of cultural method of chicken embryo cardiac muscle cell, specific preparation process are as follows：
(1) the SPF chicken embryos of 11 ages in days are chosen, eggshell is successively wiped with Iodophor and 75% alcohol；Tweezers knock egg air chamber end open,
Tear film off, tweezers pull out chicken embryo；
(2) tweezers clamp carina, and scissors is cut off from carina both sides, exposure heart；The clip apex of the heart, it is in have PBS's to be placed in
In 25mL small beakers, successively wash five times, untill PBS is substantially without color；
(3) press from both sides out the apex of the heart after above-mentioned processing to be placed in culture dish, scissors shreds to fine sand shape, every 10 hearts and adds the types of 8mL I
Clostridiopetidase A, enzyme concentration 1mg/mL；4 DEG C of digestion 14-16h；
(4) culture dish is taken, DMEM in high glucose of the 2mL containing 20vt%FBS is added and terminates digestion；Bus suction pipe, which dispels, mixes heart
Grain, filtering is suctioned out, be placed in centrifuge tube, 800r/min centrifugation 10min, abandon supernatant；
(5) it is resuspended with DMEM in high glucose of the 10mL containing 20vt%FBS, is distributed into culture dish, 37 DEG C of differential velocity adherent 50-60min, then
Caused supernatant after differential velocity adherent is transferred in centrifuge tube；
(6) after the supernatant in above-mentioned centrifuge tube is mixed with the piping and druming of bus suction pipe, according to 2 × 105It is thin that individual/ml density is added to 6 holes
On born of the same parents' plate, per hole 2mL, add BRDU and suppress fibroblast, per hole in the final concentration of 0.1Mm/L of BRDU, it is thin after 24-48 hours
Born of the same parents grow up to individual layer, are separately cultured end, observe cell growth condition.
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Citations (3)
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| EP0201784A2 (en) * | 1985-05-09 | 1986-11-20 | Yaguang Liu | Pharmaceutical composition decreasing side effects of anticancer chemotherapy and increasing the immune function |
| CN101374942A (en) * | 2006-01-31 | 2009-02-25 | 阿斯比奥制药株式会社 | Method for purifying cardiac myocyte and presumptive cardiac myocyte derived from stem cell and fetus |
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2017
- 2017-11-10 CN CN201711106879.8A patent/CN107828721A/en active Pending
Patent Citations (4)
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|---|---|---|---|---|
| EP0201784A2 (en) * | 1985-05-09 | 1986-11-20 | Yaguang Liu | Pharmaceutical composition decreasing side effects of anticancer chemotherapy and increasing the immune function |
| CN101374942A (en) * | 2006-01-31 | 2009-02-25 | 阿斯比奥制药株式会社 | Method for purifying cardiac myocyte and presumptive cardiac myocyte derived from stem cell and fetus |
| CN103409368A (en) * | 2006-01-31 | 2013-11-27 | 第一三共株式会社 | A method for purifying cardiomyocytes or programmed cardiomyocytes derived from stem cells or fetuses |
| CN104189958A (en) * | 2014-08-25 | 2014-12-10 | 中国人民解放军总医院 | Method for preparing chitosan-silk fibroin composite nano-fiber multifunctional patch for promoting myocardial tissue regeneration and monitoring stem cells |
Non-Patent Citations (7)
| Title |
|---|
| JIAO XU等: "Inhibition of heat shock protein 70 intensifies heat-stressed damage and apoptosis of chicken primary myocardial cells in vitro", 《MOLECULAR MEDICINE REPORTS》 * |
| QIN SHI-ZHEN等: "Manganese enhances the expression of the manganese superoxide dismutase in cultured primary chick embryonic myocardial cells", 《JOURNAL OF INTEGRATIVE AGRICULTURE》 * |
| XIAO-HUI ZHANG等: "Apoptosis in response to heat stress is positively associated with heat-shock protein 90 expression in chicken myocardial cells in vitro", 《J VET SCI》 * |
| 吴燕峰等: "《实用医学细胞培养技术》", 31 January 2010, 中山大学出版社 * |
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