CN106520671A - In vitro isolated culture method for rabbit melanophore - Google Patents
In vitro isolated culture method for rabbit melanophore Download PDFInfo
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Abstract
The invention relates to an isolated culture method for primary generation rabbit melanophore. According to the method, the rabbit melanophore is isolated through an enzyme digestion method, and after culturing is conducted through an M254 complete medium, an M254/HMGS culture medium is adopted through replacement for culturing till the convergence degree reaches 80%-90%, then a differential digestion method is utilized for removing other cells, and the purified melanophore is obtained. The purity of the melanophore is greatly improved, the cultured cells are identified through cell morphologic observation, L-DOPA staining, semiquantitation and an anti-S-100 immunocytochemical staining method, the result shows that the isolated primary generation melanophore is mostly in a dendritic shape and a thin and long shuttle shape, the brownish black is shown after L-DOPA staining, the RT-PCR analysis shows that the isolated cells express the marker gene of the melanophore, and the S-100 staining cytoplasm is brown.
Description
Technical field
The present invention relates to cytogeneticss and biology field, and in particular to external point of a kind of rabbit melanocyte
From cultural method.
Background technology
Mammal skin and various colors are had by hair, caused by being the melanin by stable chemical nature,
Brown-black element and eumelanin can be divided into, they are that L-Tyrosine or relevant composition are aoxidized under the catalysis of corresponding enzyme.
Gene control the formation of hair color pigment and type, so as to determine the hair color of mammal.Rabbit is used as a kind of important fur
Animal and test model, its hair color also very abundant, have various natural colours such as black, grey, yellow, blue, without the need for printing and dyeing, enjoy consumption
Person favors.But, as living animal experiment is difficult, lack suitable cell model again, people seriously hindered to family
The research of rabbit hair color genetic mechanism.This method with black rabbit as experiment material, Isolation and culture rabbit melanocyte, and by thin
The methods such as born of the same parents' dyeing are identified, can be used for exploring the expression and regulation and control of hair color related gene, be grinding for coat color gene function
Offer theory and practice basis is provided.
The content of the invention
In order to solve the problems, such as to lack suitable cell research model in rabbit color inheritance research, the invention provides one
The In-vitro separation culture method of kind of rabbit melanocyte, can separation and Culture and identification rabbit melanocyte in vitro, contribute to out
The expression regulation research work of exhibition hair color related gene, the also research for color inheritance mechanism provide theory and practice basis.
The technical solution adopted in the present invention is that a kind of In-vitro separation culture method of rabbit melanocyte, is by enzyme
Digestion method separates rabbit melanocyte, changes M254/HMGS culture medium culturings to degree of converging Jing after M254 complete medium cultures
Reach 80%～90%, recycle differential digestion method to remove other cells such as horn cell and fibroblast, substantially increase
The purity of aim cell.
The method of the invention specifically includes following steps：
1) separation of rabbit melanocyte is obtained
A) skin of 1.5cm × 1.5cm sizes is taken, pre-cooling PBS is rinsed 3 times, removes subcutaneous connective tissue；
B) skin be cut into the skin chunk of 0.2cm × 0.5cm sizes, be put in the Dispase II enzymic digestion liquid of 20mL, 4
DEG C digested overnight (14-16h)；
C) again skin is put in Dispase II enzymes, 37 DEG C of incubation 30min；Take epidermis off；
D) epidermis taken off is collected in the culture dish of 35mm dryings, adds 0.25% pancreatin, 37 DEG C of digestion of 500 μ L
8min；
E) 2mL serum is added to terminate digestion, for several times, 200 mesh filter screen filtrations, 1000rpm are centrifuged 5min for piping and druming；
F) add 2mL M254 complete mediums resuspended, insert in 35mm culture dishs；
G), after cultivating 1h, change 2mL M254/HMGS culture medium into, a subculture is changed every 2d；
2) rabbit melanocyte is passed on and purification
A) when degree of converging reaches 80%～90%, 0.25% tryptic digestive juice is added, digests 1～2min, first
The cell type that digestion splits away off is melanocyte；
B) rapidly join 2mL serum and terminate digestion, 1000rpm centrifugation 5min abandon clear liquid；
C) 3mL M254/HMGS culture medium, re-suspended cell are added；
D) according to determine cell concentration by cell suspension renewed vaccination in 60mm culture dishs, be put into CO2Cell culture
In case, 37 DEG C are continued culture, change liquid every 2d；
E) observation of cell upgrowth situation, differential digestion are obtained pure melanocyte 3 times.
Step b) of the present invention c) in, using it is cold digestion and heat digestion combine, be more prone to take the epidermis of rabbit off, rabbit
It is thicker that sub- skin compares other species skins, and epidermis and corium can be more difficult with Dispase II enzymic digestions, cold digestion overnight it
Half an hour heat digestion again, can be easier to take epidermis off afterwards.
In step f) of the present invention, M254/HMGS culture medium, this side after one hour of M254 complete mediums culture, are changed
Method can reduce the presence of other cells in addition to melanocyte.M254 complete mediums are to add tire in M254 culture medium
Ox blood serum (9:1).
The present invention in an experiment, studies the culture medium combination for knowing clearly different, as a result shows the selection of culture medium to effect shadow
Sound is very big.Fig. 5 shows：Using the effect (Fig. 5 A) that M254/HMGS culture medium is changed after DMEM/F12 culture medium culturing 1h：Carefully
Born of the same parents' number of adherent is less than using replacing M254/HMGS culture medium (Fig. 5 B) after M254 complete medium culture 1h.Fig. 6 shows,
Using direct after the effect (Fig. 6 A) of M254/HMGS culture medium being changed after M254 complete medium culture culture 1h and being separated
Using M254/HMGS culture medium (Fig. 6 B) effect difference：The quantity of melanocyte is apparently higher than directly using M254/
The melanocyte quantity of HMGS culture medium culturings.To combining cell shape by the melanocyte of the inventive method separation and Culture
The multiple technologies means such as state observation, sxemiquantitative, L-DOPA dyeing, anti-S-100 immunocytochemical stains method, identify separation and Culture
Melanocyte, as a result shows, is in dendron shape and elongated fusiform detached primary melanocyte more, and L-DOPA dyeing is in palm fibre
Black positive reaction, PCR analysis result display separation cells contain the marker gene of melanocyte, and S-100 dyeing kytoplasms are
Brown color.Illustrate that the cell cultivated under the conditions of Selective agar medium has the form of melanocyte, melanocyte can be expressed
Mark, can synthesizing black crude granule, cell model can be provided for color inheritance Mechanism Study.
Description of the drawings
Fig. 1：The form (100 ×) of the 1st, 3,5,7 generation rabbit melanocyte
A：1st generation melanocyte；B：3rd generation melanocyte；C：5th generation melanocyte D：7th generation melanin is thin
Born of the same parents.
Fig. 2：5th generation rabbit melanocyte L DOPA is dyeed
A：Before L DOPA dyeing；B：After L DOPA dyeing.
Fig. 3：Anti- 100 immunocytochemical stains of S of 5th generation rabbit melanocyte
A：Before immunocytochemical stain；B：After immunocytochemical stain.
Fig. 4:Expression of MITF, TYR, TYRP1 gene in melanocyte.
Fig. 5 is three days design sketchs afterwards of different culture media separation and Culture.A. using DMEM/F12 culture medium culturings 1h it
The effect of M254/HMGS culture medium is changed afterwards；B. using replacing M254/HMGS culture medium after M254 complete medium culture 1h
Effect.
Fig. 6 is using the effect of replacing M254/HMGS culture medium after M254 complete medium culture culture 1h and separates
M254/HMGS culture medium design sketch is used afterwards directly.A. using changing M254/ after M254 complete medium culture culture 1h
HMGS culture medium；B. M254/HMGS culture medium is directly used after separating.
Specific embodiment
Embodiment 1
1 materials and methods
1.1 experiment material
(1) laboratory animal：6 monthly ages black rabbit.
(2) test equipment：Ophthalmic tweezers, eye scissorss, stainless steel filtering net (200 mesh), scalpel etc..
(3) main agents：M254/HMGS culture medium, PBS, 0.25% tryptic digestive juice, 0.25%
Dispase II Digestive systems, SABC immunohistochemical staining test kits, DAB colour reagent boxes, Mouse Anti-S-100,0.1%
TritonX-100, PBS, hyclone (FBS), dual anti-(mycillin mixed liquor 100 ×), dimethyl sulfoxide
(DMSO)。
The separation of 1.2 rabbit melanocytes is obtained
(1) barbital sodium solution anaesthesia experiment rabbit, 75% ethanol are sterilized to sample region, and Shearing shearses shave off dorsal body setae, take one piece
1.5cm × 1.5cm skin histologies, rinse 3 times repeatedly with the PBS containing dual anti-pre-cooling.
(2), during be put into skin histology containing dual anti-PBS culture dishs, super-clean bench ophthalmic tweezers be put into and eye scissorss removal is subcutaneous
Dirty bloodstain and connective tissue, with containing dual anti-PBS flushings 3 times.
(3) it is skin edge amendment is neat with scalpel, note epidermis down, it is to avoid damage of the scalpel to epidermis.With
Skin is cut into eye scissorss the skin chunk of 0.2cm × 0.5cm sizes, is put in the Dispase II enzymic digestion liquid of 20mL,
The volume of Dispase II enzymes is ten times of skin volume, and 4 DEG C digest 14～16h (overnight).
(4) skin is taken out to be put in the PBS of 4 DEG C of pre-coolings and is rinsed 3 times, be put in dry culture dish, epidermis down, is used
Fine forcepss are first attempted taking epidermis off, if epidermis is not easy to take off, are placed again into 37 DEG C of incubation 30min in Dispase II enzymes, it
After take out skin, rinse 3 times in the PBS of pre-cooling, then epidermis taken off with finely pinching in dry culture dish, epidermis is very
The material of thin transparence, the epidermis taken off are collected in the culture dish of the drying of 35mm, add 0.25% pancreatin 37 of 500 μ L
DEG C digestion 8min, 2mL serum terminate digestion, blown and beaten with pipette tips repeatedly, 200 mesh filter screens filter, 1000rpm centrifugation 5min, add
2mLM254 complete mediums are resuspended, insert in 35mm culture dishs, after culture 1h, change 2mL M254/HMGS culture medium into, every
2d changes a subculture, and the growth conditions of observation of cell, if cell confluency degree reaches 80%～90%, carries out the pure of cell
Chemical industry is made.
1.3 rabbit melanocytes are passed on and purification
(1) cell growth status in culture plate are observed, when degree of converging reaches 80%～90%, sucks culture medium, added
500 μ L0.25% tryptic digestive juices, gently rock, using differential digestion method remove horn cell and fibroblast etc. its
His cell type, examines under a microscope cell dissociation situation, and it is melanocyte to digest the cell type for splitting away off first,
General digestion time is in 1～2min.
(2) rapidly join 2mL serum and terminate digestion, gently blow and beat repeatedly, cell suspension is collected ready in advance
In 15mL centrifuge tubes, 1000rpm centrifugation 5min, abandoning supernatant add 3mL M254/HMGS culture medium, re-suspended cell.
(3) draw a small amount of cell suspension and add trypan blue working solution, counted in instilling cell counting count board, according to survey
Cell suspension renewed vaccination in 60mm culture dishs, is put into CO by fixed cell concentration2In cell culture incubator, 37 DEG C are continued culture,
Liquid is changed every 2d.
(4) observation of cell upgrowth situation, differential digestion are obtained pure melanocyte 3 times.
The frozen and recovery of 1.4 rabbit melanocytes
1.4.1 melanocyte is frozen
(1) growth conditions of melanocyte are observed on time, when the cell density in culture plate reaches 70%～80%,
Culture medium is sucked, the pancreatin Digestive system of 1mL 0.25% is added, 2min is digested.
(2) under inverted microscope observation of cell metamorphosis, when cell kytoplasm form generation retraction and be rounded, carefully
Gap between born of the same parents becomes big, and when gently rocking culture plate and having a large amount of cell detachments, the serum for rapidly joining 2mL terminates digestion, instead
It is multiple lightly to blow and beat culture plate bottom so as to form cell suspension, to make sure to keep in mind in piping and druming, action is soft, in order to avoid cause energetically
Cell injury, affects the growth vigor of cell.
(3) cell suspension is collected in ready centrifuge tube, 1000rpm centrifugation 5min, supernatant discarded draw 1mL
Frozen stock solution re-suspended cell, moves in cryopreservation tube, carries out labelling, is put in cell cryopreservation box (Automatic Program cooling), -80 DEG C of mistakes
Night, next day proceed to cell cryopreservation tube in liquid nitrogen and preserve, and speed is wanted in action.
1.4.1 melanocyte recovery
(1) the frozen rabbit melanocyte of a pipe is taken out from liquid nitrogen container, immerse rapidly (cryopreservation tube in 37 DEG C of water-bath
Want endways and be put into water-bath, liquid level can not have the lower edge of lid, to prevent contamination of cells), cryopreservation tube is rocked back and forth accelerates pipe
The thawing of interior frozen stock solution.
(2) after solid melts completely, rapid 75% ethanol sprays whole cryopreservation tube, puts into super after cleaning tube wall liquid
Net platform, is blown and beaten with pipettor and suction out after cell suspension several times, in adding the centrifuge tube containing 10mL M254 complete mediums,
L000rpm centrifugation 5min, supernatant discarded, PBS re-suspended cells, 1000rpm centrifugations 5min, is finally cultivated with M254/HMGS again
Base re-suspended cell, is inoculated in Tissue Culture Plate according to suitable concentration, is put into CO237 DEG C of quiescent cultures in cell culture incubator,
The adherent situation of next day observation of cell changes culture medium in good time, continues culture.
The identification of 1.5 rabbit melanocytes
1.5.1 L-DOPA dyeing
(1) the 5th generation melanocyte is taken, in exponential phase, is inoculated in 6 orifice plates, 37 DEG C, 5%CO2Culture 2d.
PBS 2 times, 4% paraformaldehyde room temperature fix 20min.
(2) pre-cooling PBS 2 times, add 0.1% L-DOPA, 37 DEG C of incubation 4h, after updating Incubating Solution, 37 DEG C of continuation
Incubation 18h, this process need lucifuge always.
(3) after the completion of to be dyed, then rinsed 3 times with PBS, basis of microscopic observation collection image.
1.5.2 RT-PCR detections
(1) when cell confluency degree reaches more than 90%, about 1～5 × 105Individual cell, is gently flushed three times with PBS, is added
1mL Trizol, extract total serum IgE according to kit specification, determine OD with nucleic acid-protein analyzer230/260And OD260/280Ratio
Value and concentration, take the quality that 1 μ LRNA, 1% agarose gel electrophoresiies detect RNA, and remaining sample is protected in being put in -80 DEG C at once
Deposit.
(2) in the reaction system for configuring reverse transcription on ice according to following ingredients：5×PrimeScriptTMBuffer4 μ L,
PrimeScriptTM1 μ L of 1 μ L of RTEnzymeMixI1 μ L, OligodTPrime (50 μM), Random6mers (100 μM),
2 μ L of TotalRNA, RnaseFreedH2O adds to 20 μ L, by total serum IgE reverse transcription into cDNA.
(3) sequence according to rabbit Tyr, the Tyrp1 and MITF gene announced in NCBI, soft using 7 design of primers of oligo
Part designs PCR primer, and primer information is as shown in table 1.Enter performing PCR amplification, reaction system：By 10 μ L 2 × Taq PCR Mix, 7 μ
L aquesterilisa, 1 μ LcDNA, each 1 μ L of upstream and downstream primer, in adding 100 μ LPCR pipes, the reaction system of 20 μ L, fully mixes altogether
After be put in instrument, detect expression of the specific gene in melanocyte.
1. primer information of table
(4) 1% agarose gel (adding the Goldview of 0.5 μ L) is prepared, thoroughly gel is softly put into electricity by solidification
In swimming groove, appropriate electrophoresis liquid is added, glue 30min is run under the voltage of 100V, after race glue is finished, glue is taken out in gel imaging system
Expose in system and adopt figure.
1.5.3 anti-S-100 immunocytochemical stains
(1) melanocyte of the 5th generation in exponential phase is taken, adjustment cell concentration is 2 × 105/ mL, is inoculated in
In six orifice plates, 37 DEG C, 5%CO2Incubator culture 2d.
(2) PBS washes 2 × 2min, and 4% paraformaldehyde room temperature fixes 20min, and PBS washes 3 × 2min.
(3) 0.1%TritonX-100 room temperature treatments 20min, PBS wash 3 × 2min.
(4) Deca 5%BSA confining liquid, room temperature, 20min suction out confining liquid, do not rinse.
(5) add anti-S-100 antibody (1:200), negative control is replaced with PBS, and 4 DEG C overnight, and PBS rinses 3 × 2min.
(6) Deca biotinylated goat antimouse IgG, 37 DEG C, 20min, PBS wash 3 × 2min.
(8) Deca reagent SABC, 37 DEG C, 20min, PBS wash 4 × 5min.
(9) DAB colour developings：Take A, B, C reagent each to drip in 1mL distilled water, after mixing, instill in 6 orifice plates, room temperature shows
The response time is controlled under color, mirror, typically between 5～30min.
2 results
By various conditions grope attempt, isolate rabbit melanocyte using enzyme digestion first, carry out identification and
Secondary Culture (see accompanying drawing 1), is dyeed by L-DOPA, it is seen that cell is dyed to brownish black (see accompanying drawing 2), detects melanin mark
Expression of the gene in cell is separated, it is found that MITF, TYR, TYRP1 gene is expressed (see accompanying drawing 3), anti-S-100 immunocytes
Chemical staining, it is seen that kytoplasm intrinsic color, in brown color (see accompanying drawing 4).As a result, it was confirmed that under the conditions of Selective agar medium, separating training
Foster cell has the form of melanocyte, can express the marker gene of melanocyte, being capable of synthesizing black crude granule.
SEQUENCE LISTING
<110>Yangzhou University
<120>A kind of In-vitro separation culture method of rabbit melanocyte
<130>
<160> 8
<170> PatentIn version 3.3
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Claims (2)
1. a kind of In-vitro separation culture method of rabbit melanocyte, it is characterised in that be that rabbit black is separated by enzyme digestion
Plain cell, Jing after M254 complete medium cultures changes M254/HMGS culture medium culturings to degree of converging and reaches 80%～90%, then
The melanocyte that other cells obtain purification is removed using differential digestion method.
2. method according to claim 1, it is characterised in that：Comprise the following steps：
1) separation of rabbit melanocyte is obtained
A) skin of 1.5cm × 1.5cm sizes is taken, pre-cooling PBS is rinsed 3 times, removes subcutaneous connective tissue；
B) skin be cut into the skin chunk of 0.2cm × 0.5cm sizes, be put in the Dispase II enzymic digestion liquid of 20mL, 4 DEG C of mistakes
Night digests 14-16h；
C) skin is put in dry culture dish and takes epidermis off, if epidermis is difficult to take off, skin is put into into Dispase again
In II enzymes, 37 DEG C of incubation 30min；
D) epidermis taken off is collected in the culture dish of 35mm dryings, adds 0.25% pancreatin, 37 DEG C of digestion 8min of 500 μ L；
E) 2mL serum is added to terminate digestion, for several times, 200 mesh filter screen filtrations, 1000rpm are centrifuged 5min for piping and druming；
F) add 2mL M254 complete mediums resuspended, insert in 35mm culture dishs；
G), after cultivating 1h, change 2mL M254/HMGS culture medium into, a subculture is changed every 2d；
2) rabbit melanocyte is passed on and purification
A) when degree of converging reaches 80%～90%, 0.25% tryptic digestive juice is added, digests 1～2min, digest first
The cell type for splitting away off is melanocyte；
B) rapidly join 2mL serum and terminate digestion, 1000rpm centrifugation 5min abandon clear liquid；
C) 3mL M254/HMGS culture medium, re-suspended cell are added；
D) according to determine cell concentration by cell suspension renewed vaccination in 60mm culture dishs, be put into CO237 in cell culture incubator
DEG C continue culture, change liquid every 2d；
E) observation of cell upgrowth situation, differential digestion are obtained pure melanocyte 3 times.
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Cited By (4)
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| CN108359633A (en) * | 2018-04-19 | 2018-08-03 | 山东农业大学 | A kind of beaver rabbit dermis of skin hair papilla cell isolated culture method |
| CN108567996A (en) * | 2017-03-07 | 2018-09-25 | 武汉北度生物科技有限公司 | A kind of preparation and its application of 3D multilayer structures cell patch |
| CN110951675A (en) * | 2019-12-23 | 2020-04-03 | 中国水产科学研究院珠江水产研究所 | Method for separating and culturing melanocytes of orange-colored double-crowned fish |
| CN115340974A (en) * | 2022-08-15 | 2022-11-15 | 元道生命科技(武汉)有限公司 | Preparation method of mouse-derived epidermal melanocytes |
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