CN109735486A - A kind of primary melanocyte cultural method causing early ageing for studying UVB irradiation - Google Patents
A kind of primary melanocyte cultural method causing early ageing for studying UVB irradiation Download PDFInfo
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Abstract
A kind of primary melanocyte cultural method causing early ageing for studying UVB irradiation, is related to radioecology, technical field of cell biology.In order to cultivate the primary melanocyte with more high sensitivity, the present invention provides following methods: foreskin sample being impregnated, excess tissue is cut off, dermatological specimens are cut into strip；It has been transferred in lid vessel, trypsin solution is added and is incubated in refrigerator；Corium and epidermis are separated, epidermis is transferred in centrifuge tube, is incubated for, washs at room temperature, centrifugation；It is resuspended and is precipitated with melanocyte culture medium, moved to and continue follow-up cultivation in carbon dioxide incubator.The method of the present invention is sensitiveer compared with Nostoc commune Vanch method, strong applicability, is that more preferably selecting for primary melanocyte experimental study is irradiated for various dose UVB.
Description
Technical field
The present invention relates to radioecologies, technical field of cell biology, and in particular to one kind is caused for studying UVB irradiation
The primary melanocyte cultural method of early ageing.
Background technique
UVB irradiation: according to different biological effects, ultraviolet light can be divided into four wave bands, medium wavelength according to wavelength
Wave band between 290-320nm is referred to as UVB wave band, also referred to as medium wave erythemal effect ultraviolet light.UVB has medium penetration power,
Although most of can be absorbed by ozone layer, long-term or excessive irradiation can make skin darkening, cause the diseases such as red and swollen, decortication
Shape has certain carcinogenicity to skin.On October 27th, 2017, international cancer research institution, the World Health Organization announce carcinogenic
In object inventory, ultraviolet radiation (wavelength 100-400nm, including UVA, UVB and UVC) is listed in a kind of carcinogenic substance.People are daily
SPF (the sun protection factor) value of used suncream mark is the ability protected for UVB, therefore is applied
UVB, which carries out corresponding research, higher practicability.
Melanocyte: melanocyte (melanocyte) is located at the basal layer of epidermis, collectively constitutes epidermis with epidermal cell
Melanocyte unit, major function are to generate melanosome skin is prevented to be damaged.These cells are located at the basal cell of skin
Layer, but its branch is located between the keratinocyte of basal cell layer or more.The major function of melanocyte is to generate melanin,
It plays a very important role the color of skin.Meanwhile melanin can protect skin not by ultraviolet radiation
Damage, also functions to certain effect in immune system.
Cell ageing: cell is the basic unit and Systemic aging basic unit of organism structure and function.Cell
Aging shows as cyto-architectural retrogression on morphology；In a physiologically show as deterioration and poor metabolism.
Cell ageing is divided into premature senescence and prematureness aging, the former is normal physiological aging；And the latter is then pathological seaility
(Cell,2017,170(4):816-816).Under normal conditions, normal physiological aging is beneficial for body, can be promoted
Into metabolism, the accumulation of harmful substance is reduced.However, pathological seaility is and the generation of clinically many diseases, development phase
Guan Eryu age unrelated life entity morphosis and physiological function regression (just true and Wang Chunge, 2014, Medical review, 20
(16): 2994-2995), a large number of studies show that, the Ultraviolet B (Ultraviolet B UVB) with medium wavelength can induce thin
Born of the same parents enter irritability early ageing state (J COSMET SCI, 2005), keep melanocyte (Melanocyte MC) period stagnant in G1
Phase, and can induce the expression of P53, promote melanocyte early ageing (Cancer Res, 1995,55 (18)).
Academic circles at present for the UVB research for irradiating primary melanocyte is that its melanin content is caused to change and explain more
Bright UVB studies the primary melanocyte of UVB irradiation and its early ageing is caused to have quite for the influencing mechanism of B16 cell relevant molecule
Important substantive significance, therefore, cultivating, there is the primary melanocyte of more high sensitivity to be of great significance.
Summary of the invention
The present invention is improved on the basis of the primary melanocyte cultural method of general applications, and a kind of use is provided
The primary melanocyte cultural method of early ageing is caused in research UVB irradiation, is included the following steps:
(1) dermatological specimens are impregnated in ethanol, residual ethanol on sample is removed after taking-up；
(2) removal fat and subcutaneous tissue, dermatological specimens have been transferred in lid vessel, and trypsin solution is added and is incubated for
Overnight；
(3) after being incubated for, digestive juice is discarded, dermatological specimens is cleaned with PBS solution, abandons waste liquid；
(4) dermis of skin and epidermis are separated, epidermis is placed in PBS solution, is incubated for 30-60min at room temperature, obtains cell
Suspension；
(5) liquid is discarded supernatant after being centrifuged cell suspension, washs precipitating with PBS solution；
(6) it is then resuspended and is precipitated with melanocyte culture medium, the re-suspension liquid of acquisition moves to culture vessel, is placed in carbon dioxide
48-72h is cultivated in incubator, obtains primary melanocyte；
(7) continue to cultivate and regularly replace culture solution；
(8) for testing or freezing after passing on.
It further limits, dermatological specimens described in step (1) are male's foreskin sample.
It further limits, dermatological specimens described in step (1) impregnate in 70% ethanol solution, time 10-
30min。
It further limits, trypsin solution concentration described in step (2) is 0.25%；The incubation refers at 4 DEG C
14-20h is incubated in refrigerator.
It further limits, dermatological specimens described in step (2) are cut into the strip of wide 1-2mm.
It further limits, when separating dermis of skin and epidermis described in step (4), it is molten that a drop PBS is added dropwise on epidermis
Liquid.
It further limits, incubation described in step (4), which refers to shake under 80-150r/min revolving speed, to be incubated for.
It further limits, the revolving speed of centrifugation described in step (5) is 1500r/min, and the time of centrifugation is 3min.
It further limits, melanocyte culture medium described in step (6) is M254 culture medium, adds 1% (final concentration)
Human melanocytes grow replenishers and 1% (final concentration) is dual anti-, described dual anti-for penicillin and streptomysin.
The melanocyte that cultural method of the present invention obtains can be used for medium wave erythemal effect ultraviolet (UV) B irradiation and cause cell
The research of early ageing.
Beneficial effect
The present invention can be used for studying various dose medium wave erythemal effect ultraviolet light (ultraviolet radiation B,
UVB) primary melanocyte is irradiated and causes its early ageing, it is limited at present for UVB photograph in radiobiological studies to expand
Penetrate the research for causing primary melanocyte B16 cell content to influence.
A kind of primary melanocyte cultural method that early ageing is caused for studying UVB irradiation that the present invention establishes, advantage exist
In compared with conventional method, the primary melanocyte turned out is sensitiveer, is more suitable for UVB irradiation causes primary melanocyte early
The various researchs to decline, while one is provided to cultivate the experimental method of the primary melanocyte for studying UVB irradiation cause early ageing
Fixed reference value is that more preferably selecting for primary melanocyte experimental study is irradiated for various dose UVB.
Detailed description of the invention
Fig. 1 inverted phase contrast microscope observes the melanin tissue isolated under 100 times；
Fig. 2 inverted phase contrast microscope, the primary melanocyte that observation culture obtains under 100 times；
Fig. 3 inverted phase contrast microscope, the passage melanocyte that observation culture obtains under 100 times；
Fig. 4 various dose UVB irradiates primary melanocyte beta galactosidase Coloration experiment as a result, being followed successively by from left to right
0、20、50、80、100mJ/cm2The corresponding experimental result of five dosage irradiations.
Specific embodiment
Heretofore described 70% ethyl alcohol refers to that volume fraction is 70% ethanol solution.
PBS solution: phosphate buffer, ingredient Na2HPO4、KH2PO4, NaCl and KCl, wherein Na2HPO4、KH2PO4It is two
Grade dissociation, for buffering PH range, NaCl and KCl are to increase salt ionic concentration, preparation method are as follows:
Potassium dihydrogen phosphate KH2PO4: 0.27g, disodium hydrogen phosphate Na2HPO4:1.42g, sodium chloride nacl: 8g and potassium chloride
KCl:0.2g adds deionized water about 800mL, dissolution is sufficiently stirred, and concentrated hydrochloric acid tune PH to 7.4 is then added, is finally settled to 1L.
After autoclave sterilization, 4 DEG C of refrigerators are saved, and 1 × PBS buffer solution is that concentration is 0.01M, and (i.e. institute in the present invention can be used directly
The concentration used), 2 × PBS buffer solution concentration is 0.02M.
0.25% trypsin solution: 4.5mL is added in the trypsin solution for being 2.5% to the mass fraction of 0.5ml
Remove Ca2+、Mg2+D-Hanks liquid.The melanocyte culture medium is that (purchase adds 1% from gibco) to M254 culture medium
(final concentration) human melanocytes grow replenishers (Human Melanocyte Growth Supplement HMGS) and 1% (eventually
Concentration) it is dual anti-, wherein human melanocytes growth replenishers are bought from gibco, article No. M-254-500；1% dual anti-purchase is from green
The skies, article No. C0222, wherein dual anti-is penicillin and streptomysin.
Other reagents or drug etc. can be bought by commercialization approach obtain unless otherwise specified.
The medium wave erythemal effect ultraviolet (UV) B irradiation that embodiment 1. is used to study various dose causes the primary melanocyte of early ageing thin
Born of the same parents' cultural method.
(1) dermatological specimens are impregnated in ethanol, residual ethanol on sample is removed after taking-up；Specifically: adult healthy male
Property foreskin 2, sample, is derived from Beijing hospital Urology Surgery.Foreskin sample is impregnated into 20min in 70% ethyl alcohol respectively, is taken out
Sample is cleaned with PBS solution afterwards, removes remaining ethyl alcohol on sample.
(2) fat and subcutaneous tissue are cut as much as possible, and dermatological specimens are cut into the strip of wide 1-2mm, strip is broken
Piece is transferred in centrifuge tube, and 0.25% trypsin solution is added, is fully immersed in skin samples in enzyme solution, covers nozzle,
It is incubated for 18 hours in 4 DEG C of refrigerators.
(3) after being incubated for, epidermis digestive juice (i.e. 0.25% trypsin solution) is discarded, cleans dermatological specimens with PBS liquid, abandoned
Waste liquid.
(4) corium and epidermis are separated with blade, discards corium, the epidermis for dripping PBS liquid on epidermis to prevent separation
Become excessively dry in air；Epidermis is transferred in the centrifuge tube containing PBS solution, so that epidermis is fully immersed in PBS molten
In liquid, at room temperature under 80-150r/min revolving speed, rotation, which is shaken, is incubated for 60min, cell suspension is obtained, wherein black containing part
Pigment tissue is shown in Fig. 1.
(5) at room temperature, cell suspension is centrifuged 3min, revolving speed 1500r/min is discarded supernatant, and it is heavy to be washed with PBS solution
It forms sediment, liquid is discarded supernatant after washing, retain precipitating, repeat above-mentioned centrifugation and washing operation 3 times.
(6) then, it is resuspended and is precipitated with melanocyte culture medium, the melanocyte culture medium is M254 culture medium, addition
1% (final concentration) human melanocytes grow replenishers (Human Melanocyte Growth Supplement, HMGS) and 1%
(final concentration) is dual anti-, and the re-suspension liquid of acquisition moves to culture bottle, is placed in carbon dioxide incubator at 37 DEG C, 5%CO2Under conditions of
48h is cultivated, that is, obtains primary melanocyte, as shown in Figure 2.
(7) centrifugation replacement culture solution carries out twice, continuing culture 7 days for one week.
(8) more and more melanocyte adherent growths, growth cycle is longer, and fritter tissues can be complete within general 15 days or so
Part dissipates adherent growth；After culture bottle covers with, is passed on and frozen；Fig. 3 show second generation cell, repeats experimental procedure and mentions
A large amount of melanocytes are taken to freeze, in case experiment uses.
The medium wave erythemal effect ultraviolet (UV) B irradiation that embodiment 2. is used to study various dose causes the primary melanocyte of early ageing thin
Born of the same parents' cultural method.
Embodiment 1 is repeated, is with the difference of embodiment 1, the step 1) dermatological specimens are baby or child's foreskin sample
This.
The medium wave erythemal effect ultraviolet (UV) B irradiation that embodiment 3. is used to study various dose causes the primary melanocyte of early ageing thin
Born of the same parents' cultural method.
Embodiment 1 is repeated, is with the difference of embodiment 1, is cultivated described in step 6) with melanocyte culture medium M254
The outstanding precipitating of base weight, the re-suspension liquid of acquisition move to culture bottle, are placed in carbon dioxide incubator at 37 DEG C, 5%CO2Under conditions of train
72h is supported, that is, obtains primary melanocyte.
By taking 1 cultural method of embodiment as an example, the present invention is utilized respectively 0,20,50,80,100mJ/cm2The UVB of dosage irradiates
The primary melanocyte 72h turned out, beta galactosidase Coloration experiment result have statistical significance, as shown in figure 4, with
The quantity of the increase of dosage, senile cell dramatically increases, it can be seen that, the cell that above method culture obtains can be used for difference
Dosage UVB irradiates the experimental study of primary melanocyte.
Claims (10)
1. a kind of primary melanocyte cultural method for causing early ageing for studying UVB irradiation, which is characterized in that this method includes such as
Lower step:
(1) dermatological specimens are impregnated in ethanol, residual ethanol on sample is removed after taking-up；
(2) removal fat and subcutaneous tissue, dermatological specimens have been transferred in lid vessel, and trypsin solution is added and was incubated for
Night；
(3) after being incubated for, digestive juice is discarded, dermatological specimens is cleaned with PBS solution, abandons waste liquid；
(4) dermis of skin and epidermis are separated, epidermis is placed in PBS solution, is incubated for 30-60min at room temperature, it is outstanding to obtain cell
Liquid；
(5) liquid is discarded supernatant after being centrifuged cell suspension, washs precipitating with PBS solution；
(6) it is then resuspended and is precipitated with melanocyte culture medium, the re-suspension liquid of acquisition moves to culture vessel, is placed in carbon dioxide culture
48-72h is cultivated in case, obtains primary melanocyte；
(7) continue to cultivate and regularly replace culture solution；
(8) for testing or freezing after passing on.
2. cultural method according to claim 1, which is characterized in that dermatological specimens described in step (1) are male's foreskin
Sample.
3. cultural method according to claim 1, which is characterized in that dermatological specimens described in step (1) are in 70% ethyl alcohol
It is impregnated in solution, time 10-30min.
4. cultural method according to claim 1, which is characterized in that trypsin solution concentration described in step (2) is
0.25%；The incubation, which refers to, is incubated for 14-20h in 4 DEG C of refrigerators.
5. cultural method according to claim 1, which is characterized in that dermatological specimens described in step (2) are cut into wide 1-
The strip of 2mm.
6. cultural method according to claim 1, which is characterized in that separation dermis of skin and epidermis described in step (4)
When, a drop PBS solution is added dropwise on epidermis.
7. cultural method according to claim 1, which is characterized in that incubation described in step (4) refers to 80-150r/min
It shakes and is incubated under revolving speed.
8. cultural method according to claim 1, which is characterized in that the revolving speed of centrifugation described in step (5) is 1500r/
Min, the time of centrifugation are 3min.
9. cultural method according to claim 1, which is characterized in that melanocyte culture medium described in step (6) is
M254 culture medium, 1% (final concentration) human melanocytes of addition growth replenishers and 1% (final concentration) are dual anti-, described dual anti-for blueness
Mycin and streptomysin.
10. the melanocyte wave erythemal effect ultraviolet light under study for action that cultural method described in claim 1-9 any one obtains
UVB irradiation causes the application in cell early ageing.
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Cited By (1)
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| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
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| CN112458040B (en) * | 2020-12-23 | 2022-07-05 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
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